Abstract 4990: Sequential tracking of PD-L1 expression and RAD50 induction in CTCs and circulating stromal cells of lung cancer patients during treatment with radiotherapy
Authors: Adams Daniel L. , Edelman Martin J. , Fang Penny , Jiang Wen , He Jianzhong , Xu Ting , Gao Hui , Reuben James M. , Qiao Yawei , Hahn Steven , Komaki Ritsuko , Liao Zhongxing , Tang Cha-Mei , Lin Steven H. 
Published: July 2016

Abstract
Background: PD-L1 expression can be induced by radiotherapy (RT) and may be an immune escape mechanism after RT, and has the potential to predict for responsiveness to immune checkpoint blockade immunotherapy. However, repetitive biopsies for monitoring PD-L1 expression in tumor and/or stroma are not feasible. Sequential assessment of PD-L1 expression on cancer associated circulating cells during treatment may be a way to measure the efficacy of combining RT and immunotherapy. RAD50 is a DNA repair gene that can be used in tracking tumor cell response to radiation. We therefore evaluated PD-L1 expression and RAD50 induction in CTCs and Cancer Associated Macrophage-Like Cells (CAMLs) in lung cancer patients (pts) before and during RT to track expression changes of these markers.
Methods: Twenty-nine pts with stage I-IV lung cancer were included in this prospective pilot study. Four pts received radiation therapy for stage I disease and 25 other pts received chemoradiation for stage II-IV disease. A baseline blood sample (7.5 ml) was drawn prior to the start of RT (T0), and a second blood sample was drawn at a treatment visit during RT (T1) for a total of 58 samples. Blood was processed using CellSieve™ microfiltration (Creatv MicroTech), stained for cytokeratin 8, 18 & 19 and CD45, and imaged. Using the QUASR (Quench, Underivatize, Amine-Strip and Restain) technique to remove fluoresce signal, all cells were restained for RAD50-AlexaFluor550 and PD-L1-AlexaFluor 488, along with DAPI nuclear stain. The RAD50 foci were quantified within nuclear regions and PD-L1 pixel intensity measured and grouped into 4 IHC groups: 0-negative (pixel average 0-215), 1-low (pixel average 216-300), 2-medium (pixel average 301-750), and 3-high (pixel average 751+).
Results: There was at least one cytokeratin positive cell (i.e. CTC or CAMLs) found in all but 2 samples (97%). CTCs were found in 69% of T0 and 72% of T1 samples, and CAMLs in 79% of T0 and 97% of T1 samples. PD-L1 expression ranged from 34-2552 pixel intensity, with an average of 285 at T0 and 525 at T1 (p = 0.07). Interestingly, 9 pts had no PD-L1 expression at T0 but an increase to 2 to 3+ at T1, 12 pts with low/no PD-L1 expression remained low at T1, and 8 pts had high PD-L1 expression that remained high or decreased at T1. RAD50 foci ranged from 0-16 per cell, with an average of 0.56 at T0 that increased to 4.68 at T1 (p&lt;0.001) during radiotherapy. There was no correlation between RAD50 induction and PD-L1 expression.
Conclusions PD-L1 expression and RAD 50 foci were quantifiable in both CTCs and CAMLs, and a correlative response to radiotherapy +/- chemotherapy was quantified. This data suggests that sequential tracking of CTCs and immune-related cells from the primary lung tumor is feasible using microfiltration and may potentially serve as a predictive biomarker for cancer therapy sensitivity.
Citation Format: Daniel L. Adams, Martin J. Edelman, Penny Fang, Wen Jiang, Jianzhong He, Ting Xu, Hui Gao, James M. Reuben, Yawei Qiao, Steven Hahn, Ritsuko Komaki, Zhongxing Liao, Cha-Mei Tang, Steven H. Lin. Sequential tracking of PD-L1 expression and RAD50 induction in CTCs and circulating stromal cells of lung cancer patients during treatment with radiotherapy. [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 4990.