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Radiosensitization by High Affinity Programmable Minor Groove Binders
Volume 87 Number 2S Supplement 2013
3172 High Throughput Clonogenic Survival Screen Identifies Novel Radiation Sensitizers for K-ras Mutant Non-Small Cell Lung Cancer S.H. Lin,1 J. Zhang,1 U. Giri,1 C. Stephan,2 M.M. Sobieski,2 N. Nguyen,2 P. Davies,2 R. Komaki,1 S.S. Yoo,3 and J.V. Heymach1; 1University of Texas MD Anderson Cancer Center, Houston, TX, 2Texas A&M Health Science Center, Houston, TX, 3National Cancer Institute, Bethesda, MD Purpose/Objective(s): Traditional clonogenic survival and high throughput colorimetric assays are inadequate for drug screens to identify novel radiation sensitizers. To bridge this gap in knowledge, we have developed a method which we coin the High Throughput Clonogenic Survival Screen (HCS) that will allow high volume screening of drug libraries to identify potent radiation sensitizers, particularly for K-ras mutant lung cancer, a current unmet need. Materials/Methods: Drugs at various concentrations or vehicle were added to cells seeded overnight in 96 well plates at low densities, and after 6 hours, plates were irradiated at various doses. Plates were kept in culture 4-6 days depending on the cell line. After incubation, colonies were stained with cresyl-violet, imaged on a high content screening system, and colony count determined using a proprietary image analysis software. Colonies achieving 50 or more cells were tallied. Traditional clonogenic survival assays (tCSA) were performed by seeding cells into 6-well plates overnight in triplicate and adding drugs or vehicle for 6 hours prior to irradiation. Media was changed after 3 days, and plates were kept in culture for 10-14 days. Drug screens were done using the K-ras mutant lung cancer cell line H460 and the Custom Clinical Collection (145 compounds). Results: We compared the HCS method to tCSA and found that HCS recapitulates a similar cell survival curve and survival fraction at 2 Gy (SF2) values for several cancer cell types. As a proof-of-principle, we tested a known sensitizer vorinostat (SAHA) in both the tCSA and HCS assays. As expected, 1 mM vorinostat caused a significant radiation sensitizing effect using tCSA, and was recapitulated, albeit at a lower concentration (100 nM), using the HCS assay. Drug screens (repeated 3 times) identified several compounds that were cytotoxic to the K-ras mutant H460 cells alone at 1 mM (inhibitors to Src/Abl, IGF1R, ROCK1, AKT, PI3K, c-MET) and at low nM concentrations (HSP90 and HDAC inhibitors). The entire class of HDAC inhibitors (5 drugs) had some sensitizing effect to radiation (a leftward shift in the IC50 curve by w10 fold). However, many drugs such as inhibitors to PARP, SRC, Chk1/2, DNA-PK, JNK, and MEK1/2, exhibited significant radiation sensitizing effect with minimal to no activity by themselves. We validated the sensitizing effect of the potent MEK1/2 inhibitor trametinib using tCSA, and found the effect was specific for K-ras mutant and not K-ras wild type cells. Conclusions: High throughput drug screening for novel radiation sensitizers for K-ras mutant lung cancer is feasible using the HCS approach. This is an enabling technology that will allow synthetic lethal drug screens with radiation and accelerate the translation of drugs into clinical testing. Author Disclosure: S.H. Lin: None. J. Zhang: None. U. Giri: None. C. Stephan: None. M.M. Sobieski: None. N. Nguyen: None. P. Davies: None. R. Komaki: None. S.S. Yoo: None. J.V. Heymach: None.
3173 Design and Optimization for Combination of 188Re-Liposome and External Beam Radiation Therapy Against Human Esophageal Adenocarcinoma Xenograft Y. Chen,1 C. Chi,1 Y. Lee,2 T. Chang,3 and C. Chang3; 1Mackay Memorial Hospital, Taipei, Taiwan, 2National Yang-Ming University, Taipei, Taiwan, 3 Institute of Nuclear Energy Research, Taoyuan, Taiwan Purpose/Objective(s): The external beam radiation therapy (EBRT) can deliver high-energy radiation beam to cover both gross tumor and potential
Poster Viewing Abstracts S651 microscopic tumor cells in tumor vicinity. Radioisotope therapy (RIT) by using isotopes emitting relatively short-distance radiation can directly eradicate gross tumor inside torso. Esophageal cancer, one of the most treatment resistant malignancies with high potential to develop local recurrence and distant metastasis, has a dismal prognosis. We proposed that combination of EBRT and RIT may deliver radiation to simultaneously treat primary tumor, regional lymphatics and distant sites at risk of systemic metastasis. Rhenium 188 (188Re)-liposome is a nanoparticle emitting gamma (for molecular imaging validation) and beta (for therapeutic RT) rays with biodistribution profile preferential to tumor. We established an experimental model of combinatory treatment and examined the effect of the EBRT and 188Re-liposome on human esophageal cancer xenograft. Materials/Methods: The 188Re-liposome with quality assurance was prepared according to previous work in clinical trial. Human esophageal cancer cell lines BE-3 (adenocarcinoma) and CE81T/VGH (squamous cell carcinoma) expressing luciferase gene for optical imaging were implanted to nude mice for xenograft. The imaging modalities used included optical and nanoSPECT/CT imaging systems to examine the tumor uptake of 188Re-liposome and treatment results. Optimal dosing for EBRT and 188Re-liposome were assessed in a clinical practiceoriented way. Results: As for quality assurance, the labeling efficiency of 188ReEMBDA (Rf Z 0) was 100%. The labeling efficiency and total activity of 188Re-liposome were 76.5% and 8731 mCi, respectively. For molecular imaging, nanoSPECT/CT scan results showed that esophageal adenocarcinoma BE-3, but not squamous cell carcinoma CE81T/VGH, xenograft could uptake the 188Re-liposome. Treatment of 188Re-liposome had no significant toxicity in mice. The EBRT alone inhibited the growth of BE-3 xenograft in a dose-dependent manner. The optimization of combinatory regimens revealed that EBRT with 3 Gy and 188Reliposome with 310 mCi might be feasible for further preclinical study. The treatment endpoints including tumor growth delay, survival and toxicity for various combinatory regimens and fractionation schedules will be presented. Conclusions: The combination of EBRT with 188Re-liposome might be a potential treatment modality for esophageal cancer by providing molecular imaging, image-guided treatment and comprehensive radiation therapy coverage simultaneously. Author Disclosure: Y. Chen: None. C. Chi: None. Y. Lee: None. T. Chang: None. C. Chang: None.
3174 Radiosensitization by High Affinity Programmable Minor Groove Binders N. Nickols,1 F. Yang,2 J.A. Ratikan,1 B.C. Li,2 W.H. McBride,1 and P.B. Dervan2; 1Department of Radiation Oncology, University of California Los Angeles Medical Center, Los Angeles, CA, 2Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA Purpose/Objective(s): Non-genotoxic small molecules of low systemic toxicity that can potentiate the effects of radiation could be valuable adjuvant therapeutics for solid tumors. Pyrrole-imidazole (Py-Im) polyamides are synthetic oligomers that bind the minor groove of DNA noncovalently with programmable sequence recognition and high affinity. We reported that a polyamide targeted to the DNA sequence 5’-WGWWCW3’ (W Z A/T) suppressed growth of prostate tumor xenografts. In cell culture, this polyamide interferes with RNA Polymerase II activity and results in a dose-dependent depletion of the RNA Polymerase II large subunit. We hypothesized that high-affinity DNA binding Py-Im polyamides may enhance the effects of ionizing radiation. Materials/Methods: We synthesized a polyamide targeting 5’WGWWCW-3’ by solid phase methods. The human laryngeal squamous cell carcinoma cell line UM-SCC-12 was treated with the polyamide at 0 (vehicle) or 20 nM for 48 hours and then irradiated to doses of 0, 2, 4,
S652
International Journal of Radiation Oncology Biology Physics
and 6 Gy. Twenty-four hours after irradiation, cells were re-plated and surviving fractions measured by clonogenic assay. Results: Polyamide treatment alone did not decrease surviving fraction in the absence of irradiation. The radiosensitization by polyamide treatment increased with radiation dose. Surviving fractions at 2 Gy were 0.660.07 and 0.550.05 (SER 1.2); at 4 Gy were 0.410.02 and 0.220.02 (SER 1.9); at 6 Gy were 0.220.07 and 0.110.01 (SER 2.0) for vehicle and polyamidetreated cells, respectively. Calculated D0 values were 4.16 Gy (vehicle) and 2.79 Gy (polyamide). SER: sensitization enhancement ratio. Errors are standard deviations (n Z 3). Conclusions: The polyamide demonstrated radiosensitization of UM-SCC12 cells in vitro. These results justify continued investigations into the therapeutic combination of irradiation and minor groove binding polyamides. Author Disclosure: N. Nickols: None. F. Yang: None. J.A. Ratikan: None. B.C. Li: None. W.H. McBride: None. P.B. Dervan: None.
3176
3175 Normal Tissue Tolerance of Microplanar Beam X-ray: A Long-term Observation N. Mukumoto,1 D. Miyawaki,1 H. Akasaka,1 M. Nakayama,1 Y. Miura,2 K. Umetani,3 N. Nariyama,3 K. Shinohara,4 T. Kondoh,5 and R. Sasaki1; 1 Division of Radiation Oncology, Kobe University Graduate School of Medicine, Kobe City, Hyogo, Japan, 2Department of Rehabilitation Science, Kobe University Graduate School of Health Sciences, Kobe City, Hyogo, Japan, 3Japan Synchrotron Radiation Research Institute, Mikazuki, Sayo, Hyogo, Japan, 4Advanced Research Institute for Science and Engineering, Waseda University, Tokyo, Japan, 5Department of Neurosurgery, Shinsuma Hospital, Kobe City, Hyogo, Japan Purpose/Objective(s): Synchrotron X-ray beams permit the delivery of very high radiation doses to tumors in a single fraction using arrays of microplanar X-ray beams. Microplanar beam (MPB) radiation is, at present, an experimental approach that has produced, in single exposures, exceptionally high tolerance in the normal tissues while preferentially damaging malignant tumors. In this study, we have challenged to evaluate normal tissue tolerance and mortality by MPB radiation to mouse brain with comparing to broad beam from the same radiation source. Materials/Methods: Synchrotron X-ray beam radiation was performed using BL28B2 beam-line at the Spring-8 facility, Hyogo, Japan. For each subgroup, male C57BL/6J mice aged 6-8 weeks that weighted approximately 20-25 g were fixed to the stereotactic metal frame. As for radiation techniques, anterior-posterior windows were set, and a broad or a 12 10 mm unidirectional array of MPB (25 micrometer width, 100-300 micrometer center-to-center spacing) were used. Delivered doses as the broad beam were 12-120 Gy, while the doses as the MPB were 120-960 Gy. Mortalities within 90 days after the irradiation were scored and plotted by using KaleidaGraph software. Histopathological evaluation by hematoxylin and eosin (HE) staining was performed using the irradiated brain tissues. Results: In the experimental setting, 50% and 100% lethal dose by the 200 micrometer spacing MPB beam were 600 Gy and 720 Gy, while those by the broad beam were 80 Gy and 96 Gy. Beam paths by MPB irradiation were recognized as bands of cell with hyperchromatic. Micro or partial cerebral hemorrhage was shown in the dead mice brain from both groups. Therefore, vascular damage might be a possible reason for the cause of death. Conclusions: Our results indicated that the MPB enable to deliver extremely high doses without loss of normal tissue function. Stereotactic multi-dimensional MPB radiation might achieve high therapeutic ratio in the future. Author Disclosure: N. Mukumoto: None. D. Miyawaki: None. H. Akasaka: None. M. Nakayama: None. Y. Miura: None. K. Umetani: None. N. Nariyama: None. K. Shinohara: None. T. Kondoh: None. R. Sasaki: None.
Elevated Serum HGF Is Associated With Radioresistance and Local Recurrence in Non-Small Cell Lung Cancer H.E. Soh, V. Bhardwaj, M.A. Cortez, P.K. Allen, J.V. Heymach, R. Meyn, D. Xia, D.R. Gomez, H.J. Skinner, and J.W. Welsh; The University of Texas MD Anderson Cancer Center, Houston, TX Purpose/Objective(s): Hepatocyte growth factor (HGF), a c-Met ligand involved in cancer tumorigenesis and metastasis, mediates resistance to radiation and drives resistance to ErbB1 inhibitors such as erlotinib. Here we tested whether plasma HGF levels correlate with outcomes after definitive radiation for non-small cell lung cancer (NSCLC) and evaluated the role of HGF in radiosensitivity of NSCLC cells with high or low c-Met expression. Materials/Methods: We retrospectively compared plasma HGF levels with survival and local tumor control in patients enrolled in a phase II trial of erlotinib + chemoradiation. Patients were concurrently treated to 63 Gy in 35 once-daily 1.8-Gy fractions and with 7 cycles of paclitaxel and carboplatin. HGF was measured in pretreatment plasma samples with Quantikine ELISA kits, and tumor progression was assessed on serial CT scans. Chi-square and t tests were used to compare groups. The effect of HGF on NSCLC cell radiosensitivity was measured with clonogenic assays and cell cycle analysis. Results: All 32 patients (21 men, 11 women; median age 63.5 y) received the full radiation dose without treatment breaks. Median pretreatment HGF levels were 703 pg/mL (vs 503 pg/mL in 5 healthy subjects, p Z 0.05). At a median follow-up time of 25.4 months, patients with high HGF levels (>503 pg/mL, n Z 26) had poorer overall survival rates (42% vs 83%, chi-square p Z 0.07) and shorter local-regional control (13.1 vs 30.1 mo, p Z 0.02) and disease-free survival intervals (10.1 vs 26.9 mo, p Z 0.09) than patients with low plasma HGF (<503 pg/mL, n Z 6). No correlation was found between HGF levels and tumor volume. In in vitro analyses, treating NSCLC cells with HGF (50 ng/mL) reduced radiosensitivity in cell lines with high c-Met expression (A549; radioprotection factor 1:1.2) and H358 (1:1.25) but not in cell lines with low c-Met levels (H460 and H1299). HGF treatment reduced cell cycle arrest at G2M in A549 cells treated with 4 Gy. Stable overexpression of HGF in A549 cells also reduced the radiosensitivity of those cells (radioprotection factor 1:1.74). Conclusions: Our results suggest that overexpression of HGF may be associated with radioresistance and poor local control in patients with NSCLC treated with radiation. The ability to identify patients at higher risk of local failure would be valuable for personalizing radiation therapy, particularly given the lack of a survival advantage from dose escalation for unselected patients with NSCLC in RTOG 0617. These results should be evaluated prospectively in future trials. Author Disclosure: H.E. Soh: None. V. Bhardwaj: None. M.A. Cortez: None. P.K. Allen: None. J.V. Heymach: None. R. Meyn: None. D. Xia: None. D.R. Gomez: None. H.J. Skinner: None. J.W. Welsh: None.
3177 Application of Silicon Nanoparticles (SI-NPs) for RadiationInduced Cytotoxicity via Generation of Reactive Oxygen Species (ROS) M.L. Kotler,1 N.I. Garabano,1 P. David Gara,2,3 O.R. Casas,3 D. Dodat,3 S.E. Finkelstein,4 C. Mantz,5 E. Fernandez,5 and M.C. Gonzalez6; 1 University of Buenos Aires, Ciudad Auto´noma de Buenos Aires, Argentina, 2INIFTA, Departamento de Quı´mica, Facultad de Ciencias Exactas, UNLP, La Plata, Argentina, La Plata, Argentina, 3CITOMA. Instituto de Terapia Radiante S.A., CIO, La Plata, Argentina, 421st Century Translational Research Consortium, Scottsdale, Arizona, Scottsdale, AZ, 521st Century Translational Research Consortium, Fort Myers, FL, 6INIFTA, Departamento de Quı´mica, Facultad de Ciencias Exactas, UNLP, La Plata, Argentina
3172 High Throughput Clonogenic Survival Screen Identifies Novel Radiation Sensitizers for K-ras Mutant Non-Small Cell Lung Cancer S.H. Lin,1 J. Zhang,1 U. Giri,1 C. Stephan,2 M.M. Sobieski,2 N. Nguyen,2 P. Davies,2 R. Komaki,1 S.S. Yoo,3 and J.V. Heymach1; 1University of Texas MD Anderson Cancer Center, Houston, TX, 2Texas A&M Health Science Center, Houston, TX, 3National Cancer Institute, Bethesda, MD Purpose/Objective(s): Traditional clonogenic survival and high throughput colorimetric assays are inadequate for drug screens to identify novel radiation sensitizers. To bridge this gap in knowledge, we have developed a method which we coin the High Throughput Clonogenic Survival Screen (HCS) that will allow high volume screening of drug libraries to identify potent radiation sensitizers, particularly for K-ras mutant lung cancer, a current unmet need. Materials/Methods: Drugs at various concentrations or vehicle were added to cells seeded overnight in 96 well plates at low densities, and after 6 hours, plates were irradiated at various doses. Plates were kept in culture 4-6 days depending on the cell line. After incubation, colonies were stained with cresyl-violet, imaged on a high content screening system, and colony count determined using a proprietary image analysis software. Colonies achieving 50 or more cells were tallied. Traditional clonogenic survival assays (tCSA) were performed by seeding cells into 6-well plates overnight in triplicate and adding drugs or vehicle for 6 hours prior to irradiation. Media was changed after 3 days, and plates were kept in culture for 10-14 days. Drug screens were done using the K-ras mutant lung cancer cell line H460 and the Custom Clinical Collection (145 compounds). Results: We compared the HCS method to tCSA and found that HCS recapitulates a similar cell survival curve and survival fraction at 2 Gy (SF2) values for several cancer cell types. As a proof-of-principle, we tested a known sensitizer vorinostat (SAHA) in both the tCSA and HCS assays. As expected, 1 mM vorinostat caused a significant radiation sensitizing effect using tCSA, and was recapitulated, albeit at a lower concentration (100 nM), using the HCS assay. Drug screens (repeated 3 times) identified several compounds that were cytotoxic to the K-ras mutant H460 cells alone at 1 mM (inhibitors to Src/Abl, IGF1R, ROCK1, AKT, PI3K, c-MET) and at low nM concentrations (HSP90 and HDAC inhibitors). The entire class of HDAC inhibitors (5 drugs) had some sensitizing effect to radiation (a leftward shift in the IC50 curve by w10 fold). However, many drugs such as inhibitors to PARP, SRC, Chk1/2, DNA-PK, JNK, and MEK1/2, exhibited significant radiation sensitizing effect with minimal to no activity by themselves. We validated the sensitizing effect of the potent MEK1/2 inhibitor trametinib using tCSA, and found the effect was specific for K-ras mutant and not K-ras wild type cells. Conclusions: High throughput drug screening for novel radiation sensitizers for K-ras mutant lung cancer is feasible using the HCS approach. This is an enabling technology that will allow synthetic lethal drug screens with radiation and accelerate the translation of drugs into clinical testing. Author Disclosure: S.H. Lin: None. J. Zhang: None. U. Giri: None. C. Stephan: None. M.M. Sobieski: None. N. Nguyen: None. P. Davies: None. R. Komaki: None. S.S. Yoo: None. J.V. Heymach: None.
3173 Design and Optimization for Combination of 188Re-Liposome and External Beam Radiation Therapy Against Human Esophageal Adenocarcinoma Xenograft Y. Chen,1 C. Chi,1 Y. Lee,2 T. Chang,3 and C. Chang3; 1Mackay Memorial Hospital, Taipei, Taiwan, 2National Yang-Ming University, Taipei, Taiwan, 3 Institute of Nuclear Energy Research, Taoyuan, Taiwan Purpose/Objective(s): The external beam radiation therapy (EBRT) can deliver high-energy radiation beam to cover both gross tumor and potential
Poster Viewing Abstracts S651 microscopic tumor cells in tumor vicinity. Radioisotope therapy (RIT) by using isotopes emitting relatively short-distance radiation can directly eradicate gross tumor inside torso. Esophageal cancer, one of the most treatment resistant malignancies with high potential to develop local recurrence and distant metastasis, has a dismal prognosis. We proposed that combination of EBRT and RIT may deliver radiation to simultaneously treat primary tumor, regional lymphatics and distant sites at risk of systemic metastasis. Rhenium 188 (188Re)-liposome is a nanoparticle emitting gamma (for molecular imaging validation) and beta (for therapeutic RT) rays with biodistribution profile preferential to tumor. We established an experimental model of combinatory treatment and examined the effect of the EBRT and 188Re-liposome on human esophageal cancer xenograft. Materials/Methods: The 188Re-liposome with quality assurance was prepared according to previous work in clinical trial. Human esophageal cancer cell lines BE-3 (adenocarcinoma) and CE81T/VGH (squamous cell carcinoma) expressing luciferase gene for optical imaging were implanted to nude mice for xenograft. The imaging modalities used included optical and nanoSPECT/CT imaging systems to examine the tumor uptake of 188Re-liposome and treatment results. Optimal dosing for EBRT and 188Re-liposome were assessed in a clinical practiceoriented way. Results: As for quality assurance, the labeling efficiency of 188ReEMBDA (Rf Z 0) was 100%. The labeling efficiency and total activity of 188Re-liposome were 76.5% and 8731 mCi, respectively. For molecular imaging, nanoSPECT/CT scan results showed that esophageal adenocarcinoma BE-3, but not squamous cell carcinoma CE81T/VGH, xenograft could uptake the 188Re-liposome. Treatment of 188Re-liposome had no significant toxicity in mice. The EBRT alone inhibited the growth of BE-3 xenograft in a dose-dependent manner. The optimization of combinatory regimens revealed that EBRT with 3 Gy and 188Reliposome with 310 mCi might be feasible for further preclinical study. The treatment endpoints including tumor growth delay, survival and toxicity for various combinatory regimens and fractionation schedules will be presented. Conclusions: The combination of EBRT with 188Re-liposome might be a potential treatment modality for esophageal cancer by providing molecular imaging, image-guided treatment and comprehensive radiation therapy coverage simultaneously. Author Disclosure: Y. Chen: None. C. Chi: None. Y. Lee: None. T. Chang: None. C. Chang: None.
3174 Radiosensitization by High Affinity Programmable Minor Groove Binders N. Nickols,1 F. Yang,2 J.A. Ratikan,1 B.C. Li,2 W.H. McBride,1 and P.B. Dervan2; 1Department of Radiation Oncology, University of California Los Angeles Medical Center, Los Angeles, CA, 2Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA Purpose/Objective(s): Non-genotoxic small molecules of low systemic toxicity that can potentiate the effects of radiation could be valuable adjuvant therapeutics for solid tumors. Pyrrole-imidazole (Py-Im) polyamides are synthetic oligomers that bind the minor groove of DNA noncovalently with programmable sequence recognition and high affinity. We reported that a polyamide targeted to the DNA sequence 5’-WGWWCW3’ (W Z A/T) suppressed growth of prostate tumor xenografts. In cell culture, this polyamide interferes with RNA Polymerase II activity and results in a dose-dependent depletion of the RNA Polymerase II large subunit. We hypothesized that high-affinity DNA binding Py-Im polyamides may enhance the effects of ionizing radiation. Materials/Methods: We synthesized a polyamide targeting 5’WGWWCW-3’ by solid phase methods. The human laryngeal squamous cell carcinoma cell line UM-SCC-12 was treated with the polyamide at 0 (vehicle) or 20 nM for 48 hours and then irradiated to doses of 0, 2, 4,
S652
International Journal of Radiation Oncology Biology Physics
and 6 Gy. Twenty-four hours after irradiation, cells were re-plated and surviving fractions measured by clonogenic assay. Results: Polyamide treatment alone did not decrease surviving fraction in the absence of irradiation. The radiosensitization by polyamide treatment increased with radiation dose. Surviving fractions at 2 Gy were 0.660.07 and 0.550.05 (SER 1.2); at 4 Gy were 0.410.02 and 0.220.02 (SER 1.9); at 6 Gy were 0.220.07 and 0.110.01 (SER 2.0) for vehicle and polyamidetreated cells, respectively. Calculated D0 values were 4.16 Gy (vehicle) and 2.79 Gy (polyamide). SER: sensitization enhancement ratio. Errors are standard deviations (n Z 3). Conclusions: The polyamide demonstrated radiosensitization of UM-SCC12 cells in vitro. These results justify continued investigations into the therapeutic combination of irradiation and minor groove binding polyamides. Author Disclosure: N. Nickols: None. F. Yang: None. J.A. Ratikan: None. B.C. Li: None. W.H. McBride: None. P.B. Dervan: None.
3176
3175 Normal Tissue Tolerance of Microplanar Beam X-ray: A Long-term Observation N. Mukumoto,1 D. Miyawaki,1 H. Akasaka,1 M. Nakayama,1 Y. Miura,2 K. Umetani,3 N. Nariyama,3 K. Shinohara,4 T. Kondoh,5 and R. Sasaki1; 1 Division of Radiation Oncology, Kobe University Graduate School of Medicine, Kobe City, Hyogo, Japan, 2Department of Rehabilitation Science, Kobe University Graduate School of Health Sciences, Kobe City, Hyogo, Japan, 3Japan Synchrotron Radiation Research Institute, Mikazuki, Sayo, Hyogo, Japan, 4Advanced Research Institute for Science and Engineering, Waseda University, Tokyo, Japan, 5Department of Neurosurgery, Shinsuma Hospital, Kobe City, Hyogo, Japan Purpose/Objective(s): Synchrotron X-ray beams permit the delivery of very high radiation doses to tumors in a single fraction using arrays of microplanar X-ray beams. Microplanar beam (MPB) radiation is, at present, an experimental approach that has produced, in single exposures, exceptionally high tolerance in the normal tissues while preferentially damaging malignant tumors. In this study, we have challenged to evaluate normal tissue tolerance and mortality by MPB radiation to mouse brain with comparing to broad beam from the same radiation source. Materials/Methods: Synchrotron X-ray beam radiation was performed using BL28B2 beam-line at the Spring-8 facility, Hyogo, Japan. For each subgroup, male C57BL/6J mice aged 6-8 weeks that weighted approximately 20-25 g were fixed to the stereotactic metal frame. As for radiation techniques, anterior-posterior windows were set, and a broad or a 12 10 mm unidirectional array of MPB (25 micrometer width, 100-300 micrometer center-to-center spacing) were used. Delivered doses as the broad beam were 12-120 Gy, while the doses as the MPB were 120-960 Gy. Mortalities within 90 days after the irradiation were scored and plotted by using KaleidaGraph software. Histopathological evaluation by hematoxylin and eosin (HE) staining was performed using the irradiated brain tissues. Results: In the experimental setting, 50% and 100% lethal dose by the 200 micrometer spacing MPB beam were 600 Gy and 720 Gy, while those by the broad beam were 80 Gy and 96 Gy. Beam paths by MPB irradiation were recognized as bands of cell with hyperchromatic. Micro or partial cerebral hemorrhage was shown in the dead mice brain from both groups. Therefore, vascular damage might be a possible reason for the cause of death. Conclusions: Our results indicated that the MPB enable to deliver extremely high doses without loss of normal tissue function. Stereotactic multi-dimensional MPB radiation might achieve high therapeutic ratio in the future. Author Disclosure: N. Mukumoto: None. D. Miyawaki: None. H. Akasaka: None. M. Nakayama: None. Y. Miura: None. K. Umetani: None. N. Nariyama: None. K. Shinohara: None. T. Kondoh: None. R. Sasaki: None.
Elevated Serum HGF Is Associated With Radioresistance and Local Recurrence in Non-Small Cell Lung Cancer H.E. Soh, V. Bhardwaj, M.A. Cortez, P.K. Allen, J.V. Heymach, R. Meyn, D. Xia, D.R. Gomez, H.J. Skinner, and J.W. Welsh; The University of Texas MD Anderson Cancer Center, Houston, TX Purpose/Objective(s): Hepatocyte growth factor (HGF), a c-Met ligand involved in cancer tumorigenesis and metastasis, mediates resistance to radiation and drives resistance to ErbB1 inhibitors such as erlotinib. Here we tested whether plasma HGF levels correlate with outcomes after definitive radiation for non-small cell lung cancer (NSCLC) and evaluated the role of HGF in radiosensitivity of NSCLC cells with high or low c-Met expression. Materials/Methods: We retrospectively compared plasma HGF levels with survival and local tumor control in patients enrolled in a phase II trial of erlotinib + chemoradiation. Patients were concurrently treated to 63 Gy in 35 once-daily 1.8-Gy fractions and with 7 cycles of paclitaxel and carboplatin. HGF was measured in pretreatment plasma samples with Quantikine ELISA kits, and tumor progression was assessed on serial CT scans. Chi-square and t tests were used to compare groups. The effect of HGF on NSCLC cell radiosensitivity was measured with clonogenic assays and cell cycle analysis. Results: All 32 patients (21 men, 11 women; median age 63.5 y) received the full radiation dose without treatment breaks. Median pretreatment HGF levels were 703 pg/mL (vs 503 pg/mL in 5 healthy subjects, p Z 0.05). At a median follow-up time of 25.4 months, patients with high HGF levels (>503 pg/mL, n Z 26) had poorer overall survival rates (42% vs 83%, chi-square p Z 0.07) and shorter local-regional control (13.1 vs 30.1 mo, p Z 0.02) and disease-free survival intervals (10.1 vs 26.9 mo, p Z 0.09) than patients with low plasma HGF (<503 pg/mL, n Z 6). No correlation was found between HGF levels and tumor volume. In in vitro analyses, treating NSCLC cells with HGF (50 ng/mL) reduced radiosensitivity in cell lines with high c-Met expression (A549; radioprotection factor 1:1.2) and H358 (1:1.25) but not in cell lines with low c-Met levels (H460 and H1299). HGF treatment reduced cell cycle arrest at G2M in A549 cells treated with 4 Gy. Stable overexpression of HGF in A549 cells also reduced the radiosensitivity of those cells (radioprotection factor 1:1.74). Conclusions: Our results suggest that overexpression of HGF may be associated with radioresistance and poor local control in patients with NSCLC treated with radiation. The ability to identify patients at higher risk of local failure would be valuable for personalizing radiation therapy, particularly given the lack of a survival advantage from dose escalation for unselected patients with NSCLC in RTOG 0617. These results should be evaluated prospectively in future trials. Author Disclosure: H.E. Soh: None. V. Bhardwaj: None. M.A. Cortez: None. P.K. Allen: None. J.V. Heymach: None. R. Meyn: None. D. Xia: None. D.R. Gomez: None. H.J. Skinner: None. J.W. Welsh: None.
3177 Application of Silicon Nanoparticles (SI-NPs) for RadiationInduced Cytotoxicity via Generation of Reactive Oxygen Species (ROS) M.L. Kotler,1 N.I. Garabano,1 P. David Gara,2,3 O.R. Casas,3 D. Dodat,3 S.E. Finkelstein,4 C. Mantz,5 E. Fernandez,5 and M.C. Gonzalez6; 1 University of Buenos Aires, Ciudad Auto´noma de Buenos Aires, Argentina, 2INIFTA, Departamento de Quı´mica, Facultad de Ciencias Exactas, UNLP, La Plata, Argentina, La Plata, Argentina, 3CITOMA. Instituto de Terapia Radiante S.A., CIO, La Plata, Argentina, 421st Century Translational Research Consortium, Scottsdale, Arizona, Scottsdale, AZ, 521st Century Translational Research Consortium, Fort Myers, FL, 6INIFTA, Departamento de Quı´mica, Facultad de Ciencias Exactas, UNLP, La Plata, Argentina