High Level of CD8+PD-1+ Cells in Patients with Chronic Myeloid Leukemia Who Experienced Loss of MMR after Imatinib Discontinuation

Abstract

Treatment-free remission (TFR) is achieved in approximately half of chronic myeloid leukemia (CML) patients treated with tyrosine kinase inhibitors. The mechanisms responsible for TFR maintenance remain elusive. This study aimed to identify immune markers responsible for the control of residual CML cells early in the TFR (at 3 months), which may be the key to achieving long-term TFR and relapse-free survival (RFS) after discontinuation of imatinib. Our study included 63 CML patients after imatinib discontinuation, in whom comprehensive analysis of changes in the immune system was performed by flow cytometry, and changes in the BCR::ABL1 transcript levels were assessed by RQ-PCR and ddPCR. We demonstrated a significant increase in the percentage of CD8⁺PD-1⁺ cells in patients losing TFR. The level of CD8⁺PD-1⁺ cells is inversely related to the duration of treatment and incidence of deep molecular response (DMR) before discontinuation. Analysis of the ROC curve showed that the percentage of CD8⁺PD-1⁺ cells may be a significant factor in early molecular recurrence. Interestingly, at 3 months of TFR, patients with the e13a2 transcript had a significantly higher proportion of the PD-1-expressing immune cells compared to patients with the e14a2. Our results suggest the important involvement of CD8⁺PD-1⁺ cells in the success of TFR and may help in identifying a group of patients who could successfully discontinue imatinib.

Keywords: chronic myeloid leukemia (CML); discontinuation of imatinib; immune biomarker; treatment-free remission (TFR).

Figure 1

Immunological characteristics of all patients enrolled in the study. Differences in the percentages of immune populations at the moment of stopping imatinib and at 3 months after discontinuation in analyzed patients (Wilcoxon signed-rank test). (A) DC subpopulations; mDC, median: 0.20 vs. 0.28%; pDC, median: 0.12 vs. 0.15%; mDC PD-1⁺, median: 35.81 vs. 32.23%. (B) NKT-like and NK cells; NKT-like cells, median: 14.45 vs. 12.80%; NKT-like PD-1⁺; median: 21.02 vs. 21.28%; CD56^brightCD16-PD-1⁺, median: 2.31 vs. 2.08%; CD56^dimCD16⁺PD-1⁺, median: 3.71 vs. 3.49%. (C) CD4⁺ T cells and regulatory T cells; Treg CD4⁺CD25⁺CD127^dimFOXP3⁺, median: 3.00 vs. 2.89%; CD4⁺PD-1⁺, median: 21.59 vs. 19.34%. Red color indicates statistical significance p < 0.05, blue color indicates tendency p < 0.1.

Figure 2

Changes in immune populations in patients with stable MMR or lost MMR at 3 months after withdrawal. Differences in the percentages of immune populations between patient groups were analyzed with the Mann–Whitney U test. (A) CD8⁺PD-1⁺ cells, median: 17.70 vs. 27.13%. (B) mDC PD-1⁺, median: 31.55 vs. 42.88%. (C) pDC PD-1⁺, median: 33.42 vs. 46.76%. Red color indicates statistical significance p < 0.05, blue color indicates tendency p < 0.1.

Figure 3

Comparison of ROC curves showing an analysis of potential immunological biomarkers by age, duration of DMR, length of TKI treatment before TFR, and percentages of CD8⁺PD-1⁺, mDC PD-1⁺, and pDC PD-1⁺. Among the clinical data evaluated, ROC analysis indicates CD8⁺PD-1⁺ percentage as the strongest factor in early molecular reoccurrence (lost MMR) (p < 0.01). The statistically significant marker of lost MMR is the duration of DMR before stopping treatment (p < 0.05) (ROC curves).

Figure 4

Correlation between percentage of CD8⁺PD-1⁺ in patients at 3 months after withdrawal, and duration of treatment and DMR before TFR trial. The percentage of CD8⁺PD-1⁺ is inversely related to the treatment of imatinib (R = −0.3227, 95% CI: −0.5618 to −0.03378) and DMR duration (R = −0.3118, 95% CI: −0.5535 to −0.02176) (Spearman r). Red color indicates statistical significance p < 0.05, green color indicates the correlation coefficient.

Figure 5

Analysis of changes in the immune system of patients depending on the transcript type of BCR::ABL1. Higher percentage of the population expressing PD-1 in patients with e13a2 compared to patients with e14a2 (Mann–Whitney U test). Differences between patients with e13a2 vs. patients with e14a2: CD56^dimCD16⁺PD-1⁺, median: 4.89 vs. 3.12%; CD19⁺PD-1⁺, median: 16.20 vs. 10.48%; CD8⁺PD-1⁺, median: 23.15 vs. 14.91%. Red color indicates statistical significance p < 0.05, blue color indicates tendency p < 0.1.