Abstract 1582: Isolation of circulating tumor cells and evaluation of PD-L1 expression in metastatic lung cancer
Authors: Dhar Manjima , Che James , Wong Jessica M. , Pao Edward , Yu Victor SH , Matsumoto Melissa , Goldman Jonathan , Garon Edward , Sollier Elodie , Kulkarni Rajan , Di Carlo Dino 
Published: August 2015

Abstract
INTRODUCTION
Metastatic lung tumors have adopted methods to evade the immune system via overexpression of programmed cell death ligand 1 (PD-L1). PD-L1 binds to PD-1 on T-cells and suppresses its activity. Studies have shown immunotherapy with PD-1 and PD-L1 inhibitors induce clinical response as well as an association with response and tumor PD-L1 level. Thus, tumor PD-L1 expression is a screening criterion for some studies of anti-PD-1 or PD-L1 drugs. Current methods of evaluating PD-L1 expression involve an invasive tumor biopsy. The biopsy itself can be difficult to perform, especially for lung tumors. Biopsies only show one section of the tumor, which may not represent tumor heterogeneity. We show that circulating tumor cells (CTCs) express a wide range of PD-L1 levels, this can aid in screening patients for anti-PD-1/PD-L1 drugs. CTCs slough off from tumors, and can give us a better picture of response that takes into account the heterogeneity at many tumor sites.
METHODS
We have developed a microfluidic device for rapid, size-based capture of circulating tumor cells (CTCs) from blood. The Vortex HT chip contains parallel channels and rectangular trapping reservoirs. At high flow rate (8mL/min), large cells (&gt;15μm diameter) experience lift forces and become stably trapped in fluid vortices in the reservoirs. Small blood cells do not experience sufficient lift force and can be washed away. We release the trapped cells by lowering the flow rate to dissipate the vortices and collect the cells in a concentrated volume (∼300μL).
We use Vortex HT to assess utility of capturing CTCs from patients with non-small cell lung cancer (NSCLC). After collecting the CTCs (30 patient samples), we evaluated the expression of PD-L1 using immunofluorescence with positive controls A549 and H1703, and red blood cells as negative control. We measure cell size, intensity levels of cytokeratin (CK), and CD45 on the collected cells. These metrics allow us to distinguish between CTCs and large leukocytes. Cells larger than 16μm, with high levels of CK, and no CD45 are classified as CTCs. We use a semi-automated algorithm to quantify fluorescence.
RESULTS
We found significant heterogeneity in PD-L1 expression levels across CTCs from the same patient at one time point. Overall for all samples the PDL1 levels on CTCs ranges from 0 to 4000 total intensity per cell normalized by intensity of PDL1 on H1703. On an individual scale, a sample with 8.5 CTCs/mL had PD-L1 intensities ranging from 300 to 4000. While in another sample with 7 CTCs/ml, the PD-L1 intensities ranged from 0 to 1000. For the CTCs analyzed in these samples, the CD45 levels normalized by CD45 on healthy leukocytes were lower than 0.08 intensity.
CONCLUSION
Further study can lead to simple, non-invasive methods to predict patient response to immunotherapy. Comparison of CTC PD-L1 levels alongside tumor biopsy results could aid in identifying patients likely to respond to therapy.
Citation Format: Manjima Dhar, James Che, Jessica M. Wong, Edward Pao, Victor SH Yu, Melissa Matsumoto, Jonathan Goldman, Edward Garon, Elodie Sollier, Rajan Kulkarni, Dino Di Carlo. Isolation of circulating tumor cells and evaluation of PD-L1 expression in metastatic lung cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1582. doi:10.1158/1538-7445.AM2015-1582