ESTRO 36 Abstract Book

S529 ESTRO 36 _______________________________________________________________________________________________

involved in senescence process were measured by RT- qPCR. Results While the clonogenic assay showed very similar survival fraction curves for both conditions, we found highly significant differences between the two conditions of irradiation, when considering other biological outputs when cell were irradiated at confluence. Cell number and survival, morphological changes, cell cycle analysis, molecular footprinting and β-galactosidase activity were measured for doses up to 20 Gy. For all the assays, we observed and demonstrated stronger effects on HUVECs irradiated with the LINAC (4 MV) compared to the same irradiation performed with the SARRP (220 kV). Conclusion All together these results strongly support the fact that the clonogenic assay is not sufficient alone and that we need to implement new models with multi-parametric biological outputs to estimate a RBE that accurately predicts the biological cellular fate. Such approach could be useful for radiation protection but also for conditions such as stereotactic body radiation therapy where the LQ- model is inappropriate. Purpose or Objective Preclinical in vivo models are indispensable for radiobiological investigations. However, their application needs to follow the basic guidelines of animal studies (reduction, refinement, replacement) and such research should thus be supplemented by exploitation of suitable alternatives, e.g. in vitro model systems. Three- dimensional (3D) organotypic culture systems have been shown to more accurately reflect the in vivo cell situation as compared to standard 2D monolayer cell cultures. The present study was initiated to generate and characterize in vitro reconstituted human normal and malignant squamous epithelia. These will then be applied for analyses of the response to photon and ion irradiation, as a basis for the design of subsequent in vivo studies. Relevant damage processing pathways (including their dependence on radiation quality) will be identified, and biological targeting strategies will be screened. Also, dedicated RBE studies at different positions in the ion beam track for various endpoints will be performed. Material and Methods The 3D organotypic squamous epithelial tissues consist of epithelial cells cultured on top of “dermal matrices”, i.e. collagen gels formed from a collagen I solution populated by metabolically active fibroblasts. Epithelial cells are then seeded on top and cultured submerged. After 4 days of submerged culture the gels are lifted so that the epithelial cell monolayer is placed at the air-medium interface and further cultured for 10 days until stratification is complete. To reproduce skin equivalents, HaCaT cells were seeded onto human skin fibroblasts gels as mentioned above. To reconstruct normal and malignant oral epithelia we will use immortalized normal oral and FaDu squamous carcinoma cells, respectively. In radiobiological studies, endpoints to be compared to the in vivo situation will include morphology, differentiation, DNA damage/repair (e.g. yH2AX, micronuclei) and various radiation response-related signaling pathways, e.g of inflammation through IL-6, TGF-ß and pro-MMP1. Single dose as well as, importantly, daily fractionated irradiation protocols will be applied for both photons and ions. Results Our preliminary efforts to reconstruct squamous epithelia using HaCaT cells indicate the formation of a stratified PO-0957 Radiobiological studies in in vitro reconstituted squamous epithelia G. Zemora 1 , W. Dörr 1 1 Medical University of Vienna, Department of Radiotherapy- ATRAB - Applied and Translational Radiobiology, Vienna, Austria

epithelium on top of a fibroblast-populated matrix. Our results show positive IHC staining for the proliferating cells expressing Ki67 located at the stratum basale, as well as for the late differentiation proteins (involucrin and loricrin). The experiments for the reconstruction of 3D oral mucosa are ongoing. Conclusion In vitro reconstituted squamous epithelia are suitability models, with intermediate complexity between 2D cell cultures and tissues in vivo , for in vitro radiobiological studies. Prospectively, macrophages will be integrated in the 3D reconstructs to study radiation effects on the immune system. PO-0958 Radiogenomics: role of non-coding RNA genes in increased radiotherapy sensitivity L. Duran-Lozano 1 , V. Reyes 2 , M. Mollà 2 , M.J. Fuentes- Raspall 3 , M. Altabas 2 , T. Ramón y Cajal 4 , A. Barnadas 4 , O. Diez 1,5 , J. Giralt 2 , S. Gutiérrez-Enríquez 1 1 Vall d'Hebron Institute of Oncology-VHIO, Oncogenetics Group, Barcelona, Spain 2 Vall d'Hebron University Hospital, Department of Radiation Oncology, Barcelona, Spain 3 Hospital de la Santa Creu i Sant Pau, Department of Radiation Oncology, Barcelona, Spain 4 Hospital de la Santa Creu i Sant Pau, Medical Oncology Department, Barcelona, Spain 5 Vall d'Hebron University Hospital, Area of Clinical and Molecular Genetics, Barcelona, Spain Purpose or Objective Breast cancer (BC) is the first cause of cancer-related mortality of Spanish women and most common cancer in women worldwide. It is frequently treated with radiotherapy (RT), which can cause early and late side- effects that impact negatively on quality-of-life of cancer survivors. MicroRNAs and long non-coding RNAs (lncRNAs) modulate key cellular pathways in response to radiation. Single nucleotide polymorphisms (SNPs) in these two types of non-coding RNAs can alter their function and consequently modify the expression of genes that regulate, affecting the respective biological activities. As part of a long-term ongoing study, our aims were to test genetic association of SNPs in microRNAs and lncRNAs with late radiotherapy-induced toxicity and to characterize the expression of lncRNAs in blood cells of BC treated patients. Our final goal is to discover new genetic endpoints to predict individuals with increased susceptibility to radiotherapy side effects. Material and Methods DNA samples and clinical data were collected from 198 prospectively and 72 retrospectively recruited BC patients treated with RT in two hospitals. All patients were followed at least between two and six years after RT. 34 SNPs in microRNAs and lncRNA genes related to radiation response were genotyped using iPLEX® Gold with MassArray Agena Bioscience (Sequenom). RNA was obtained from blood before and after radiotherapy of 19 BC patients from the prospective cohort. Eight lncRNAs (FAS-AS1, MALAT1, TP53TG1, HOTAIR, PANDA, MEG3, ANRIL and LINC00467) involved in radiation cell response were assessed by RT-PCR, agarose gels and direct sequencing. A semiquantitative capillary electrophoresis of fluorescent amplicons was performed to estimate the proportion of total transcripts. Results The first analysis showed an association of overall long term toxicity after radiotherapy with rs4559081 A/A genotype of LINC00336 (OR=3.91 95%CI = 1.34-11.37), and grade ≥ 2 late radiation skin toxicity (fibrosis or telangiectasias) with rs17762938 A/C or CC of PCAT1 (OR=2.63 95% CI=1.00-6.86) and rs2910164 C/G of miR- 146a (OR=0.27 95%CI = 0.008-0.94). The expression and presence of different isoforms of all lncRNAs evaluated, except ANRIL and HOTAIR, were observed in blood cells.