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A Potential Role for Nontargeted Biological Effects in Cellular Inactivation After Ionizing Irradiation With Large Single Doses
S776
International Journal of Radiation Oncology Biology Physics
the dose giving 10% surviving fraction, was calculated by fitting the surviving fractions to the linear-quadratic model; surviving fraction Z exp(-aD-bD2), where D is the dose. The HMGB1 level in the culture supernatant of the cells receiving the D10 of X-ray or carbon-ion beam was assessed by ELISA, 72 and 96 hours after the irradiation. Results: In TE2, KYSE70, A549, NCI-H460 and WiDr cells, the D10 for X-ray was 2.1, 6.7, 8.0, 4.8 and 7.1 Gy, respectively, while that for carbonion beam was 0.9, 2.5, 2.7, 1.8 and 3.5 Gy, respectively. Seventy-two hours after the irradiation for D10, both X-ray and carbon-ion beam significantly increased the HMGB1 level in the culture supernatant in KYSE70, A549, NCI-H460, WiDr cells. Furthermore, 96 hours after the irradiation for D10, both X-ray and carbon-ion beam significantly increased the HMGB1 level in all the 5 cell lines tested. There was no significant difference in the increase in the HMGB1 level between X-ray and carbon-ion beam in each data point except at 96 hours in NCI-H460 cells, where carbon-ion beam was significantly superior to X-ray in the induction of HMGB1. Conclusions: Our results indicate that carbon-ion beam as well as X-ray increases the release of HMGB1 from various types of human cancer. The comparable levels of HMGB1 detected after the irradiation with iso-survival doses between X-ray and carbon-ion beam indicated that carbon-ion radiation therapy is capable of activating anti-tumor immunity at least equivalent to X-ray radiation therapy. Author Disclosure: Y. Suzuki: None. Y. Yoshimoto: None. T. Oike: None. N. Okonogi: None. K. Ando: None. H. Sato: None. S. Noda: None. M. Isono: None. K. Mimura: None. K. Kono: None. T. Nakano: None.
Author Disclosure: M. Veldwijk: None. B. Zhang: None. F. Wenz: A. Employee; University Medical Center Mannheim, University of Heidelberg. E. Research Grant; Elekta, Zeiss. F. Honoraria; Elekta, Zeiss, Celgene, Roche, Lilly, Ipsen. G. Consultant; Elekta. I. Travel Expenses; Elekta, Zeiss, Celgene, Roche, Lilly, Ipsen. K. Advisory Board; Elekta, Celgene. Q. Patent/License Fee/Copyright; Zeiss. C. Herskind: None.
3438 A Potential Role for Nontargeted Biological Effects in Cellular Inactivation After Ionizing Irradiation With Large Single Doses M. Veldwijk, B. Zhang, F. Wenz, and C. Herskind; Department of Radiation Oncology, Universita¨tsmedizin Mannheim, Medical Faculty Mannheim, University of Heidelberg, Mannheim, Germany Purpose/Objective(s): The rise of novel radiation therapy techniques as stereotactic body radiation therapy (SBRT) and intra-operative radiation therapy (IORT) have resulted in an increasing use of very large dose fractions. It has been argued that the biological effect of large dose fractions may differ from that of conventional fraction sizes. Therefore, the biological effect of large single doses was investigated. Material/Methods: Clonogenic cell survival of MCF7 and MDA-MB-231 breast cancer cells was determined after direct X-ray irradiation, irradiation of feeder cells, or transfer of conditioned medium (CM). CM was collected 24h after irradiation and directly used. Cell-cycle distributions and the apoptotic fraction (sub-G1) were measured by flow cytometry, cytokines in the CM were quantified by a cytokine antibody array. gH2AX foci (surrogate marker for DNA double-strand breaks) were detected by immunofluorescence microscopy. Results: An increase in density of MCF7 cells from 10 to 50x103 cells per flask resulted in an 8.5-fold decrease in clonogenic survival after irradiated with a single 12 Gy fraction (P<0.0001). Likewise, seeding of unirradiated MCF7 cells on 20 Gy irradiated MCF7 feeder cells resulted in a 28% reduction in colony formation (P<0.0001). A part of this effect could be attributed to a transferrable factor, as a dose-dependent reduction in colony formation was also observed in the 5-15 Gy dose range, when unirradiated or moderately (4 Gy) irradiated cells were treated with CM from irradiated cells. These observations were confirmed in MDA-MB-231 cells. While no effect of CM on apoptosis and cell cycle distribution was observed, and no differentially expressed cytokine could be identified, the transferable factor induced a significant, prolonged expression of gH2AX DNA repair foci at 1-12h. Conclusions: A dose-dependent non-targeted effect of irradiation on clonogenic cell survival was observed in the dose range of 5-15 Gy. The dependence of the surviving fraction on cell numbers at high doses would represent a “cohort effect” in vivo. These results support the hypothesis that non-targeted effects may contribute to the efficacy of very large dose fractions in radiation therapy.
3439 Radiation Therapy Induces an Adaptive Upregulation of PD-L1 on Tumor Cells Which May Limit the Efficacy of the Anti-Tumor Immune Response But Can Be Circumvented by Anti-PD-L1 T. Illidge,1 G. Lipowska-Bhalla,1 E. Cheadle,1 J. Honeychurch,1 E. Poon,2 M. Morrow,2 R. Stewart,2 R. Wilkinson,2 and S. Dovedi1; 1Manchester University, Manchester, United Kingdom, 2Medimmune Limited, Granta Park, Cambridge, United Kingdom Purpose/Objective(s): Radiation therapy (RT) plays a definitive part of anti-cancer therapy for the majority of common cancers but for many patients metastatic disease and local recurrence are common and the outlook remains poor. New more effective RT combination approaches are urgently required that decrease local and distant recurrence to improve outcomes. Recent evidence suggests that in addition to direct tumoricidal effects, RT can modulate the tumor cell phenotype, increasing antigenicity and sensitivity to immune-mediated killing and induce the release of DAMP, which enhance immunogenicity, rendering tumor cells more “visible” for host immune attack via recruitment and activation of DC. Despite the immunogenicity of RT induced tumor cell death, RT delivered to tumors in the clinic, rarely generates therapeutic systemic anti-cancer immune responses or “abscopal effects.” Materials/Methods: Established syngeneic murine tumors models and matched in vitro tumor cells lines treated RT. Results: RT leads to upregulation of tumor cell expression of PD-L1 in vivo but not when cells are irradiated in vitro. Furthermore tumor infiltrating CD8+ cytotoxic T lymphocytes (CTL) have increased expression of PD-1 following RT in vivo. Using depleting antibodies we determined that the depletion of CD8+ T cells but not CD4+ T cells or NK cells could abrogate this RT-induced increase in tumor cell expression of PD-L1 in vivo. Furthermore, silencing of IFNgR1 using ShRNA confirmed that this process was dependent on CD8+ T cell production of IFNg suggesting an adaptive upregulation of PD-L1 following RT occurs in response to CTL activation. Taken together these findings suggest that the immunogenicity of RT may be limited via the PD-L1/PD-1 signalling axis and may contribute to treatment failure. We next sought to determine whether blockade of the PD-1/PD-L1 signalling axis could enhance the therapeutic response to RT. Administration of either an anti-PD-1 or anti-PD-L1 mAb in combination with RT led to substantially improved survival when compared to either monotherapy alone with approximately 60% of treated mice becoming long term survivors. Importantly our data reveal that combination therapy with RT and anti-PD1/ PDL1 generates long-term immunological memory protecting against tumor rechallenge in mice. Conclusions: The upregulation of tumor cell PD-L1 expression in response to RT which appears to be a mechanism of adaptive resistance tumor cells limiting anti-tumor immune response after RT. RT and anti-PD-L1 mAb combination therapy has the potential to overcome this resistance and to increase the efficacy of RT and improve outcome further and warrants well designed clinical trial evaluation. Author Disclosure: T. Illidge: L. Funding Other; Medimmune industrial grant supported research. G. Lipowska-Bhalla: None. E. Cheadle: None. J. Honeychurch: None. E. Poon: A. Employee; Medimmune Limited. M. Morrow: A. Employee; Medimmune Limited. R. Stewart: A. Employee; Medimmune Limited. R. Wilkinson: A. Employee; Medimmune Limited. S. Dovedi: E. Research Grant; Medimmune funding.
3440 BVES Loss Is Protective in Radiation Enteritis V. Reddy,1 B. Parang,1 S.V. Poindexter,1 C.W. Barrett,1 A. Bradley,1 E. Harris,1 Y.A. Choksi,1 M.K. Mittal,1 K. Singh,1 R. Chaturvedi,1
International Journal of Radiation Oncology Biology Physics
the dose giving 10% surviving fraction, was calculated by fitting the surviving fractions to the linear-quadratic model; surviving fraction Z exp(-aD-bD2), where D is the dose. The HMGB1 level in the culture supernatant of the cells receiving the D10 of X-ray or carbon-ion beam was assessed by ELISA, 72 and 96 hours after the irradiation. Results: In TE2, KYSE70, A549, NCI-H460 and WiDr cells, the D10 for X-ray was 2.1, 6.7, 8.0, 4.8 and 7.1 Gy, respectively, while that for carbonion beam was 0.9, 2.5, 2.7, 1.8 and 3.5 Gy, respectively. Seventy-two hours after the irradiation for D10, both X-ray and carbon-ion beam significantly increased the HMGB1 level in the culture supernatant in KYSE70, A549, NCI-H460, WiDr cells. Furthermore, 96 hours after the irradiation for D10, both X-ray and carbon-ion beam significantly increased the HMGB1 level in all the 5 cell lines tested. There was no significant difference in the increase in the HMGB1 level between X-ray and carbon-ion beam in each data point except at 96 hours in NCI-H460 cells, where carbon-ion beam was significantly superior to X-ray in the induction of HMGB1. Conclusions: Our results indicate that carbon-ion beam as well as X-ray increases the release of HMGB1 from various types of human cancer. The comparable levels of HMGB1 detected after the irradiation with iso-survival doses between X-ray and carbon-ion beam indicated that carbon-ion radiation therapy is capable of activating anti-tumor immunity at least equivalent to X-ray radiation therapy. Author Disclosure: Y. Suzuki: None. Y. Yoshimoto: None. T. Oike: None. N. Okonogi: None. K. Ando: None. H. Sato: None. S. Noda: None. M. Isono: None. K. Mimura: None. K. Kono: None. T. Nakano: None.
Author Disclosure: M. Veldwijk: None. B. Zhang: None. F. Wenz: A. Employee; University Medical Center Mannheim, University of Heidelberg. E. Research Grant; Elekta, Zeiss. F. Honoraria; Elekta, Zeiss, Celgene, Roche, Lilly, Ipsen. G. Consultant; Elekta. I. Travel Expenses; Elekta, Zeiss, Celgene, Roche, Lilly, Ipsen. K. Advisory Board; Elekta, Celgene. Q. Patent/License Fee/Copyright; Zeiss. C. Herskind: None.
3438 A Potential Role for Nontargeted Biological Effects in Cellular Inactivation After Ionizing Irradiation With Large Single Doses M. Veldwijk, B. Zhang, F. Wenz, and C. Herskind; Department of Radiation Oncology, Universita¨tsmedizin Mannheim, Medical Faculty Mannheim, University of Heidelberg, Mannheim, Germany Purpose/Objective(s): The rise of novel radiation therapy techniques as stereotactic body radiation therapy (SBRT) and intra-operative radiation therapy (IORT) have resulted in an increasing use of very large dose fractions. It has been argued that the biological effect of large dose fractions may differ from that of conventional fraction sizes. Therefore, the biological effect of large single doses was investigated. Material/Methods: Clonogenic cell survival of MCF7 and MDA-MB-231 breast cancer cells was determined after direct X-ray irradiation, irradiation of feeder cells, or transfer of conditioned medium (CM). CM was collected 24h after irradiation and directly used. Cell-cycle distributions and the apoptotic fraction (sub-G1) were measured by flow cytometry, cytokines in the CM were quantified by a cytokine antibody array. gH2AX foci (surrogate marker for DNA double-strand breaks) were detected by immunofluorescence microscopy. Results: An increase in density of MCF7 cells from 10 to 50x103 cells per flask resulted in an 8.5-fold decrease in clonogenic survival after irradiated with a single 12 Gy fraction (P<0.0001). Likewise, seeding of unirradiated MCF7 cells on 20 Gy irradiated MCF7 feeder cells resulted in a 28% reduction in colony formation (P<0.0001). A part of this effect could be attributed to a transferrable factor, as a dose-dependent reduction in colony formation was also observed in the 5-15 Gy dose range, when unirradiated or moderately (4 Gy) irradiated cells were treated with CM from irradiated cells. These observations were confirmed in MDA-MB-231 cells. While no effect of CM on apoptosis and cell cycle distribution was observed, and no differentially expressed cytokine could be identified, the transferable factor induced a significant, prolonged expression of gH2AX DNA repair foci at 1-12h. Conclusions: A dose-dependent non-targeted effect of irradiation on clonogenic cell survival was observed in the dose range of 5-15 Gy. The dependence of the surviving fraction on cell numbers at high doses would represent a “cohort effect” in vivo. These results support the hypothesis that non-targeted effects may contribute to the efficacy of very large dose fractions in radiation therapy.
3439 Radiation Therapy Induces an Adaptive Upregulation of PD-L1 on Tumor Cells Which May Limit the Efficacy of the Anti-Tumor Immune Response But Can Be Circumvented by Anti-PD-L1 T. Illidge,1 G. Lipowska-Bhalla,1 E. Cheadle,1 J. Honeychurch,1 E. Poon,2 M. Morrow,2 R. Stewart,2 R. Wilkinson,2 and S. Dovedi1; 1Manchester University, Manchester, United Kingdom, 2Medimmune Limited, Granta Park, Cambridge, United Kingdom Purpose/Objective(s): Radiation therapy (RT) plays a definitive part of anti-cancer therapy for the majority of common cancers but for many patients metastatic disease and local recurrence are common and the outlook remains poor. New more effective RT combination approaches are urgently required that decrease local and distant recurrence to improve outcomes. Recent evidence suggests that in addition to direct tumoricidal effects, RT can modulate the tumor cell phenotype, increasing antigenicity and sensitivity to immune-mediated killing and induce the release of DAMP, which enhance immunogenicity, rendering tumor cells more “visible” for host immune attack via recruitment and activation of DC. Despite the immunogenicity of RT induced tumor cell death, RT delivered to tumors in the clinic, rarely generates therapeutic systemic anti-cancer immune responses or “abscopal effects.” Materials/Methods: Established syngeneic murine tumors models and matched in vitro tumor cells lines treated RT. Results: RT leads to upregulation of tumor cell expression of PD-L1 in vivo but not when cells are irradiated in vitro. Furthermore tumor infiltrating CD8+ cytotoxic T lymphocytes (CTL) have increased expression of PD-1 following RT in vivo. Using depleting antibodies we determined that the depletion of CD8+ T cells but not CD4+ T cells or NK cells could abrogate this RT-induced increase in tumor cell expression of PD-L1 in vivo. Furthermore, silencing of IFNgR1 using ShRNA confirmed that this process was dependent on CD8+ T cell production of IFNg suggesting an adaptive upregulation of PD-L1 following RT occurs in response to CTL activation. Taken together these findings suggest that the immunogenicity of RT may be limited via the PD-L1/PD-1 signalling axis and may contribute to treatment failure. We next sought to determine whether blockade of the PD-1/PD-L1 signalling axis could enhance the therapeutic response to RT. Administration of either an anti-PD-1 or anti-PD-L1 mAb in combination with RT led to substantially improved survival when compared to either monotherapy alone with approximately 60% of treated mice becoming long term survivors. Importantly our data reveal that combination therapy with RT and anti-PD1/ PDL1 generates long-term immunological memory protecting against tumor rechallenge in mice. Conclusions: The upregulation of tumor cell PD-L1 expression in response to RT which appears to be a mechanism of adaptive resistance tumor cells limiting anti-tumor immune response after RT. RT and anti-PD-L1 mAb combination therapy has the potential to overcome this resistance and to increase the efficacy of RT and improve outcome further and warrants well designed clinical trial evaluation. Author Disclosure: T. Illidge: L. Funding Other; Medimmune industrial grant supported research. G. Lipowska-Bhalla: None. E. Cheadle: None. J. Honeychurch: None. E. Poon: A. Employee; Medimmune Limited. M. Morrow: A. Employee; Medimmune Limited. R. Stewart: A. Employee; Medimmune Limited. R. Wilkinson: A. Employee; Medimmune Limited. S. Dovedi: E. Research Grant; Medimmune funding.
3440 BVES Loss Is Protective in Radiation Enteritis V. Reddy,1 B. Parang,1 S.V. Poindexter,1 C.W. Barrett,1 A. Bradley,1 E. Harris,1 Y.A. Choksi,1 M.K. Mittal,1 K. Singh,1 R. Chaturvedi,1