ESTRO 2020 Abstract Book

S246 ESTRO 2020

Research Center for Environmental Health GmbH, Clinical Cooperation Group 'Personalized Radiotherapy in Head and Neck Cancer', Neuherberg, Germany ; 4 Helmholtz Center Munich- German Research Center for Environmental Health GmbH, Research Unit Radiation Cytogenetics, Neuherberg, Germany ; 5 Institute of Cell Biology Cancer Research- Medical Faculty- University of Duisburg-Essen, Department of Molecular Cell Biology, Essen, Germany ; 6 Humboldt University of Berlin, IRI Life Sciences, Berlin, Germany ; 7 University Medical Centre- Georg-August University Goettingen, Department of Medical Statistics, Goettingen, Germany Purpose or Objective Comprehensive therapy of locally advanced head and neck squamous cell carcinomas (HNSCC) includes (neo- )adjuvant radiotherapy with concomitant chemotherapy. Despite extensive treatment regimens with doses above 70 Gy, 5-year overall survival is as low as 50% for locally advanced, HPV-negative cases. Inherent radioresistance of HNSCC tumors is a major determinant of therapeutic failure. However, underlying pathomechanisms remain elusive. In this project, cellular parameters of radioresistance were characterized via multidimensional statistical analyses in a cell line panel of known radiosensitivities. Unexpectedly, a link between radioresistance and mechanisms of cellular senescence was identified. This was validated in vivo and in retrospective clinical analyses. Material and Methods In vitro, dimensionality reduction of clonogenic survival data of a cell line panel of five HPV-negative and two HPV- positive cell lines enabled extraction of radioresistance scores. These scores were used for correlation studies with different data qualities, including gene expression data, data on senescence induction, DNA damage response, and apoptosis induction. In vivo, a mouse-xenograft model with varying treatment regimens was established. Endpoints of analysis were tumor volume and senescence- associated cytokine production. Clinical relevance was validated using gene expression analyses of in silico data (The Cancer Genome Atlas) and a retrospective HNSCC cohort (LMU-KKG). Results In the HNSCC cell line panel a significant positive correlation was observed for inherent radioresistance with radiation-induced senescence and an associated cytokine release (CXCR2 ligands). The functional relevance of the cytokines for the development of radioresistance, was demonstrated via medium-transfer and inhibition of cytokine release in vitro . In vivo , irradiation-induced release of CCL5, CXCL12, CXCL1, IL1a, IL1b and IL6 dominated in HPV-negative tumors, while CXCL10 and CXCL11 were induced in HPV-positive tumors. Pharmacological inhibition of cytokine production affected tumor growth upon radiotherapy. Retrospective clinical analyses confirmed an association of expression of CXCR2 and its ligands with overall- and disease-specific survival. Conclusion Our study identifies irradiation-induced senescence and the associated production of cytokines as critical drivers of radioresistance in HNSCC. Therapeutic targeting of these mechanisms may open new treatment perspectives for a stratified subgroup of patients as shown in retrospective clinical analyses. Further experiments are under way to validate the clinical relevance of these findings. Acknowledgments : The project was funded by BMBF, ZiSStrans (NUK047)

and a potential benefit from combined radio- immunotherapy. A previous study [1] showed increased calreticulin expression from high energy proton irradiation compared to X-irradiation. Here, we investigate calreticulin expression in a lung cancer and glioblastoma cell line following low-energy proton irradiation at two positions around the Bragg peak. [1] Gameiro et al . Int J Radiat Oncol Biol Phys 2016: 95:120–130 Material and Methods A low energy proton beam line (16 MeV) was used for irradiation of A549 lung cancer cells and T98G glioblastoma cells. Irradiations were carried out at both the proximal and distal side of the Bragg peak. Estimated linear energy transfers were about 10 and 40 keV/mm, respectively. Dosimetry was ensured using a parallel plate advanced Markus ion chamber with a thin window. Delivered doses ranged from 2 to 16 Gy. Samples were analyzed for calreticulin expression 48 hours post- irradiation using flow cytometry (BD Biosciences, US). Controls, barcoded with DyLight 650, were added to irradiated samples, with the mix being further split into samples receiving either the primary calreticulin antibody (ab2907) plus secondary fluorescent antibody (Alexa Fluor 488) or the secondary antibody only. A fluorescence metric was calculated, taking unspecific binding of secondary antibody and background control levels into account. Signed and unsigned rank tests and linear regression was used for statistical analysis. Results Irradiated cells showed a significantly higher fluorescence compared to controls (P < 0.005). Taking the fluorescence ratio from cells irradiated with the same dose at the distal to the proximal side of the Bragg peak, a ratio of 1.3 was found overall for both cell lines (P < 0.02). Linear regression using dose and LET as independent variables and fluorescence level as dependent variable rendered both dose and LET highly significant (P< 0.001 for both cell lines). A slight fluorescence signal saturation was observed at the highest dose (16 Gy). The dose and LET dependence appeared stronger for A549 cells compared to T98G cells, although the difference was not significantly different. Conclusion Proton irradiation increases calreticulin levels at the cell surface compared to unirradiated controls. The calreticulin levels increase significantly with delivered dose. Calreticulin expression for cells irradiated at the distal side of the Bragg peak was higher than for cells irradiated at the proximal side, and a significant increase in expression levels with LET was found. This implies that calreticulin expression, and potentially immune response, can be maximized if proton track ends are deposited within the tumor during proton therapy.

Proffered Papers: Proffered papers 22: Radiobiological resistance mechanisms

OC-0446 Senescence and associated cytokines are critical drivers of inherent radioresistance in HNSCC U. Schötz 1,2,3 , S. Shnayien 1 , S. Spörl 1 , L. Kinzel 1 , C. Maihöfer 1,3 , U. Ganswindt 1,3 , J. Hess 3,4 , K. Unger 3,4 , H. Zitzelsberger 3,4 , D. Klein 5 , V. Jendrossek 5 , B. Klinger 6 , A. Sieber 6 , N. Blüthgen 6 , C. Belka 1,3 , S. Unkel 7 , K. Lauber 1,3 1 University Hospital- LMU München, Department of Radiation Oncology, Marburg, Germany ; 2 Philipps- University- University Hospital Giessen and Marburg, Department of Radiotherapy and Radiooncology, Marburg, Germany ; 3 Helmholtz Center Munich- German