Abstract 5697: Simultaneous assessment of PD-L1 and IFR1 expression on breast cancer circulating tumor cells
Authors: U'Ren Lance , Ericson Nolan , Huston Ryan , Mahen Elisabeth , Gadi VK , Blau C. Anthony , Kaldjian Eric 
Published: August 2018

Abstract
Background: There is a need for noninvasive predictive biomarkers of response to anti-PD1/PDL1 therapies. Assessment of circulating tumor cells (CTCs) is a rational approach to noninvasive sampling of tumors to understand the potential response or nonresponse to anti-PD1/PD-L1 therapies. IFN-gamma signals through the JAK/STAT cascade to induce PD-L1 via the Interferon Regulatory Factor-1 (IRF1) transcription factor, and is a potent inducer of PD-L1 expression in tumor cells. Recent studies have shown that low or absent IRF1 expression can identify melanomas that lack IFN-gamma responsiveness, and that IRF1 can have a higher predictive value of response to anti-PD1/PD-L1 therapy than PD-L1 itself. Using CTC models and the RareCyte platform, we developed a multiparameter assay that allows simultaneous PD-L1 and IRF1 assessment after CTC identification. We used this assay to better understand the relationship between IRF1 and PD-L1 expression in CTC model systems and applied this assay to blood samples from breast cancer patients.
Materials and Methods: Peripheral blood from normal donors or cancer patients under an IRB-approved protocol was collected into RareCyte blood collection tubes. PD-L1(+) and PD-L1(-) CTC models were created by culturing A549 overnight with or without 10ng/mL INF-gamma. The A549 cells were spiked into normal donor blood and buffy coats isolated from 7.5mL of blood by AccuCyte® separation and spread onto slides. MDA-MB-231 cells were used as a model to represent breast cancer cells that express PD-L1 in the absence of IFN-gamma signaling. Slides were stained with a 6-marker panel that included antibodies to pan-cytokeratin (CK), EpCAM, CD45, PDL1, IRF1, and a nuclear dye on the Leica Bond Rx auto-stainer. Slides were scanned with CyteFinder® and CTCs identified by CK and/or EPCAM positivity and negative CD45 staining. Confirmed CTCs were then assessed for expression of PD-L1 and IRF1; cellular compartment was recorded for IRF1 staining.
Results: Nuclear expression of IRF1 correlated with PD-L1 expression in IFN-gamma stimulated A549 cells. Unstimulated MDA-MB-231 cells expressed high levels of PD-L1, but this did not correlate with nuclear IRF1 expression. Breast cancer patient samples were identified having CTCs that expressed PD-L1. A PDL1 mean fluorescence intensity (MFI) threshold was set at the upper 95% CI of unstimulated A549 cells to define “high” and “low” PD-L1 expression in the patient samples. The population of high-PD-L1 CTCs had higher nuclear IRF1 MFI than low-PD-L1 CTCs, with mean fold-increase of 2.7 (range from 1.4 to 3.3).
Conclusions: Nuclear IRF1 expression correlates with PD-L1 expression in breast cancer CTCs. Using CTCs to evaluate expression of IRF1 and PD-L1 together with assessment of CTC mutational load could be a powerful noninvasive assessment of response to anti-PD1/PD-L1 therapies.
Citation Format: Lance U'Ren, Nolan Ericson, Ryan Huston, Elisabeth Mahen, VK Gadi, C. Anthony Blau, Eric Kaldjian. Simultaneous assessment of PD-L1 and IFR1 expression on breast cancer circulating tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5697.