OC-0487: Genetically engineered breast cancer mouse models to explore and target radioresistance

S190 B02) on undifferentiated cells to investigate the effect of inhibition on radiosensitivity. Results: GSC expressed higher levels of Rad51 compared to normal human astrocytes (NHA). When these cells were differentiated the levels of Rad51 were reduced in parallel to a reduction in established stem cell markers (SOX2, nestin). Both Rad51 inhibitors reduced Rad51 foci formation following irradiation in these cells, but did not affect induction of γH2AX foci. Inhibiting Rad51 with either agent in combination with single radiation doses between 1 and 5 Gy produced radiosensitisation, with dose modifying factors between 1.33 and 1.46. Conclusions: We have shown that Rad51 expression is increased in GSC and that pharmacological inhibition of Rad51 sensitises them to radiation. This suggests that targeting Rad51 may be a specific target in GSC and that targeting this repair pathway may lead to improved response in GBM. OC-0485 Abstract withdrawn OC-0486 G2 checkpoint signalling in hypoxic cells G. Hasvold1, C. Lund-Andersen1, M. Lando1, H. Lyng1, R.G. Syljuasen1 1 DNR - Norwegian Radium Hospital, Department of Radiotherapy and Radiobiology, Oslo, Norway Purpose/Objective: Tumour hypoxia is associated with poor prognosis for cancer patients, correlating with aggressive disease, resistance to standard treatments such as radiation therapy, and increased genomic instability. This genomic instability has been coupled to hypoxia-induced down-regulation of DNA repair pathways and increased levels of replication stress. However, little is known about the effects of hypoxia on the regulation of the DNA damage-induced G2 checkpoint, one of the main processes safeguarding genomic integrity. Our aim was to explore the effects of hypoxia on the levels of key G2 checkpoint regulators and examine subsequent consequences for checkpoint stringency. Materials and Methods: Hypoxia treatments (20-72 hours) were performed in an InVivO2200 Hypoxic Workstation (Ruskinn), with oxygen set-points of 0.0% (≤0.03%) or 0.2% (0.16-0.24%) O2. U2OS and HeLa cells were grown in plastic dishes or flasks for all experiments. Gene expression levels were assessed by Illumina bead arrays, protein levels by Western immunoblotting or flow cytometry with barcoding. Flow cytometry was also used for determination of cell cycle profiles and mitotic fractions. Residual DNA damage in cells entering mitosis after ionizing radiation was measured by live-cell imaging of U2OS cells expressing mCherry-53BP1 and by γH2AX levels in mitotic (pSer10-H3 positive) cells. Clonogenic survival assays were performed to measure cell survival. Results: Microarray gene expression analysis and Western immunoblotting of asynchronous U2OS and HeLa cells exposed to 20-24 hours hypoxia indicated a hypoxia-induced down-regulation of several G2 checkpoint regulators, including cyclin A and B, Plk1 and Chk2, and an up-regulation of p21. However, as hypoxia alters the cell cycle distribution, we subsequently used flow cytometry analysis to compare protein levels of individual G2 phase cells. This analysis showed that while protein levels of cyclin B, Plk1 and Chk2 were decreased in hypoxic G2 phase cells,cyclin A levels were unaltered compared to normoxic G2 phase cells. Consistent with low levels of cyclin B and Plk1 in G2 phase cells, G2 checkpoint activation appeared to be enhanced in cells irradiated after prolonged hypoxia. However, in keeping with impaired DNA repair, these cells appeared to exit the checkpoint with slightly more residual damage as measured by 53BP1 foci before mitosis and γH2AX levels in mitotic cells. Conclusions: Tumour hypoxia leads to alterations in the levels of several G2 checkpoint regulators, which may modify the stringency of the checkpoint and influence genome maintenance. This may also alter the responses to G2 checkpoint inhibitors currently in clinical trials, such as those targeting Plk1, Wee1 and Chk1. OC-0487 Genetically engineered breast cancer mouse models to explore and target radioresistance G. Borst1, C. Coackley2, H. Yucel2, C. Guyader3, A. Berlin2, J.J. Sonke1, M. Verheij1, J. Jonkers3, S. Rottenberg3, R. Bristow2 1 The Netherlands Cancer Institute, Radiotherapy, Amsterdam, The

ESTRO 33, 2014 Netherlands Princess Margaret Cancer Centre, Radiobiology, Toronto, Canada Netherlands Cancer Institute, Molecular Biology, Amsterdam, The Netherlands 2 3

Purpose/Objective: Our purpose is to define and target pathways involved in genetic- and hypoxia-mediated radioresistance. Resistant hypoxic cells may also be DNA repair-deficient and could define a new strategy for targeting using the concept of 'contextual synthetic lethality'. We therefore tested the effect of neoadjuvant and concurrent PARPi treatment in genetically- and contextually-mediated synethetic lethality models. Materials and Methods: We used clinically-relevant spontaneous p53-/;Brca1-/- and p53-/-; Brcawt murine breast tumours (syngeneic and orthotopic) in which single (12Gy) and fractionated RT doses were delivered to determine initial response in treatment-naïve vs. radiorecurrent or metastatic tumours. Using both growth delay and ex vivo clonogenic assays we have compared the efficacy of differential irradiation schedules alone or in combination with PARPi. Neoadjuvant PARPi (Olaparib, i.p. injection of 50mg/kg,BID, 7 days; in which irradiation took place 48 hours after end of PARPi dosing to preclude radiosensitization) was also tested. Changes in tumour hypoxia is being assessed throughout using PET-FAZA and EF5 staining in situ. Results: We observed in the p53-/-; Brca1-/-model that pre-irradiated and metastatic tumors were relatively resistant associated with larger volumes and shorter growth delay when compared to de novo tumour response (p<0.05). This resistance was offset using PARPi treatment. Both growth delay and clonogenic survival supported a role for improved tumour cell clonogen killing with neoadjuvant PARPI treatment (p<0.05). Hypoxia and DNA repair-correlative studies will be presented. Conclusions: Pre-irradiated and metastatic lesions were radioresistant compared to RT-naïve tumours de novo and can be offset with the use of PARPi. DNA and RNA sequencing are ongoing to understand the molecular mechanisms underlying this observation to guide future experiments exploring underlying mechanisms. Neoadjuvant PARPi can improve radiotherapy response supporting the concept of 'contextual synthetic lethality' and should be tested in Phase II clinical trials.

PROFFERED PAPERS: CLINICAL 9: BREAST CANCER (2) OC-0488 17 year results of the randomized boost versus no boost EORTC 22881-10882 trial in early breast cancer H. Bartelink1, P. Maingon2, P.M. Poortmans3, C. Weltens4, A. Fourquet5, J.J. Jager6, D.A.X. Schinagl7, C.C. Rodenhuis8, S. Collette9, L. Collette9 1 The Netherlands Cancer Institute -, Department of Radiation Oncology, Amsterdam, The Netherlands 2 Centre Georges – François Leclerc, Department of Radiation Oncology, Dijon, France 3 Dr. Bernard Verbeeten Instituut, Department of Radiation Oncology, Tilburg, The Netherlands 4 UZ Leuven, Department of Radiation Oncology, Leuven, Belgium 5 Institut Curie, Department of Radiation Oncology, Paris, France 6 Maastro Clinic, Department of Radiation Oncology, Maastricht, The Netherlands 7 Radboud University Nijmegen Medical Centre, Department of Radiation Oncology, Nijmegen, The Netherlands 8 University Medical Center Utrecht, Department of Radiation Oncology, Utrecht, The Netherlands 9 EORTC, Statistics Department, Brussels, Belgium Purpose/Objective: To investigate the long-term impact of a boost radiation dose of 16 Gy on local control, fibrosis, and overall survival for patients with stage I and II breast cancer who underwent breastconserving therapy. Materials and Methods: A total of 5,318 patients with microscopically complete excision followed by whole-breast irradiation of 50 Gy were randomly assigned to receive either a boost dose of 16 Gy (2,661 patients) or no boost dose (2,657 patients) (Baseline characteristics see: Bartelink et al. NEJM 2001; 348:1378-87). The patient cohort had a median follow-up of 17.2 years at the time of this intent-to-treat analysis (2-sided significance level 5%). Results: The median age was 55 years. Local recurrence was reported as the first treatment failure in 354 patients with no boost versus 237 patients with boost; at 20 years, the cumulative incidence of local recurrence was 16.4% (CI: 14.7-18.2) versus 12% (CI: 10.3-13.8) for the