ESTRO 2021 Abstract Book

S298

ESTRO 2021

for the validation of common and rare gene mutations that impact radiation sensitivity, thereby revealing new insights into intrinsic determinants of tumor sensitivity to radiotherapy.

OC-0401 Avoidance of DNA Replication Stress Leads to Decreased Cytosolic DNA in Breast Cancer Stem Cells F. Meyer 1 , A. Engel 2 , A. Krause 2 , T. Wagner 2 , L. Poole 2 , A. Dubrovska 3 , C. Peitzsch 3 , C. Petersen 4 , K. Rothkamm 2 , K. Borgmann 2 1 University Medical Center Hamburg-Eppendorf, Lab of Radiation Biology and Experimental Radiooncology, Hamburg, Germany; 2 University Medical Center Hamburg-Eppendorf, Lab of Radiobiology and Experimental Radiooncology, Hamburg, Germany; 3 Technical University Dresden, OncoRay, Dresden, Germany; 4 University Medical Center Hamburg-Eppendorf, Clinic for Radiotherapy, Hamburg, Germany Purpose or Objective Cancer stem cells (CSC) are a major cause for the failure of tumortherapy. This is mainly attributed to an increased DNA repair capacity and immune escape. Recent studies showed that functional DNA repair via Homologous recombination (HR) avoids radiation-induced accumulation of DNA in the cytoplasm, thus inhibiting an innate immune response. Yet, it is unclear how CSC contribute to an innate immune response after irradiation. Materials and Methods The investigations were performed in four breast cancer cell lines, their respective radioresistant subclones which were selected by repeated irradiation (10x4Gy) and isolated, ALDH1-positive CSC. Expression of Homologous Recombination (HR)-related and stem cell factors was determined, general markers for DNA- repair (53BP1-foci), DNA replication stress (yH2AX/RPA foci) and HR-functionality (plasmid reporter assay, RAD51 foci) were analyzed. Replication processes were assessed by DNA fiber assay, cytosolic dsDNA formation by PicoGreen™-assay and cGAS/STING activation via Western Blot. Radiosensitization was investigated by inhibition of the ATR/CHK1 signaling pathway in colony assays. Results An increased activity of ALDH1 and increased expression of stem cell markers was observed in all radioresistant clones and their isolated, ALDH1-positive BCSC. After irradiation, survival in the clonal subpopulations was significantly increased and the number of residual 53BP1 foci significantly decreased. This was especially apparent after irradiation in S-Phase (p<0.0001), indicating improved DNA repair via HR. This was supported by a stronger activation of the S phase kinases ATR and CHK1, an increased HR capacity and replication fork stability after irradiation. This resulted in significantly decreased yH2AX- and RPA foci after irradiation (p<0.001). This HR-mediated avoidance of radiation-induced replication stress resulted in a significantly lower accumulation of cytosolic DNA and decreased cGAS/STING activation. Indeed, the proportion of ALDH1-positive cells correlated significantly with the amount of cytosolic dsDNA after irradiation (p<0.001). The inhibition of ATR and CHK1 each led to a distinct radiosensitization of the radioresistant BCSC; the most radioresistant cell line was most strongly sensitized by ATR-inhibition (EF=3) and disrupted HR-repair. Furthermore, ATR-inhibition led to a significant increase of cytosolic dsDNA after irradiation in the radioresistant, isolated ALDH1-positive BCSC (p<0.0001). Conclusion The results show that BCSC are radiation resistant and minimize the formation of cytosolic DNA after irradiation through enhanced DSB repair and protection of replication forks by HR. Disruption of the S-phase damage response by ATR inhibition leads to a significant radiation sensitization and increase of immunogenic, cytosolic DNA. Therefore, we hypothesize that inhibitors to generally inactivate the S-phase DNA damage response, such as ATR or CHK1, may be used to further develop existing therapies in the future. OC-0402 Metabolomics as predictor of treatment response in neoadjuvant chemoradiation for rectal cancer C. Rosa 1,2 , F.C. Di Guglielmo 1 , L. Gasparini 1 , L. Caravatta 1 , M. Di Tommaso 3 , D. Pieragostino 4 , P. Del Boccio 5 , I. Cicalini 4 , D. Genovesi 1,6 1 SS. Annunziata Hospital, Department of Radiation Oncology, Chieti, Italy; 2 G. D'Annunzio, Department of Neuroscience, Imaging and Clinical Sciences, Chieti, Italy; 3 SS. Annunziata Hospital, Department of Radiation Oncology, Department of Radiation Oncology, Chieti, Italy; 4 G. D’Annunzio University, Analitical Biochemistry and Proteomics Unit, Research Centre on Aging (Ce.S.I), Chieti, Italy; 5 G. D’Annunzio University, Analitical Biochemistry and Proteomics Unit, Research Centre on Aging (Ce.S.I) , Chieti, Italy; 6 G. D'Annunzio University, Department of Neuroscience, Imaging and Clinical Science, Chieti, Italy Purpose or Objective Neoadjuvant chemoradiotherapy (CRT) is a standard of care in locally advanced rectal cancer (LARC) patients. In order to perform a treatment personalization, the identification of potential biomarkers able to predict tumor response to CRT is an interesting and promising topic. The collection of metabolites from blood, with simple and non-invasive procedures, could be well accepted by patients thanks to it feasibility. The aim of our study is the identification of potential metabolites able to predict tumor response. Materials and Methods An update of our previous study was performed, analysing ceramides in addition to aminoacids and acyl- carnitines in a larger population. Between March 2013 and January 2019, 40 LARC patients (26 males and 14 females, mean age: 68 years; range: 39–88 years) were treated with neoadjuvant CRT at the Radiotherapy Department. Sera (7.0 µL) were collected during routine chemistry tests before starting treatment (T0), at day 14 (T14), at day 25 (T25) of CRT and after finish CRT, at day 45 (T45). They were subjected to a targeted tandem mass spectrometry (MS/MS) analysis for the detection of the analytes. Tumor response was evaluated according to Mandard tumor regression grade (TRG). Results