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.
Table 1.
CTC PD-L1 expression at timepoint-1.
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®.
Table 2.
Platelet PD-L1 distribution in all samples.
Table 3.
Association of platelet PD-L1 score and CTC enumeration and PD-L1 expression at timepoint-1.
Table 4.
Multivariable results of factors of interest with CTC PD-L1 positive rate a and Platelet PD-L1 positivity b.
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 (<100 PD-L1 positive platelets). A. Patients (N = 10) that maintained platelet PD-L1 positive from timepoint-1 to subsequent timepoints. B. Patients (N = 2) that converted from having platelet PD-L1 positive to platelet PD-L1 negative at subsequent timepoints. C. Patient (N = 1) that had platelet PD-L1 expression fluctuating among multiple timepoints. D. Patients (N = 15) that converted from platelet PD-L1 negative at timepoint-1 to platelet PD-L1 positive at subsequent timepoints.