Cellular and Molecular Effects of Suspending Ibrutinib Treatment in Patients with Chronic Lymphocytic Leukemia in Sustained Partial Remission
Authors: Palma Marzia , Andersson Maria Lisa , Mulder Tom Adriaan , Heimersson Kia , Berglöf Anna , Peña Perez Lucia , Del Peso Santos Teresa , Alvarado Valero Yesid , Zain-Luqman Rula , Månsson Robert , Rosenquist Richard , Smith Edvard C.I. , Österborg Anders , Lundin Jeanette 
Published: December 2024

Background: In an academic clinical trial, we are investigating an alternative strategy for ibrutinib dosing in patients with chronic lymphocytic leukemia (CLL) who have received at least 6 months (mo) of therapy and are in stable partial remission (PR). Briefly, treatment is suspended and patients followed off therapy until early signs of progressive disease (PD), at which ibrutinib is re-instituted. Such ‘ON-OFF’ ibrutinib cycles are repeated until resistance and need of alternative therapy. In the phase 1b part of the study, we showed that ibrutinib can safely be suspended (Lundin et al., 2021). A spin-off study runs in parallel with the trial to characterize the cellular and molecular changes induced by this alternative dosing of ibrutinib.

Methods. Peripheral blood (PB) samples were collected from 20 patients right before the first treatment interruption (i.e. start of OFF-phase) and at 2 weeks, 1 mo, 3 mo, 6 mo and 12 mo after treatment interruption and before re-start. Pre-treatment samples (i.e. at primary start of ibrutinib) were also available for 11/20 patients while samples taken before re-start of treatment (i.e. start of ON-phase) were available for 10/20 patients. Flow cytometry analysis of the different T- and NK-cells subsets was performed and plasma inflammation-related biomarkers were assessed by a proximity extension assay. Quantitative digital-droplet PCR is ongoing to detect the occurrence of mutations within BTK and PLCG2 on samples taken before treatment stop and at re-start.

Results: After treatment interruption, CLL cells remained stable until time for re-start when they increased (p&lt;0.0005). The same dynamics were observed for CD8+ and CD4+ cells (p=0.03 and p=0.01, respectively) and for Th1 and Th2 cells (p=0.001, respectively). Naïve CD4+ T cells (CCR7+CD45RA+) remained stable throughout the OFF phase until re-start. CD4+ central memory (CM) T cells (CCR7+CD45RA-) started increasing at mo 6 (p=0.03) and were still higher at re-start (p=0.007). Effector memory (EM) CD4+ T cells (CCR7-CD45RA-) remained stable until re-start (p=0.03). CD4+ T effector memory re-expressing CD45RA (TEMRA; CCR7-CD45RA+) decreased at mo 3 after stop (p=0.01) until mo 12 (p=0.004) but increased again at re-start. Remarkably, regulatory T cells (Tregs) started increasing earlier compared to the other cell populations, from 3 mo (p= 0.02) and progressively more significantly until re-start (p= 0.001). NK cells decreased initially (p= 0.04 at wk 2) and later increased from 12 mo to restart (p=0.003).

The expression of all exhaustion markers on T cells increased at re-start: CD4+PD-1+ (p=0.005), CD8+PD-1+ (p=0.002), CTLA-4+CD4+ (p=0.005), CTLA-4+CD8+ (p=0.02), TIGIT+CD4+ (p=0.002), TIGIT+CD8+ (p=0.002).

When analyzing 92 inflammatory plasma protein biomarkers, we observed the following patterns: 1) some markers which had decreased during treatment, increased again in the OFF-phase, such as CCL3 and CCL4; 2) some markers which had increased during ibrutinib treatment, significantly decreased in the OFF-phase, such as AREG, TNFSF13, EDAR, CST5; 3) some markers which had decreased during treatment, stayed at low levels in the OFF-phase, such as VIM, NT3, NF2, IRAK4, BACH1 and FGF2. The markers in the first group were the majority. Changes were considered statistically significant when the adjusted p-value was &lt;0.05.

Conclusions. After interruption of ibrutinib, the immune cell phenotype achieved after long-term treatment seems to remain substantially stable until PD occurs, at which the phenotype reverts to the pre-treatment one.Similarly, the majority of the changes in plasma protein biomarkers acquired with treatment gradually revert to the pre-treatment status. However, a minority of them remained unchanged despite PD, for reasons to be investigated.