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with head and neck squamous cell carcinoma (HNSCC) treated with definitive radiotherapy combined with cisplatin (CRT) or cetuximab (BRT). Material and Methods Immunohistochemistry for CD68/CD163 (double staining) and human leukocyte antigen (HLA) class I as well as p16 expression were performed on pretreatment tissue samples of the enrolled HNSCC patients (treated with CRT vs BRT matched 2:1) and 95 patients’ results were evaluable and included in the analysis. Density of CD163+ cells and CD68+CD163- cells in stromal and intraepithelial compartment were evaluated semi-quantitatively by two pathologists, as well as HLA class I expression in tumor cells. The correlations between biomarker expressions and clinicopathological characteristics and treatment outcomes were analyzed. Results Positive p16 expression was significantly associated with high intraepithelial macrophage density (IEMD) (p<0.001) and low HLA class-I expression (p=0.003); while p16- associated with high M2/Macrophages in the stroma (p=0.002). Multivariate analysis showed that the phenotype of TAM or HLA class I downregulation was not independent prognostic factors for survival or tumor control. Subgroup analysis showed that in p16+ population, patients with high IEMD had significantly improved outcome vs. patients with low IEMD in terms of 5-year progression-free survival (PFS, 81.2% vs. 25.0%, p<0.001), locoregional control (LRC, 95.5% vs. 50.0%, p=0.003) and distant control (DC, 89.5% vs. 33.3%, p<0.001), while in p16- population, no difference was observed (5-year PFS: 39.0% vs. 37.3%, p=0.59; LRC: 65.9% vs. 60.8%, p=0.76; DC: 69.1% vs. 76.4%, p=0.42). In subgroup analysis according to treatment option, 5-year PFS of patients with low CD163+ density in tumor stroma treated with CRT was significantly improved vs. those with BRT (54.5% vs. 36.1%, p=0.03), while in tumors with high CD163+ density, there was no significantly difference of PFS between the two treatment subgroups (p=0.67). Conclusion The phenotype of TAM or HLA class I downregulation didn’t independently predict survival in HNSCC patients. However, IEMD expression may play different roles in patients with p16+ vs. p16- disease. CD163+ cells density in stroma may provide information for selecting suitable patients for concurent cetuximab or cisplatin with radiotherapy. Further validation in external cohort is warranted. OC-0490 Targeting PARP1 and the intra-S/G2 checkpoints for highly effective radiosensitization of HPV+ HNSCC T. Rieckmann 1 , C. Busch 2 , K. Hintelmann 1 , T. Berenz 1 , M. Kriegs 3 , A. Münscher 2 , C. Petersen 4 , K. Rothkamm 3 1 University Medical Center Hamburg - Eppendorf UKE, Laboratory for Radiobiology & Department of Otorhinolaryngology, Hamburg, Germany 2 University Medical Center Hamburg - Eppendorf UKE, Department of Otorhinolaryngology and Head and Neck Surgery, Hamburg, Germany 3 University Medical Center Hamburg - Eppendorf UKE, Laboratory for Radiobiology and Experimental Radiation Oncology, Hamburg, Germany 4 University Medical Center Hamburg - Eppendorf UKE, Department of Radiotherapy and Radiation Oncology, Hamburg, Germany Purpose or Objective The enhanced radiation sensitivity of HPV+ HNSCC is also observed on the cellular level when comparing HPV+ and HPV- HNSCC cell lines. We could show that the underlying mechanism is a defect in DNA double-strand break repair associated with a profound and sustained G2-arrest. This defect can be exploited by molecular targeting

approaches additionally compromising the DNA damage response of these cells to further enhance their radiation sensitivity. We now tested a novel approach of combined targeting of PARP1 and the DNA-damage induced intra-S and G2 cell cycle checkpoints to achieve highly efficient radiosensitization. Material and Methods Mechanistic proof of efficacy of the various inhibitors and functional analyses were performed using Western blot, immunofluorescence microscopy, assessment of cell cycle distribution and flow cytometric assessment of γH2AX. PARP1 was inhibited using olaparib; intra-S/G2 checkpoint inhibition was performed using the Wee1- inhibitor AZD1775 or a combination of AZD1775 and the Chk1-Inhibitor prexasertib. Radiosensitization was assessed using colony formation assay. HPV+ HNSCC cells: UD-SCC-2, UM-SCC-47, UPCI-SCC-154. Results Enhancing CDK1/2 activity through the Wee1-inhibitor AZD1775 resulted in reduced proliferation rates and severe replication stress in HPV+ HNSCC cells. The latter was apparent from an accumulation of cells in the S- phase as well as a strong increase of the γH2AX marker especially in S-phase cells. Additional PARP-inhibition through olaparib resulted in an only modest further reduction of proliferation and had little effect on cell cycle disturbances or γH2AX levels except for UD-SCC-2 cells. When combined with radiation, both olaparib as well as AZD1775 induced moderate radiosensitization as observed previously. Combined inhibition resulted in highly effective radiosensitization of all three cell lines. Alternative checkpoint targeting through combined Wee1/Chk1-inhibition using severely reduced doses of both inhibitors to limit toxicity yielded highly similar results when combined with PARP-inhibition, which further demonstrates the robustness of the approach. Conclusion The combined inhibition of PARP1 and the intra-S/G1 checkpoint is a highly effective approach for the radiosensitization of HPV+ HNSCC cells. It may therefore represent a viable alternative for the current standard of concomitant cisplatin-based chemotherapy and may even allow for a reduction in radiation dose. Further functional studies are ongoing and in-vivo experiments are currently being planned. OC-0491 CRISPR-Cas9 screen of DNA damage response reveals novel radiosensitizers for head and neck cancers R. Dok 1 , M. Glorieux 1 , M. Bamps 1 , A. Sablina 2 , S. Nuyts 3 1 KU Leuven, Oncology, Leuven, Belgium 2 VIB-KU Leuven Center for Cancer Biology, Oncology, Leuven, Belgium 3 UH Leuven-KU Leuven, Oncology, Leuven, Belgium Purpose or Objective Local recurrences of head and neck squamous cell carcinoma (HNSCC) after radiotherapy can be due to the high DNA repair capacity of cancer cells. Inhibiting these DNA repair mechanisms in combination with radiotherapy (RT) could increase tumor control. Several studies fortify that imbalances in DNA repair pathways and cell cycle regulation are different in Human Papilloma Virus related (HPV+) and unrelated (HPV-) HNSCC, suggesting that different treatment approaches should be used for HPV+ and HPV- HNSCC. Material and Methods To unravel the most efficient radiosensitizers for different HNSCC groups, we performed an array-based screen using a CAS9/CRISPR library targeting different classes of DNA repair genes . We validated these results with commercially available drugs by clonogenic assays in 6 HNSCC cell lines. We investigated the repair kinetics by gH2AX and RAD51 foci formation and the effect on cell cycle by flow cytometry. We assessed the therapeutic