Abstract 1973: The expression of PD-L1 in circulating tumor cells is correlated with positive estrogen receptor status and higher CTC counts in HER2-negative metastatic breast cancer

Background: Hormone therapies alter the tumor microenvironment with the potential to sensitize estrogen receptor (ER)-positive metastatic breast cancer (MBC) to immunotherapy. Current trials are ongoing investigating the combination of hormone therapies, including aromatase inhibitors or selective ER modulators, with immune checkpoint inhibitors that target PD-1/PD-L1. We previously reported a significant correlation between tumor markers (e.g., ESR1) and circulating tumor cells (CTCs) regarding therapy resistance in MBC (2023 ASCO, 2024 SABCS). Herein, we sought to evaluate the if PD-L1 expression is associated with ER status, and CTC enumeration in patients with HER2-negative MBC. Methods: This study included 23 HER2-negative MBC patients enrolled between 2017 and 2022 at Robert H. Lurie Comprehensive Cancer Center of Northwestern University. Whole blood sample (7.5 mL/each) was collected from each patient. CTC enumeration and PD-L1 detection was conducted using the FDA-approved CellSearch® System (Menarini). CTCs were identified as CK+/DAPI+/CD45-. We assessed the association between PD-L1 expression, ER status, and CTC enumeration using Kruskal-Wallis test for statistics. Results: Among 23 patients, 12 patients were ER-positive and 11 were ER-negative, as determined through pathological identification. CTC counts of ≥5 were detected in 16 patients, while 7 patients had CTC counts <5. In the ER-positive group, the mean total CTC count was 68.9, with a mean of 25.1 PD-L1+ CTCs and 10 CTC clusters. In contrast, the ER-negative group had a significantly lower mean total CTC count of 48.5, with an average of 2.4 PD-L1+ CTCs and no CTC clusters (p <0.0001). These findings indicate that PD-L1 expression in CTCs is strongly associated with ER-positive status in MBC patients. Additionally, PD-L1+ CTCs were found in 16 patients, who exhibited a mean total CTC count of 61.4 and 6.5 CTC clusters, significantly higher than the 7 PD-L1-negative patients who had a mean total CTC count of 49.1 and no CTC clusters (p<0.05). Among patients with CTC counts ≥5, four had a PD-L1+ CTCs/total CTCs ratio above 50%, with an average of 87 total CTCs per patient, which was significantly higher than the average of 80.5 total CTCs seen in the other 12 patients with a ratio below 50% (p < 0.05). Conclusions: Our findings indicate that ER-positive, HER2-negative MBC is associated with significantly higher PD-L1 expression in CTCs compared to ER-negative, HER2-negative MBC. This increased PD-L1 expression in CTCs may contribute to immune evasion by affecting immune cell infiltration and response, potentially reducing the effectiveness of immunotherapy in ER-positive cases and predicting a more aggressive metastatic potential. Adding ER status and CTC enumeration when assessing PD-L1 expression in CTCs could be considered for treatment stratification in MBC. Citation Format: Qiang Zhang, Justin W. Zhang, Andrew A. Davis, Lorenzo Gerratana, Jian Jiao, Paolo D'Amico, Weijun Qin, Xinkun Wang, Akhil Chawla, Lisa Flaum, Leonidas C. Platanias, William J. Gradishar. The expression of PD-L1 in circulating tumor cells is correlated with positive estrogen receptor status and higher CTC counts in HER2-negative metastatic breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1973.