ESTRO 38 Abstract book

S596 ESTRO 38

might introduce new useful biomarkers for treatment choices in the future. PO-1073 Biomathematical modeling of fractionated irradiation on immunogenic cell death induction in vitro F. Eckert 1 , L. Bardoscia 1,2 , P. Hausmann 1 , Z. Daniel 1 , D. Thorwarth 3 , S.M. Huber 4 1 University Hospital Tübingen, Radiation Oncology, Tübingen, Germany ; 2 University of Brescia, Radiation Oncology, Brescia, Italy ; 3 University Hospital Tübingen, Section for Biomedical Physics- Department of Radiation Oncology, Tübingen, Germany ; 4 University Hospital Tübingen, Experimental Radiation Oncology, Tübingen, Germany Purpose or Objective Immunogenic cell death (ICD) is counted among the crucial steps of eliciting effective anti-tumor immune responses after irradiation. Most publications argue in favor of hypofractionated regimens. However, systematic analyses of radiobiological parameters such as fractionation sensitivity are scarce for ICD induction. Several clinical settings such as the treatment of glioblastoma including large volumes of normal brain tissue rely on normofractionated radiotherapy regimens. Thus, we aimed to evaluate ICD induction systematically after different radiation regimens and modeled the data with biomathematical methods. Material and Methods A standard glioblastoma cell line (U87MG) and two patient-derived, stem cell enriched glioblastoma cell lines (LKVII and LKXVII) were analyzed for their membrane exposure of Calreticulin (CRT) 24h after irradiation (determined as a suitable time point in an initial experiment for the kinetics of CRT exposure after irradiation with 15 Gy) with single doses (2-24 Gy) and daily fractionated treatment (5 x 2 Gy, 3 Gy and 4 Gy, respectively). Percentage of cells with irradiation-induced CRT exposure after single dose irradiation was fitted with a non-linear least square fit to a Hill function. Equivalent single fraction irradiation doses were calculated for membranous CRT after fractionated irradiation using the equation derived from the data fit. Immunogenic cell death induction was confirmed by HGMB1 release (ELISA), normalized to number of viable cells 48h after single dose irradiation for U87MG and LKVII. Results The three cell lines showed a vast difference in basal CRT positivity in unirradiated controls of 6.9±0.24%, 9.5±0.09% and 15.3±0.18% for U87MG, LKVII and LKXVII, respectively. All cell lines showed significant ICD induction after irradiation with ≥8Gy single dose and 5 x 3 Gy and 4 Gy. The data were well fitted by a Hill function (R 2 >0.98). For both patient-derived cell lines, normofractionated irradiation of 10 Gy significantly increased membrane exposure of CRT. Corresponding single doses for fractionated regimens were higher for LKVII and LKXVII compared to U87MG. Significant HMGB1 release into the medium was observed for 8 Gy irradiation for LKVII and for 16 Gy for both tested cell lines (8.9±0.9 pg/10 6 cells vs.21.1±1.5 pg/10 6 cells, 4.0±0.7 pg/10 6 cells vs. 47.4±9.35 pg/10 6 cells for LKVII and U87MG, respectively). Conclusion Glioblastoma cells, a highly radioresistant tumor entity, shows ICD after single dose of 8 Gy or higher and fractionated irradiation. Daily fractionated irradiation seems to lead to accumulation of cellular damage leading to immunogenic cell death. Even a clinically low dose of 5 x 2 Gy induces ICD in patient-derived, stem cell enriched cultures. Thus, at least concerning the initial step of effective anti-tumor immune response, normofractionated radiotherapy might be a possible partner for immunotherapy strategies in the clinic.

Conclusion In the present study we indicated a common biological processes activated in MDA-MB-468 cell line stimulated with WF collected from patients after IORT treatment and cells treated with WF collected from BCS patients together with RIBE medium. Therefor we confirmed the role of the radiation-induced bystander effect in altering the biological properties of the wound fluids.

Poster: Radiobiology track: Immuno-radiobiology

PO-1072 INTRABEAM: precision hypo-fractionated radiotherapy with a systemic immune response. I. Linares 1 , M.Á. Berenguer 1 , E. Martínez 1 , M. Laplana 1 , R. Cañas 2 , S. Comas 3 , H. Pérez-Montero 1 , M. Ventura 1 , F. Guedea 1 1 Institut Català d'Oncologia, Radiation Oncology, Barcelona, Spain ; 2 Institut Català d'Oncologia., ONCOBELL Program-IDIBELL., Barcelona, Spain ; 3 Institut Català d'Oncologia, Radiation Oncology, Badalona, Spain Purpose or Objective To evaluate the changes in immune-cell phenotype in peripheral blood following intraoperative radiotherapy (IORT) in breast cancer patients. Material and Methods 45 patients were classified in three groups of treatment as follows: Group A (Lumpectomy and Intrabeam exclusive), Group B (Lumpectomy and Intrabeam followed by EBRT 40.05 Gy in 15 fractions of 2.67 Gy) and Group C (Lumpectomy and EBRT 40.05 Gy in 15 fractions of 2.67 Gy +/- EBRT boost 9 Gy in 3 fractions). For each group, peripheral blood mononuclear cells (PBMCs) were isolated from heparinized venous blood samples collected before treatment and during different time points after treatment: before lumpectomy, 48 hours after IORT or EBRT, and 3 and 10 weeks after radiation treatment was completed. Peripheral blood populations of cytotoxic T- cells (CTL), helper T-cells, Natural Killer cells (NK), regulatory T-cells (Treg) and Myeloid Derived Suppressor cells (MDSC) were measured using flow cytometry. Cell phenotypes were evaluated using the FACS Navios system (BeckmanCoulter). Data were analyzed using FlowJo software (Tree Star Inc., Ashland, OR, USA). Results 30 patients were included: 11, 15 and 4 patients for Group A, B and C respectively. For group A and B, the number of CTL increased three weeks after IORT (60.20% basal vs 67.10%) and EBRT (66.50% basal vs 71.30%) respectively. In contrast, for the control group (group C), a decrease in CTL was seen (64.35% vs 61.50%). In group A the number of NK cells increased after treatment (46.20% basal vs 59.20%), while in group B (42.90% basal vs 36.35%) and group C (56.80% basal vs 38.40%), we observed a NK decrease. For Treg we had mixed results which were hard to interpret. For Group A we saw a decrease during treatment (1.54% basal vs 1.44%) while for Group B we observed an increase of these cells (2.0% basal vs 2.75%). After 3 weeks, this tendency was reverted. For Group C, we observed an increase during treatment (1.45% basal vs 2.87%). For the MDSC panel, for granulocytes we observed a decrease in group A (6.44% basal vs 5.78%) and an increase in both group B (7.90% basal vs 10.31%) and group C (7.65% basal vs 10.20%) after 10 weeks. For Monocytes, in group A we observed the number of activated monocytes stable (8.95% basal 9.13%), whereas in group B and C we saw an increase after EBRT. Conclusion These results suggest that high doses per fraction would play an important role in CT, NK cells but not on the Treg and monocytes immunosuppression cells. Deciphering immune responses to treatment in breast cancer patients