ESTRO 36 Abstract Book

S213 ESTRO 36 _______________________________________________________________________________________________

Radiotherapy and Radiation Oncology, Muenchen, Germany

of Radiation Oncology, Shanghai, China 3 Institut Gustave Roussy, Department of Pathology, Villejuif, France 4 Institut Gustave Roussy, Department of Head and Neck Oncology, Villejuif, France Purpose or Objective The aim of the present study was to investigate the prognostic value of CD8+, FoxP3+ tumor infiltrating lymphocytes (TILs) and PD-L1 expression in patients with head and neck squamous cell carcinoma (HNSCC) treated with definitive radiotherapy combined with cisplatin or cetuximab. Material and Methods Immunohistochemistry for CD8, FoxP3 was performed on formalin or formalin-acetic acid-alcohol fixed, paraffin- embedded, pretreatment tissue samples of 77 patients from a previously reported HNSCC cohort. PD-L1 results were evaluable in 38 patients only. The Kaplan–Meier method, univariate and multivariate analyses (MVA), were used to analyze the correlations of these biomarkers expression with patient/ tumor characteristics and treatment outcomes. Results CD8+ and FoxP3+ TIL densities differed significantly by HPV/p16 status, primary tumor location, T stage, and N stage. High CD8+ TIL level was identified in MVA as an independent prognostic factor for improved PFS (HR=0.3, 95%CI 0.1-0.7, p = 0.01) and there was a non-significant trend for better OS (HR=0.4, 95%CI 0.2-1.1, p = 0.07). High FoxP3+ TILs and PD-L1+ correlated with a favorable OS in the UVA, but not in the MVA. In p16+ subgpopulation, high CD8+TILs patients had improved 5-year OS compared with low CD8+TILs patients (100% vs. 88.9%, p=0.049), and PD- L1+ patients had improved 3-year OS compared with PD- L1- ones (100% vs. 50.0%, p=0.01). In low CD8+ TILs tumors, 5-year LRC of patients treated with CRT was improved vs. those with BRT (92.3% vs. 51.0%, p=0.01), while in high CD8+ TILs, no significant difference with CRT vs. BRT. Conclusion Immune factors in the tumor microenvironment correlated with HNSCC clinicopathological features and survival outcomes. CD8+ TILs were independently correlated with an improved clinical outcome in HNSCC patients and may potentially be correlated with different treatment outcome by cisplatin or cetuximab. Further work is warranted to validate these findings in a larger independent cohort and investigate the potentially associated causal mechanisms. SP-0401 Grid therapy: past, present, and future A. Siegbahn 1 1 Stockholm University, Department of Physics, Stockholm, Sweden Irradiation of tumours with grids of x-ray beams was proposed already about a decade after W.C. Röntgen's original discovery of x rays. Grid therapy was thereafter used in the clinic during the first half of the past century, mainly to reduce the incidence of radiation-induced skin reactions. The beam elements (forming the grid) were approximately 1 cm wide and were separated with a similar distance to form a chessboard-shaped irradiation pattern at the patient surface. The dose distribution in the target volume was alternating between very high peak- and low valley-doses. Later, it was realized that the toxicity was also reduced for other organs located beneath the skin, if leaving tissue volumes, in-between the radiation beam elements, unirradiated. In recent years, a large cohort of head-and-neck, thoracic and abdominal Symposium: Experimental therapies

Purpose or Objective Radio(chemo)therapy is a crucial treatment modality for head and neck squamous cell carcinoma (HNSCC) and radioresistance is a major reason for therapy failure and is related to tumour recurrence and poor prognosis. However, the underlying molecular mechanisms are largely unknown. In order to gain knowledge on this fundamental and clinically relevant feature, we established an in vitro model of CAL-33 HNSCC cell line subclones with different radiosensitivity. The main aim of the study was to identify signalling pathways and key regulators of radioresistance by static and dynamic global mRNA expression analyses followed by gene association network (GAN) reconstruction and pathway enrichment analyses. Material and Methods Subclones with altered radiosensitivity were generated by fractionated irradiation of the parental CAL-33 cells and colony formation assays were performed to confirm the differences in radiosensitivity. Selected subclones were characterized at the genome and transcriptome levels by SKY, array CGH, and mRNA-microarray analyses. Temporally differentially expressed genes upon irradiation were identified using natural cubic spline regression modelling on time-course transcriptome data. Moreover, early and late responding genes were determined. GANs were reconstructed using a partial correlation-based approach. Pathway enrichment analyses were conducted employing the Reactome pathway database. Microarray gene expression data were technically validated by qRT- PCR. Results The characterization of two subclones with enhanced radiation resistance (RP) and enhanced radiosensitivity (SP) revealed distinct genomic and transcriptomic changes compared to the parental cells. Interestingly, the expression of the endogenous retrovirus ERV3-1 in response to irradiation has been observed in all of the CAL- 33 cells, suggesting a role in the radiation response of HNSCC cells. Differentially expressed genes after irradiation shared by both subclones pointed to important pathways of the early and late radiation response, including senescence, apoptosis, DNA repair, Wnt, PI3K/AKT, and Rho GTPase signalling. The analysis of the most important nodes of the GANs revealed pathways specific to the radiation response in different phenotypes of radiosensitivity. Exemplarily, for the RP subclone the senescence-associated secretory phenotype (SASP) together with GPCR ligand binding were considered as crucial. Conclusion Our study presents comprehensive gene expression data of CAL-33 subclones with different radiation sensitivity. Based on these data GANs have identified known to be linked to the radiation response phenotypes. The resulting GANs and pathways associated with the resistant phenotype are of special interest and include SASP together with GPCR ligand binding. The identified pathways may represent key players of radioresistance, which could serve as potential targets for molecularly designed, therapeutic intervention. OC-0400 Prognostic impact of tumor infiltrating lymphocytes and PD-L1 expression in head and neck cancers D. Ou 1,2 , J. Adam 3 , I. Garberis 3 , P. Blanchard 1 , F. Nguyen 1 , A. Levy 1 , O. Casiraghi 3 , P. Gorphe 4 , I. Breuskin 4 , F. Janot 4 , S. Temam 4 , J. Scoazec 3 , E. Deutsch 1 , Y. Tao 1 1 Institut Gustave Roussy, Department of Radiation Oncology, Villejuif, France

2 Fudan University Shanghai Cancer Center, Department