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Targeted Inhibition of Rad51 Renders Non–small Cell Lung Cancer Cells More Sensitive to Proton Therapy
Volume 99 Number 2S Supplement 2017 Affiliated Hospital of Soochow University, Institute of Radiotherapy & Oncology, Soochow University, Suzhou Key Laboratory for Radiation Oncology Suzhou, Suzhou, China Purpose/Objective(s): Radiation-induced neurogenesis impairment in hippocampus was considered to play a key role in cognitive deficits arise from exposure to ionizing radiation, but the mechanism remains elusive. This study aimed to investigate the role of TrkA、TrkB in radiationinduced hippocampal neurogenesis impairment. Materials/Methods: A singal dose of 10Gy 4MV electron beam were given to young male Sprague-Dawley rats (50e60g, day 21). Western blot was performed and the brain sections were immunohistochemical stained. Q-PCR were applied to detect mRNA levels.The alterations of TrkA/TrkB protein and mRNA levels and hippocampal neurogenesis were observed and analyzed. Golgi staining was used to observe the dendritic spine of hippocampus.To evaluate the consequence of whole brain irradiation on hippocampus-dependent memory formation, Morris water maze test, Novel object recognition and open field test were performed. Results: The results show that compared with control group, the numbers of dendritic spine significantly decreased after irradiation and its shape change obviously. Immunofluorescence showed a significant decrease in neural precursors prolifration comparing with control group (53%). Protein levels of TrkA expressions increased significantly (32%) while the levels of TrkB expressions decreased significantly (43%); the mRNA levels of TrkA expressions increased significantly (27%) while the levels of TrkB expressions decreased significantly (36%). In Morris water maze test, passive-avoidance test and open field test, we confirmed whole brain irradiation lead to notable memory impairment at day 30 after irradiation. Conclusion: As a signaling pathways downstream of NGF and BDNF, TrkA,TrkB may play an important role in radiation-induced neurogenesis impairment. Future studies are necessary to determine the strong association between TrkA and TrkB after whole brain irradiation. Author Disclosure: W.H. Hao: None. S. Ji: None. D. Xin: None. Y. Tian: None.
3416 Esophageal Dose, Clinical Factors, and Cytokines: Predicting Radiation-Induced Esophagitis in Nonesmall Cell Lung Cancer P.G. Hawkins,1 P.S. Boonstra,1 S. Hobson,1 J.W.D. Hearn,1 J.A. Hayman,1 R.K. Ten Haken,2 M.M. Matuszak,3 P. Stanton,1 G.P. Kalemkerian,1 T.S. Lawrence,1 M. Schipper,1 F.M. Kong,4 and S. Jolly1; 1University of Michigan, Ann Arbor, MI, 2University of Michigan, Department of Radiation Oncology, Ann Arbor, MI, 3Department of Radiation Oncology, University of Michigan, Ann Arbor, MI, 4Department of Radiation Oncology, Simon Cancer Center, Indiana University School of Medicine, Indianapolis, IN Purpose/Objective(s): Radiation esophagitis (RE) is a common and occasionally dose-limiting adverse event associated with definitive radiotherapy for non-small-cell lung cancer (NSCLC). Prediction of RE is currently driven by dosimetric parameters, but patient-specific factors may also influence risk. While plasma cytokine measurements have been shown to predict other forms of radiation-induced toxicity, their ability to predict RE has been less well-studied. Materials/Methods: We analyzed data from 126 patients treated from 2004 to 2013 with definitive radiotherapy, with or without chemotherapy, for non-small-cell lung cancer on 4 prospective institutional clinical trials. The primary endpoint for this analysis was grade 3 or greater esophagitis, which was defined according to CTCAE v3.0. Plasma levels of 30 cytokines were measured prior to treatment. Logistic regression models based on combinations of dosimetric factors (maximum dose to 2 cubic cm (D2cc) and generalized equivalent uniform dose (gEUDaZ5)), clinical variables, and log-transformed pretreatment cytokine measurements were developed. All esophageal doses were biologically corrected to 2 Gy-perfraction equivalent doses (EQD2) using the linear quadratic model (a/
Poster Viewing E595 bZ10). Cross-validated estimates of area under the receiver operating characteristic curve (AUC) were used to assess prediction accuracy. Results: The incidence of grade 3+ esophagitis in this population was 10.3% (13/126). Dose-only models predicted grade 3+ RE with crossvalidated AUC values of 0.750 (D2cc) and 0.727 (gEUD). Combining clinical factors with D2cc increased the cross-validated AUC to 0.779. Incorporating pretreatment cytokine measurements, modeled first as direct associations with RE, with D2cc and clinical factors yielded a lower crossvalidated AUC of 0.758. A separate analysis modeling cytokine levels as interactions with the dose-esophagitis association produced a cross-validated AUC of 0.773. D2cc and gEUD correlated with incidence of grade 3+ RE with odds ratios (OR) of 1.094/Gy and 1.096/Gy, respectively. When analyzed in conjunction with dose, two clinical factors were associated with RE, namely gender and age. Female gender was associated with higher risk of RE, with ORs of 1.09 and 1.112 in the D2cc and gEUD models, respectively. Older age was associated with decreased risk of RE, with ORs of 0.992/year and 0.991/year in the D2cc and gEUD models, respectively. Conclusion: In this analysis, esophageal dose, both in terms of D2cc and gEUD, correlated with incidence of grade 3+ RE in patients receiving definitive radiotherapy for NSCLC. Combining dosimetric variables with clinical factors improved prediction, while incorporating pretreatment plasma cytokine levels did not. Modeling these dose metrics and clinical factors in this manner may prove useful in directing radiation treatment planning. Identification of biologic or other factors predictive of RE may further improve prediction. Author Disclosure: P.G. Hawkins: None. P.S. Boonstra: None. S. Hobson: None. J.W. Hearn: None. J.A. Hayman: None. R.K. Ten Haken: Research Grant; NIH-NCI. Travel Expenses; Varian Medical Systems Inc. M.M. Matuszak: Employee; William Beaumont Hospital. P. Stanton: None. G.P. Kalemkerian: Research Grant; Merck, OncoMed, Pfizer, Astex, GlaxoSmithKline, Millennium. T.S. Lawrence: royalties; Lippincott, Williams and Wilkins. Honoraria; Massachusetts General Hospital, Pfizer Oncology Innovation Summit, Sidney Kimmel Foundation for Cancer Research. Consultant; Pfizer Oncology Innovation Summit. Advisory Board; ASTRO Radiation Oncology Institute, Dana Farber Cancer Institute, Massachusetts General Hospital, Sidney Kimm. M. Schipper: Consultant; Armune Bioscience and Hygieia Sciences. F.(. Kong: Research Grant; NCI/NIH, Varian. ; Sino-American Network for Therapeutic Radiology, American Association of Women Radiologists (AAWR), Association for Chinese Professors, Chinese-American Network for Hematology and Oncolo, SinoAmerican Network for Therapeutic Radiation On. S. Jolly: None.
3417 Targeted Inhibition of Rad51 Renders Nonesmall Cell Lung Cancer Cells More Sensitive to Proton Therapy T.S. Herman,1 D. Andrade,2 M. Mehta,2 J. Griffith,2 H. Jin,2 S. Ahmad,2 O. Algan,2 and A. Munshi2; 1University of Oklahoma Health Science Center, Stephenson Cancer Center, Department of Radiation Oncology, Oklahoma City, OK, 2University of Oklahoma Health Sciences Center, Oklahoma City, OK Purpose/Objective(s): Radiation therapy using proton beams is an emerging therapy that is being used for cancer treatment due to its superior physical dose distribution, higher RBE and increased effectiveness, leading to greater clinical gains. A recent study has shown a preferential involvement of the homologous recombination (HR) pathway in proton mediated radiosensitization. Rad51, a key protein in the HR pathway of DNA double-strand break repair, is overexpressed in various cancers and plays an essential role in radioresistance. Therefore, this study was designed to evaluate the impact of targeting Rad51on the RBE of protons and photons in NSCLC cell lines. Materials/Methods: The effects of Rad51 inhibition by genetic (Rad51 siRNA) and pharmacologic (BO2, a small molecule Rad51 inhibitor) approaches on radiosensitization of H1299 and A549 NSCLC cells were determined in vitro. The RBE of protons and photons following siRad51
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International Journal of Radiation Oncology Biology Physics
treatment was compared using clonogenic cell survival assay when protons were delivered with doses of 2, 4 and 6 Gy at the mid-spread out Bragg peak (SOBP) using a reference beam of 15 g/cm2 range and 10 g/ cm2modulation. Involvement of the DNA damage response was assessed by western blot analysis for DNA repair proteins and immunofluorescence staining for g-H2AX foci. Results: Rad51 depletion by siRNA sensitized H1299 and A549 cells to protons and photons. However, both H1299 and A549 cell lines were significantly more sensitive to proton irradiation compared to photon. Survival fraction at 2 Gy (SF2) for H1299 cells exposed to proton beam was reduced from (53.7 0.49) % in siScr to (21.8 2.28) % in siRad51treated group. Similarly, SF2 for A549 cells was reduced from (59.1 1.16) % in siScr to (29.6 4.9) % following siRad51-treatment. Survival enhancement ratios (SER) were calculated at 10% cell survival by dividing proton radiation dose of the siScr to that of siRad51 for that survival. The SER for H1299 cells at 10% survival was 1.9 and for A549 was 1.33, respectively. Similar results were obtained with the Rad51 small molecule inhibitor, BO2. Our results reveal that this enhanced efficacy of protons is associated with increased ATM and Chk1 phosphorylation as well as increased H2AX phosphorylation. Further, Rad51 knockdown led to persistent radiation-induced DNA damage as reflected by g-H2AX foci staining. Conclusion: Our results demonstrate that Rad51 inhibition significantly increased the radiosensitivity of NSCLC cells, and that this increased radiosensitivity was mediated by the suppression of DNA repair. The effect of siRad51 on radiosensitization to protons was more significant than to photons. Author Disclosure: T.S. Herman: None. D. Andrade: None. M. Mehta: None. J. Griffith: None. H. Jin: None. S. Ahmad: None. O. Algan: None. A. Munshi: None.
used as internal controls) were purchased from Applied Biosystems. qRTPCR was performed using 7500 Fast Real-Time PCR System with tripleset per sample for each measurement and two independent qRT-PCR experiments were performed. The relative value for the differentially expressed miRNAs were analyzed between patients with grade 3 toxicities and patients without toxicities, using paired ttests. Results: Characteristics of the patient population include 27/28 patients with an oropharyngeal primary; 1 patient had a hypopharynx primary. 16 (57%) of patients had grade 3 acute toxicities (including hematologic toxicities). Two (7%) of patients had grade 3 late toxicities. The relative values of (miR-6821 - miR-1290) were significantly lower in patients with grade 3 or higher toxicities (including acute 14 acute and 2 late toxicity) versus the patients without toxicity (PZ 0.002). The qRT-PCR data further confirmed the significant changes in the miRNAs. Conclusion: Lower levels of circulating pre-treatment miRNA value (miR6821-miR-1290) from SCCHN patients were associated with higher rates of acute and late radiation induced toxicities. These results suggest that pre-treatment circulating miRNA could be used as biomarker to predict for radiation-induced toxicities for locally advanced SCCHN. Further studies are needed to validate these results. Author Disclosure: K.A. Higgins: None. N.F. Saba: None. D.M. Shin: None. J.J. Beitler: None. G. Chen: None. C. Xiao: None. D. Wang: None. S. Tang: None. Y. Wang: None.
3418 Circulating Pre-treatment miRNAs as Potential Biomarkers to Predict Radiation Toxicity K.A. Higgins,1 N.F. Saba,2 D.M. Shin,3 J.J. Beitler,1 G. Chen,2 C. Xiao,4 D. Wang,2 S. Tang,1 and Y. Wang1; 1Department of Radiation Oncology and Winship Cancer Institute, Emory University, Atlanta, GA, 2Department of Hematology and Medical Oncology, Winship Cancer Institute, Emory University, Atlanta, GA, 3Department of Hematology and Medical Oncology, Winship Cancer Institute, Emory University, Atlanta, GA, 4Nell Hodgson Woodruff School of Nursing, Winship Cancer Institute at Emory University, Atlanta, GA Purpose/Objective(s): Toxicities from radiation therapy for locally advanced head and neck cancer patients remain significant; however, no predictive biomarkers currently exist. miRNAs are highly stable and mainly derived from blood leukocytes, which represent one of the most highly radiation sensitive cells. We hypothesized that circulating miRNAs may reflect normal tissue radiation response and could be identified before treatment as a biomarker to predict radiation toxicities. Materials/Methods: With IRB approval, blood samples were collected from 28 patients with squamous cell carcinoma of the head and neck (SCCHN) receiving definitive chemoradiation from 2013-2015. All patients received 70 Gy radiation with IMRT/VMAT technique and concurrent chemotherapy with either cisplatin or carboplatin and paclitaxel. Acute and late treatment-related toxicities were graded using CTCAE version 4.0. Twenty microliters of serum per sample were used for directly detecting whole transcriptome panel (including 2083 miRNAs in total) with HTG molecular Inc. platform on the EdgeSeq and 300 microliters of serum per sample were extracted with RNA using the miRNeasy Serum/ Plasma Kit and Taqman MicroRNA Reverse Transcription Kit for later real-time PCR verification. The raw data of miRNA expression were further analyzed by combining clinical phenotypes with or without toxicities (including acute or late toxicities). The top two changed miRNAs (miR-1290 and miR-6821) between non-toxicity and toxicity groups were further detected using real time PCR (qRT-PCR). Primers for the changed miRNAs and additional three miRNAs (miR16, miR-223 and miR-486
3419 Paradigm Shift in Tumor Microenvironment: RT Alone Versus Combination of Local Interleukin-12 Treatment and RT J.H. Hong,1,2 C.F. Yu,1 F.H. Chen,1,3 and C.S. Chiang4; 1Radiation Biology Research Center, Institute for Radiological Research, Chang Gung University / Chang Gung Memorial Hospital at Linkou, Taoyuan, Taiwan, 2 Department of Radiation Oncology, Chang Gung Memorial Hospital, Linkou, Taoyuan, Taiwan, 3Department of Medical Imaging and Radiological Sciences, Chang Gung University, Taoyuan, Taiwan, 4 Department of Biomedical Engineering and Environmental Sciences, National Tsing Hua University, Hsinchu 30013, Taiwan Purpose/Objective(s): In animal tumor models, high dose radiotherapy (RT) was shown to decrease the tumor microvascular density (MVD) and render tumors into chronic hypoxia with macrophage aggregation. Combination of immunotherapy with RT is a new direction for cancer treatment, but its effects on radiation-induced microenvironment is unclear. This study is aimed to study the effects of combination of local IL-12 and RT on tumor control, toxicities and tumor microenvironment. Materials/Methods: TRAMP-C1 tumors derived from a murine prostate cancer were either no treatment (control), or irradiated with high single dose (SiRT, 25Gy) or fractionation irradiation (FxRT, 8.9 Gy for continued 5 days), or intra-tumorally injected with Ad-sc-IL12 virus (1108 pfu) (IL12 alone), or treated with both modalities (IL-12 + RT) at the tumor size of 4mm. Ad-sc-IL12 virus was intra-tumorally injected immediately after SiRT or the first treatment in FxRT. Tumor sizes were measured at the indicated time points and in some experiments tumors were taken at 7 days after treatment for a variety of examinations including immunohistochemistry stain and flow cytometry analysis. Results: RT (SiRT or FxRT) or IL-12 alone did not shrink the tumor, but caused tumor growth delay around 7 and 12 days, respectively. IL-12+RT significantly shrunk the tumor to < 1mm and extended the tumor growth delay up to 20 days. Although IL-12 alone delayed tumor growth, it did not show the effects on tumor microenvironment in terms of MVD, and percentage of areas of hypoxia and necrosis when compared with control tumors. Strikingly combination of local IL-12 and RT did not decrease the MVD as RT alone did, and this subsequently cause less hypoxic and necrotic regions. CD8+ T cells were significantly increased in IL-12+RT tumors and infiltrating T cells in IL-12+RT tumors were presented with activated morphology, as compared with other groups. The serum IFN-g (Th1 response) was highly detectable in IL-12 alone and IL-12+RT groups. For toxicity evaluation, the serum levels of IL-12 at day 7 were
3416 Esophageal Dose, Clinical Factors, and Cytokines: Predicting Radiation-Induced Esophagitis in Nonesmall Cell Lung Cancer P.G. Hawkins,1 P.S. Boonstra,1 S. Hobson,1 J.W.D. Hearn,1 J.A. Hayman,1 R.K. Ten Haken,2 M.M. Matuszak,3 P. Stanton,1 G.P. Kalemkerian,1 T.S. Lawrence,1 M. Schipper,1 F.M. Kong,4 and S. Jolly1; 1University of Michigan, Ann Arbor, MI, 2University of Michigan, Department of Radiation Oncology, Ann Arbor, MI, 3Department of Radiation Oncology, University of Michigan, Ann Arbor, MI, 4Department of Radiation Oncology, Simon Cancer Center, Indiana University School of Medicine, Indianapolis, IN Purpose/Objective(s): Radiation esophagitis (RE) is a common and occasionally dose-limiting adverse event associated with definitive radiotherapy for non-small-cell lung cancer (NSCLC). Prediction of RE is currently driven by dosimetric parameters, but patient-specific factors may also influence risk. While plasma cytokine measurements have been shown to predict other forms of radiation-induced toxicity, their ability to predict RE has been less well-studied. Materials/Methods: We analyzed data from 126 patients treated from 2004 to 2013 with definitive radiotherapy, with or without chemotherapy, for non-small-cell lung cancer on 4 prospective institutional clinical trials. The primary endpoint for this analysis was grade 3 or greater esophagitis, which was defined according to CTCAE v3.0. Plasma levels of 30 cytokines were measured prior to treatment. Logistic regression models based on combinations of dosimetric factors (maximum dose to 2 cubic cm (D2cc) and generalized equivalent uniform dose (gEUDaZ5)), clinical variables, and log-transformed pretreatment cytokine measurements were developed. All esophageal doses were biologically corrected to 2 Gy-perfraction equivalent doses (EQD2) using the linear quadratic model (a/
Poster Viewing E595 bZ10). Cross-validated estimates of area under the receiver operating characteristic curve (AUC) were used to assess prediction accuracy. Results: The incidence of grade 3+ esophagitis in this population was 10.3% (13/126). Dose-only models predicted grade 3+ RE with crossvalidated AUC values of 0.750 (D2cc) and 0.727 (gEUD). Combining clinical factors with D2cc increased the cross-validated AUC to 0.779. Incorporating pretreatment cytokine measurements, modeled first as direct associations with RE, with D2cc and clinical factors yielded a lower crossvalidated AUC of 0.758. A separate analysis modeling cytokine levels as interactions with the dose-esophagitis association produced a cross-validated AUC of 0.773. D2cc and gEUD correlated with incidence of grade 3+ RE with odds ratios (OR) of 1.094/Gy and 1.096/Gy, respectively. When analyzed in conjunction with dose, two clinical factors were associated with RE, namely gender and age. Female gender was associated with higher risk of RE, with ORs of 1.09 and 1.112 in the D2cc and gEUD models, respectively. Older age was associated with decreased risk of RE, with ORs of 0.992/year and 0.991/year in the D2cc and gEUD models, respectively. Conclusion: In this analysis, esophageal dose, both in terms of D2cc and gEUD, correlated with incidence of grade 3+ RE in patients receiving definitive radiotherapy for NSCLC. Combining dosimetric variables with clinical factors improved prediction, while incorporating pretreatment plasma cytokine levels did not. Modeling these dose metrics and clinical factors in this manner may prove useful in directing radiation treatment planning. Identification of biologic or other factors predictive of RE may further improve prediction. Author Disclosure: P.G. Hawkins: None. P.S. Boonstra: None. S. Hobson: None. J.W. Hearn: None. J.A. Hayman: None. R.K. Ten Haken: Research Grant; NIH-NCI. Travel Expenses; Varian Medical Systems Inc. M.M. Matuszak: Employee; William Beaumont Hospital. P. Stanton: None. G.P. Kalemkerian: Research Grant; Merck, OncoMed, Pfizer, Astex, GlaxoSmithKline, Millennium. T.S. Lawrence: royalties; Lippincott, Williams and Wilkins. Honoraria; Massachusetts General Hospital, Pfizer Oncology Innovation Summit, Sidney Kimmel Foundation for Cancer Research. Consultant; Pfizer Oncology Innovation Summit. Advisory Board; ASTRO Radiation Oncology Institute, Dana Farber Cancer Institute, Massachusetts General Hospital, Sidney Kimm. M. Schipper: Consultant; Armune Bioscience and Hygieia Sciences. F.(. Kong: Research Grant; NCI/NIH, Varian. ; Sino-American Network for Therapeutic Radiology, American Association of Women Radiologists (AAWR), Association for Chinese Professors, Chinese-American Network for Hematology and Oncolo, SinoAmerican Network for Therapeutic Radiation On. S. Jolly: None.
3417 Targeted Inhibition of Rad51 Renders Nonesmall Cell Lung Cancer Cells More Sensitive to Proton Therapy T.S. Herman,1 D. Andrade,2 M. Mehta,2 J. Griffith,2 H. Jin,2 S. Ahmad,2 O. Algan,2 and A. Munshi2; 1University of Oklahoma Health Science Center, Stephenson Cancer Center, Department of Radiation Oncology, Oklahoma City, OK, 2University of Oklahoma Health Sciences Center, Oklahoma City, OK Purpose/Objective(s): Radiation therapy using proton beams is an emerging therapy that is being used for cancer treatment due to its superior physical dose distribution, higher RBE and increased effectiveness, leading to greater clinical gains. A recent study has shown a preferential involvement of the homologous recombination (HR) pathway in proton mediated radiosensitization. Rad51, a key protein in the HR pathway of DNA double-strand break repair, is overexpressed in various cancers and plays an essential role in radioresistance. Therefore, this study was designed to evaluate the impact of targeting Rad51on the RBE of protons and photons in NSCLC cell lines. Materials/Methods: The effects of Rad51 inhibition by genetic (Rad51 siRNA) and pharmacologic (BO2, a small molecule Rad51 inhibitor) approaches on radiosensitization of H1299 and A549 NSCLC cells were determined in vitro. The RBE of protons and photons following siRad51
E596
International Journal of Radiation Oncology Biology Physics
treatment was compared using clonogenic cell survival assay when protons were delivered with doses of 2, 4 and 6 Gy at the mid-spread out Bragg peak (SOBP) using a reference beam of 15 g/cm2 range and 10 g/ cm2modulation. Involvement of the DNA damage response was assessed by western blot analysis for DNA repair proteins and immunofluorescence staining for g-H2AX foci. Results: Rad51 depletion by siRNA sensitized H1299 and A549 cells to protons and photons. However, both H1299 and A549 cell lines were significantly more sensitive to proton irradiation compared to photon. Survival fraction at 2 Gy (SF2) for H1299 cells exposed to proton beam was reduced from (53.7 0.49) % in siScr to (21.8 2.28) % in siRad51treated group. Similarly, SF2 for A549 cells was reduced from (59.1 1.16) % in siScr to (29.6 4.9) % following siRad51-treatment. Survival enhancement ratios (SER) were calculated at 10% cell survival by dividing proton radiation dose of the siScr to that of siRad51 for that survival. The SER for H1299 cells at 10% survival was 1.9 and for A549 was 1.33, respectively. Similar results were obtained with the Rad51 small molecule inhibitor, BO2. Our results reveal that this enhanced efficacy of protons is associated with increased ATM and Chk1 phosphorylation as well as increased H2AX phosphorylation. Further, Rad51 knockdown led to persistent radiation-induced DNA damage as reflected by g-H2AX foci staining. Conclusion: Our results demonstrate that Rad51 inhibition significantly increased the radiosensitivity of NSCLC cells, and that this increased radiosensitivity was mediated by the suppression of DNA repair. The effect of siRad51 on radiosensitization to protons was more significant than to photons. Author Disclosure: T.S. Herman: None. D. Andrade: None. M. Mehta: None. J. Griffith: None. H. Jin: None. S. Ahmad: None. O. Algan: None. A. Munshi: None.
used as internal controls) were purchased from Applied Biosystems. qRTPCR was performed using 7500 Fast Real-Time PCR System with tripleset per sample for each measurement and two independent qRT-PCR experiments were performed. The relative value for the differentially expressed miRNAs were analyzed between patients with grade 3 toxicities and patients without toxicities, using paired ttests. Results: Characteristics of the patient population include 27/28 patients with an oropharyngeal primary; 1 patient had a hypopharynx primary. 16 (57%) of patients had grade 3 acute toxicities (including hematologic toxicities). Two (7%) of patients had grade 3 late toxicities. The relative values of (miR-6821 - miR-1290) were significantly lower in patients with grade 3 or higher toxicities (including acute 14 acute and 2 late toxicity) versus the patients without toxicity (PZ 0.002). The qRT-PCR data further confirmed the significant changes in the miRNAs. Conclusion: Lower levels of circulating pre-treatment miRNA value (miR6821-miR-1290) from SCCHN patients were associated with higher rates of acute and late radiation induced toxicities. These results suggest that pre-treatment circulating miRNA could be used as biomarker to predict for radiation-induced toxicities for locally advanced SCCHN. Further studies are needed to validate these results. Author Disclosure: K.A. Higgins: None. N.F. Saba: None. D.M. Shin: None. J.J. Beitler: None. G. Chen: None. C. Xiao: None. D. Wang: None. S. Tang: None. Y. Wang: None.
3418 Circulating Pre-treatment miRNAs as Potential Biomarkers to Predict Radiation Toxicity K.A. Higgins,1 N.F. Saba,2 D.M. Shin,3 J.J. Beitler,1 G. Chen,2 C. Xiao,4 D. Wang,2 S. Tang,1 and Y. Wang1; 1Department of Radiation Oncology and Winship Cancer Institute, Emory University, Atlanta, GA, 2Department of Hematology and Medical Oncology, Winship Cancer Institute, Emory University, Atlanta, GA, 3Department of Hematology and Medical Oncology, Winship Cancer Institute, Emory University, Atlanta, GA, 4Nell Hodgson Woodruff School of Nursing, Winship Cancer Institute at Emory University, Atlanta, GA Purpose/Objective(s): Toxicities from radiation therapy for locally advanced head and neck cancer patients remain significant; however, no predictive biomarkers currently exist. miRNAs are highly stable and mainly derived from blood leukocytes, which represent one of the most highly radiation sensitive cells. We hypothesized that circulating miRNAs may reflect normal tissue radiation response and could be identified before treatment as a biomarker to predict radiation toxicities. Materials/Methods: With IRB approval, blood samples were collected from 28 patients with squamous cell carcinoma of the head and neck (SCCHN) receiving definitive chemoradiation from 2013-2015. All patients received 70 Gy radiation with IMRT/VMAT technique and concurrent chemotherapy with either cisplatin or carboplatin and paclitaxel. Acute and late treatment-related toxicities were graded using CTCAE version 4.0. Twenty microliters of serum per sample were used for directly detecting whole transcriptome panel (including 2083 miRNAs in total) with HTG molecular Inc. platform on the EdgeSeq and 300 microliters of serum per sample were extracted with RNA using the miRNeasy Serum/ Plasma Kit and Taqman MicroRNA Reverse Transcription Kit for later real-time PCR verification. The raw data of miRNA expression were further analyzed by combining clinical phenotypes with or without toxicities (including acute or late toxicities). The top two changed miRNAs (miR-1290 and miR-6821) between non-toxicity and toxicity groups were further detected using real time PCR (qRT-PCR). Primers for the changed miRNAs and additional three miRNAs (miR16, miR-223 and miR-486
3419 Paradigm Shift in Tumor Microenvironment: RT Alone Versus Combination of Local Interleukin-12 Treatment and RT J.H. Hong,1,2 C.F. Yu,1 F.H. Chen,1,3 and C.S. Chiang4; 1Radiation Biology Research Center, Institute for Radiological Research, Chang Gung University / Chang Gung Memorial Hospital at Linkou, Taoyuan, Taiwan, 2 Department of Radiation Oncology, Chang Gung Memorial Hospital, Linkou, Taoyuan, Taiwan, 3Department of Medical Imaging and Radiological Sciences, Chang Gung University, Taoyuan, Taiwan, 4 Department of Biomedical Engineering and Environmental Sciences, National Tsing Hua University, Hsinchu 30013, Taiwan Purpose/Objective(s): In animal tumor models, high dose radiotherapy (RT) was shown to decrease the tumor microvascular density (MVD) and render tumors into chronic hypoxia with macrophage aggregation. Combination of immunotherapy with RT is a new direction for cancer treatment, but its effects on radiation-induced microenvironment is unclear. This study is aimed to study the effects of combination of local IL-12 and RT on tumor control, toxicities and tumor microenvironment. Materials/Methods: TRAMP-C1 tumors derived from a murine prostate cancer were either no treatment (control), or irradiated with high single dose (SiRT, 25Gy) or fractionation irradiation (FxRT, 8.9 Gy for continued 5 days), or intra-tumorally injected with Ad-sc-IL12 virus (1108 pfu) (IL12 alone), or treated with both modalities (IL-12 + RT) at the tumor size of 4mm. Ad-sc-IL12 virus was intra-tumorally injected immediately after SiRT or the first treatment in FxRT. Tumor sizes were measured at the indicated time points and in some experiments tumors were taken at 7 days after treatment for a variety of examinations including immunohistochemistry stain and flow cytometry analysis. Results: RT (SiRT or FxRT) or IL-12 alone did not shrink the tumor, but caused tumor growth delay around 7 and 12 days, respectively. IL-12+RT significantly shrunk the tumor to < 1mm and extended the tumor growth delay up to 20 days. Although IL-12 alone delayed tumor growth, it did not show the effects on tumor microenvironment in terms of MVD, and percentage of areas of hypoxia and necrosis when compared with control tumors. Strikingly combination of local IL-12 and RT did not decrease the MVD as RT alone did, and this subsequently cause less hypoxic and necrotic regions. CD8+ T cells were significantly increased in IL-12+RT tumors and infiltrating T cells in IL-12+RT tumors were presented with activated morphology, as compared with other groups. The serum IFN-g (Th1 response) was highly detectable in IL-12 alone and IL-12+RT groups. For toxicity evaluation, the serum levels of IL-12 at day 7 were