Prolonged lymphocytosis during ibrutinib therapy is associated with distinct molecular characteristics and does not indicate a suboptimal response to therapy

Abstract

The Bruton's tyrosine kinase (BTK) inhibitor ibrutinib has outstanding activity in patients with chronic lymphocytic leukemia. Most patients experience lymphocytosis, representing lymphocyte egress from nodal compartments. This resolves within 8 months in the majority of patients, but a subgroup has lymphocytosis lasting >12 months. Here we report a detailed characterization of patients with persistent lymphocytosis during ibrutinib therapy. Signaling evaluation showed that while BTK is inhibited, downstream mediators of B-cell receptor (BCR) signaling are activated in persistent lymphocytes. These cells cannot be stimulated through the BCR and do not show evidence of target gene activation. Flow cytometry for κ and λ expression, IGHV sequencing, Zap-70 methylation, and targeted gene sequencing in these patients are identical at baseline and later time points, suggesting that persistent lymphocytes do not represent clonal evolution. In vitro treatment with targeted kinase inhibitors shows that they are not addicted to a single survival pathway. Finally, progression-free survival is not inferior for patients with prolonged lymphocytosis vs those with traditional responses. Thus, prolonged lymphocytosis is common following ibrutinib treatment, likely represents the persistence of a quiescent clone, and does not predict a subgroup of patients likely to relapse early.

Figure 1

Protein and gene expression of BCR signaling components in persistent lymphocytes compared with baseline. Phosphorylated and total protein expression of various signaling molecules of the BCR pathway and gene expression of BTK and PLCγ2 were investigated in patients with persistent lymphocytosis. Serial samples were taken at baseline and at 6 or 9 months after initiation of therapy. Each immunoblot shown is representative of multiple patients evaluated, and statistics presented in the text show the mean change of all evaluated patients. Number of patients evaluated was determined by amount of sample available. (A) pBTK (Y223), as well as (C) pPLCγ2 (Y759) decrease in the majority of patients after ibrutinib therapy. (E) pERK (Thr202/Tyr204), (F) pMEK1/2 (Ser217/221), and (G) pAKT (Ser473) increase in the majority of patients with prolonged lymphocytosis during ibrutinib therapy. Gene expression of (B) BTK and (D) PLCγ2 does not change over time in the majority of patients.

Figure 2

Nuclear and cytoplasmic localization of BTK, ERK, and AKT in persistent lymphocytes following ibrutinib therapy and ability of persistent lymphocytes to stimulate. Nuclear and cytoplasmic localization of BCR signaling pathway proteins was evaluated using nuclear and cytoplasmic lysates from patients at baseline and after 6 months of ibrutinib therapy. In these representative immunoblots, there is no evidence of a shift toward nuclear localization of either phosphorylated or total (A) BTK, (B) ERK, or (C) AKT. (D) Patient samples at baseline and after 9 months of therapy were used to evaluate whether persistent lymphocytes can be stimulated through the BCR (15-minute exposure to plate-immobilized IgM; BD Pharmigen), TLR9 (3.2 μM CpG; Eurofins MWG Operon), or distal to BTK (1 μg/mL CD40L; PeproTech). (D) In this representative immunoblot, mild stimulation with CpG is seen at baseline along with robust stimulation following CD40L. After 9 months of ibrutinib therapy, CD40L alone is able to stimulate CLL cells.

Figure 3

Real-time PCR of BCR pathway–associated genes in persistent lymphocytes. Patient samples at baseline and 12 months of ibrutinib therapy were evaluated for gene expression of proximal signaling molecules and target genes of ERK, AKT, and NFκB. There is no consistent change in gene expression of either (A) proximal BCR genes or (B) BCR target genes.

Figure 4

Persistent lymphocytes are not addicted to a single signaling pathway. To determine whether persistent lymphocytes were dependent on a single signaling pathway for survival, cells at baseline and 9 months from patients with persistent lymphocytosis were treated with various inhibitors of the BCR signaling pathway. AEB-071 was dosed at 1 μM, Dasatinib at 5 μM, MK2206 at 1 μM, IPI-145 at 1 μM, CI-1040 at 1 μM, and Dinaciclib at 1 μM. Dinaciclib was washed out at 2 hours. Annexin V/propidium iodide staining and flow cytometry were used to identify viable cells (Annexin negative/PI negative) after 72 hours. No single drug produced more effective cytotoxicity at a late time point compared with baseline. Dinaciclib, a cyclin-dependent kinase inhibitor, induced robust cytotoxicity both at baseline and at 9 months of ibrutinib therapy.

Figure 5

PFS of patients with persistent lymphocytosis is not inferior to those achieving complete or PR by 12 months. This is a landmark analysis at day 365 comparing patients with PR-L at 12 months vs those with CR/PR at 12 months. There is no statistical difference between these groups, although there is a trend toward improved survival in patients with PR-L.