Abstract Background: Predictive biomarkers for response to anti-PD1 therapy are lacking. Because therapy with checkpoint inhibitors is cost intensive, noninvasive tools for prediction of responders are of major interest. Methods: The “Liquid Biopsy’’in head and neck squamous cell carcinoma (HNSCC) project involved the isolation of Circulating Tumor Cells (CTC) from patients with HNSCC at baseline, at different time points during treatment and at relapse. Herein, we assessed gene expression of PD-L1 on CTCs in a prospective fashion in a cohort of locally advanced inoperable HNSCC patients treated with curative intent (n = 61), in a second cohort of recurrent/metastatic HNSCC patients (n = 18) and in 20 healthy individuals, used as normal controls. For the quantification of PD-L1mRNA in CTCs, we developed a highly sensitive, specific, and robust RT-qPCR assay that was firstly analytically validated prior to its application in HNSCC patients. Results: In patients with locally advanced disease, 54/61(88.5%) samples were evaluable for CTCs at baseline. Twenty four samples were obtained after induction chemotherapy (IC) and 34(55.7%) at the end of concurrent chemo-radiation. At baseline 22/54(40.7%) pts were found to be positive for PD-L1 overexpression, at the post-IC samples 12/24(50%) patients were positive for PD-L1overexpressionand at the end of treatment, 11/34 (32.4%) patients were positive for PD-L1 overexpression. Patients with PD-L1 positive CTCs at the end of treatment had shorter progression-free survival (PFS) (p = 0.011) and overall survival (OS) (p = 0.004). Multivariate analysis showed that PD-L1 overexpression in patients at the end of treatment was independent prognostic factor of PFS (HR = 2422.4; p = 0.014) and OS (HR = 32.23; p = 0.014). Five R/M patients were found to be positive for PD-L1 out of the 18 (27.8%) at baseline. Conclusions: We demonstrate for the first time that detection of PD-L1+ CTCs at the end of treatment in patients with locally advanced disease is associated with shorter PFS and OS. Serial PD-L1 expression assessment has potential to select and monitor pts for PD-1 checkpoint inhibitors. These data support testing of PD-1 inhibitors in the adjuvant setting in patients with locally advanced HNSCC in whom PD-L1 positive CTCs are detected at the end of treatment. Citation Format: Areti Strati, George Koutsodontis, Ilias Angelidis, Clarence Sasaki, Margaritis Avgeris, Amanda Psyrri, Evi S. Lianidou. PD-L1 expressing circulating tumor cells (CTCs) in patients with head and neck squamous cell carcinoma (HNSCC). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3108.