PD-L1 expression on circulating tumor cells and platelets in patients with metastatic breast cancer

Abstract

Background: Immune checkpoint inhibition is effective in several cancers. Expression of programmed death-ligand 1 (PD-L1) on circulating tumor or immune effector cells could provide insights into selection of patients for immune checkpoint inhibition.

Methods: Whole blood was collected at serial timepoints from metastatic breast cancer patients and healthy donors for circulating tumor cell (CTC) and platelet PD-L1 analysis with a phycoerythrin-labeled anti-human PD-L1 monoclonal antibody (Biolegend clone 29E.2A3) using the CellSearch® assay. CTC PD-L1 was considered positive if detected on at least 1% of the cells; platelet PD-L1 was considered positive if ≥100 platelets per CellSearch frame expressed PD-L1.

Results: A total of 207 specimens from 124 metastatic breast cancer patients were collected. 52/124 (42%) samples at timepoint-1 (at or close to time of progressive disease) had ≥5 CTC/7.5ml whole blood. Of those, 21 (40%) had positive CTC PD-L1. In addition, platelet PD-L1 expression was observed in 35/124 (28%) at timepoint-1. Platelet PD-L1 was not detected in more than 70 specimens from 12 healthy donors. Platelet PD-L1 was associated with ≥5 CTC/7.5ml whole blood (p = 0.0002), less likely in patients with higher red blood cell counts (OR = 0.72, p<0.001) and a history of smoking tobacco (OR = 0.76, p<0.001). Platelet PD-L1 staining was not associated with tumor marker status, recent procedures or treatments, platelet-affecting drugs, or CTC PD-L1 expression.

Conclusion: PD-L1 expression was found in metastatic breast cancer patients on both CTC and platelets in an independent fashion. Inter-patient platelet PD-L1 expression was highly heterogeneous suggesting that it is a biological event associated with cancer in some but not all patients. Taken together, our data suggest that CTC and platelet PD-L1 expression could play a role in predicting which patients should receive immune checkpoint inhibition and as a pharmacodynamics biomarker during treatment.

Conflict of interest statement

I have read the journal’s policy and the authors of this manuscript have the following competing interests: C.P. reports non-financial support (travel paid), contracts, and research support from Menarini Silicon Biosystems (MSB), the manufacturer of CellSearch® and, outside the submitted work, research funding from AstraZeneca, and Pfizer. C.P. is currently an employee of Eisai, Inc. University of Michigan (UM) has received funding to support research on behalf of D.F.H. and from Menarini/Silicon Biosystems, manufacturer of CellSearch. UM has submitted a patent application related to CTC and platelet PDL1 testing on which D.F.H., C.P., E.P.D. and E.M.D. are named inventors/co-inventors.UM holds patent US 8,790,878 B2 for which D.F.H. is designated as inventor, and that is licensed to MSB with annual royalties through January 2021. Outside the submitted work D.F.H. holds stock options from InBiomotion, and serves on advisory boards for Cepheid, Freenome, CellWorks, Lexent Bio, EPIC Science, Salutogenic Innovations, L-Nutra, BioVeca, OncoCyte, Turnstone Biologics, Predictus BioSciences, Tempus, Guardant and UM has received funding to support research on behalf of D.F.H. from Astra Zeneca, Merrimack, Eli Lilly, Puma Biotechnology, Pfizer. Outside the submitted work M.H. serves as a consultant and receives research support from Veralox Therapeutics. Outside the submitted work E.F.C. has served advisory/consulting roles for AstraZeneca, Biotheranostics, Ayala Pharmaceuticals and Athenex Oncology. The remaining co-authors have declared that no competing interests exist.

Figures

Fig 1. CTC Enumeration and CTC PD-L1 Expression at timepoint-1.

Whole blood (WB) specimens from 124 patients with metastatic breast cancer were processed using the CellSearch® system. Circulating tumor cell (CTC) enumeration and PD-L1 expression are displayed for the 52 (42%) subjects who had elevated CTC (≥5 CTC/7.5ml WB) at timepoint-1. Numbers under each bar represent subject study designations. A. Distribution of CTC enumeration. Each bar represents the number of CTC in an individual patient sample. B. Distribution of CTC PD-L1 staining. Colors represent the percent of CTC that expressed PD-L1, on a scale of 0-3+ (see Methods for details). 0 (■ white), 1+ (■ gray), and 2+ (■ black). No patients had 3+ staining for CTC PD-L1.

Fig 2. PD-L1 expression on platelets.

Whole Blood, a pure platelet pellet, and platelet poor plasma from two patients, one with positive platelet PD-L1 (A) and one with negative platelet PD-L1 (D), was processed through CellSearch® in parallel with PD-L1 staining in the 4th channel. A. Fluorescent image of positive platelet PD-L1 (PE conjugated) expression obtained from initial whole blood processed through CellSearch® using the classic method. B. Fluorescent images of PD-L1 (PE conjugated), cytokeratin (FITC-conjugated), CD-45 (APC conjugated), and DNA (DAPI) for platelet pellet from patient in panel A with positive platelet PD-L1 processed through CellSearch®. C. Fluorescent images of PD-L1 (PE conjugated), cytokeratin (FITC-conjugated), CD-45 (APC conjugated), and DNA (DAPI) for platelet poor plasma from patient in panel A with positive platelet PD-L1 processed through CellSearch®. See Methods for details. D. Florescent image of negative platelet PD-L1 (PE conjugated) expression obtained from initial whole blood processed through CellSearch® using the classic method. E. Florescent images of PD-L1 (PE conjugated), cytokeratin (FITC-conjugated), CD-45 (APC conjugated), and DNA (DAPI) for platelet pellet from patient in panel D with negative platelet PD-L1 processed through CellSearch®. F. Florescent images of PD-L1 (PE conjugated), cytokeratin (FITC-conjugated), CD-45 (APC conjugated), and DNA (DAPI) for platelet poor plasma processed from patient in panel D with negative platelet PD-L1 through CellSearch®.

Fig 3. CTC PD-L1 expression at serial timepoints.

Whole blood from serial specimens from the same patients was processed through CellSearch® with PD-L1 staining in the 4th channel. Each stacked bar represents distribution of CTC PD-L1 staining as described in Fig 1. Patients in each category are separated by a vertical line. A. Patients (N = 5) that maintained CTC PD-L1 positivity from timepoint-1 to subsequent timepoints. B. Patients (N = 9) that converted CTC PD-L1 negative to positive from timepoint-1 to subsequent timepoints. C. Patients (N = 4) that converted CTC PD-L1 positive to negative from timepoint-1 to subsequent timepoints. D. Patients (N = 3) that had fluctuating CTC PD-L1 expression among multiple timepoints.

Fig 4. Platelet PD-L1 expression at serial timepoints.

Whole blood from serial specimens from the same patients was processed through CellSearch® with PD-L1 staining in the 4th channel. Each line represents platelet PD-L1 staining from separate patients. Any data point that falls at or below the solid black line is considered platelet PD-L1 negative (