PD-1 Expression Identifies Proliferating Chronic Lymphocytic Leukemia Cells and Informs Response and Resistance to BTK Inhibitor Therapy
Authors: Chang Andres , Pelletier Adam , McGuire Donald J , Tsagiopoulou Maria , Karipidou Maria , Ayers Amy , Leal Alyssa , Churnetski Michael , O'Leary Colin B , Switchenko Jeffrey M. , Davis Carl , Frank David A. , Koff Jean L. , Cohen Jonathon B , Sekaly Rafick-Pierre , Stamatopoulos Kostas , Flowers Christopher R. , Ahmed Rafi 
Published: December 2024

Chronic lymphocytic leukemia (CLL) treatment has changed dramatically with the introduction of covalent Bruton's tyrosine kinase inhibitors (BTKi) and venetoclax, but biomarkers that predict response or early progression for these treatments are lacking. In our preliminary studies, we observed that some malignant CLL B cells in the blood express PD-1, a critical immune checkpoint that is normally expressed in T cells and regulates their function. However, the mechanism for the counterintuitive expression of PD-1 in CLL cells and its clinical and biological significance are unknown. Here, we characterized the subset of CLL cells that express PD-1, defined the factors that promote its expression, and showed that high proportion of circulating PD-1+ CLL cells while on BTKi therapy may be an early indicator of BTKi resistance.
Peripheral blood (PB) from 73 treatment-naïve CLL patients was collected and analyzed by flow cytometry. Median age was 65 years and 40% had unmutated IGHV. A subset of circulating CLL cells that expressed PD-1 was detected in all patients. The percentage of circulating PD-1+ CLL cells was variable and ranged from 10% to 85% (mean 43%). Importantly, nearly all the CLL cells that expressed the proliferation marker Ki-67 also expressed PD-1 (median &gt;96%). PD-1+ CLL cells also had higher expression of CD5 and lower expression of CXCR4 (MFI fold change 1.49 and 0.49, respectively, p&lt;0.0001), in line with recently divided status. RNA sequencing analysis on flow-sorted cells with a PD-1 MFI &gt;90th percentile (PD-1hi) and PD-1- cells revealed 6580 differentially expressed genes (FDR 0.05). These genes were associated with B-cell activation, proliferation, migration, signaling, apoptosis, and metabolism. Notably, pathways associated with cell activation, cell cycle, oxidative phosphorylation, and B-cell receptor (BCR), toll-like receptor (TLR), and PD-1 signaling were all upregulated in PD-1hi cells.
We hypothesized that PD-1 expression in CLL cells might be induced through BCR and TLR signaling. To test this, we stimulated CLL cells ex vivo either through the BCR using anti-IgM antibodies, or through TLR9 with CpG and quantified PD-1 expression over time. The percentage of PD-1+ cells significantly increased within 24 hours after stimulation with either agent. Notably, concomitant inhibition of BTK-dependent signaling by ibrutinib resulted in near-complete abrogation of the effect of anti-IgM, and a marked decrease in CPG's effect on PD-1 expression.
To confirm the effect of BTKi on PD-1 expression in vivo, we analyzed the PB of 40 patients receiving either frontline or later line ibrutinib or acalabrutinib therapy for ≥1 month (median age: 65 years, 55% unmutated IGHV). In patients adherent to therapy, PD-1 was expressed in &lt;10% of remaining circulating CLL cells. The percentage of circulating PD-1+ cells decreased in all patients within 2 weeks after treatment initiation and remained low while on therapy. Importantly, 13-90% of circulating CLL cells expressed PD-1 in all 18 patients who were found to have clinical progression from BTKi therapy within 3 months of sample acquisition (median age: 66 years, 44% unmutated IGHV). Circulating PD-1+ cells that re-emerged at the time of BTKi progression overexpressed genes associated with cell activation, cell cycle, oxidative phosphorylation, cytokine response, and PD-1, BCR, and TLR signaling. Additional clinical characteristics and biomarkers for all cohorts will be reported.
Overall, our data indicate that PD-1 identifies a subset of circulating CLL cells that are transcriptionally more active and have recently undergone proliferation. We showed that PD-1 expression is induced via BCR and TLR signaling through BTK, implying that PD-1 could be a marker of CLL cell activation. Moreover, increased circulating PD-1+ CLL cells in patients on BTKi therapy could serve as an early marker of BTKi resistance.