Molecular Composition and Kinetics of B Cells During Ibrutinib Treatment in Patients with Chronic Lymphocytic Leukemia

Abstract

Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of B cells due to constitutive B-cell receptor (BCR) signaling, leading to apoptosis resistance and increased proliferation. This study evaluates the effects of the Bruton Tyrosine Kinase (BTK) inhibitor ibrutinib on the molecular composition, clonality, and kinetics of B cells during treatment in CLL patients. Employing a multi-omics approach of up to 3.2 years of follow-up, we analyzed data from 24 CLL patients, specifically focusing on nine patients treated with ibrutinib monotherapy. In this study, clonal stability was observed within the ibrutinib-treated group following an effective initial clinical response, where clonotype frequencies of residual CLL cells remained high and stable, ranging from 74.9% at 1.5 years to 87.7% at approximately 3 years. In contrast, patients treated with the B-cell lymphoma 2 (BCL2) inhibitor venetoclax exhibited substantial reductions in clonal frequencies, approaching molecular eradication. Deep whole-exome sequencing revealed minimal genomic progression in the ibrutinib group, maintaining somatic drivers and variant allele frequencies (VAF) above 0.2 throughout treatment. At the single-cell level, the NF-κB pathway inhibition and apoptotic signals were detected or even augmented during treatment in ibrutinib-treated patients. These findings may corroborate the role of ibrutinib in stabilizing the genomic landscape of CLL cells, preventing significant genomic evolution despite maintaining a high clonal burden within the residual B-cell compartment.

Keywords: chronic lymphocytic leukemia; clonal stability; ibrutinib; lymphocytosis; molecular profiling; treatment efficacy.

Figure 1

Clonal kinetics following administration of ibrutinib (Ibr) or rituximab–venetoclax (R–v). Total lymphocyte counts before treatment in ibrutinib-treated versus R–v (p_ranksum = 0.21), with significant (signed-rank) median reductions in lymphocyte counts from 20.90 × 10⁹/L (n = 9) to 2.98 × 10⁹/L (n = 9) for ibrutinib and from 43.99 × 10⁹/L (n = 14) to 0.95 × 10⁹/L (n = 12) for rituximab–venetoclax (R–v) at follow-up of 0.5 to 1.5 years (A). The monoclonal burden was 75% at first follow-up in the ibrutinib group (n = 9) compared with 1.99% in the R–v group (n = 12), with residual disease undetectable in 4 of 12 patients in the R–v group at 0.5 to 1.5 years (B). Estimated monoclonal CLL cell counts from flow cytometry: 16.2 × 10⁹/L (n = 9) for ibrutinib and 28.2 × 10⁹/L (n = 15) for R–v before treatment, decreasing to 0.6 × 10⁹/L for ibrutinib (n = 9) and near the detection limit for R–v (n = 11) during follow-up of 0.5 to 1.5 years (C). Abbreviations *: p < 0.05, ***: p < 0.005, ns: not significant. Dotted lines denote separate treatment groups or follow-up intervals.

Figure 2

Genomic stability following ibrutinib treatment. Correlation between somatic variants in CD19+ cells prior to treatment and at first follow-up ((A), ρ_Pearson = 0.86, slope 0.98, p_t-statistics < 10⁻⁴, mean follow-up 11 months, n = 7), between first and second follow-up ((B), ρ = 0.95, slope 0.99, p < 10⁻⁴, 20 months, n = 7), and between second and third follow-up ((C), ρ = 0.94, slope 0.98, p < 10⁻⁴, 34 months, n = 5). Cytogenetic markers del(13q14) and trisomy 12 following rituximab–venetoclax (R–v) treatment and copy-number alterations (CNAs) for ibrutinib-treated patients (D). NOTCH1 frameshift mutation c.7541–7542del (p.P2514Rfs*4, COSV53024776, GRCh38, 9:136496197 CAG>C) was detected with a variant allele frequency of 0.21 in R–v patient 923 at first follow-up (marked *). The same mutation was detected with a variant allele frequency decreasing to 0.03 in R–v patient 748 (*). Allelic imbalances and CNAs on chromosome 15q and 20q, including the BIRC3 mutation, were found for patient 620. The clonal burden of chromosome 15q deletion increased from less than 50% to more than 50% during treatment (E,F) and copy-neutral loss of heterozygosity at 20q during follow-up (solid lines separate chromosome 15q and 20q, dotted lines denote the theoretical ratios for disomy, 1.0, and deletion, 0.5). Allelic imbalance on chromosome 20 was evaluated at 30% burden ((G), a dotted line separates chromosomal p and q arm).

Figure 3

Transcriptional profile of ibrutinib treatment responses. Transcriptional landscape of single cells at early therapeutic follow-up (cyan: mean follow-up of 14 months) and prolonged administration (pale red: 36 months) (A–E). Significant expressional changes were identified in genes relating to the NF-κB inhibitor family, stress and apoptotic response, or mitochondrial oxidative phosphorylation (B). Increased expression or number of cells expressing NFKBIA and other NF-κB inhibitors after prolonged ibrutinib administration (C). Increased expression of PPP1R15, IER5, TP53INP1, and BCL2L11—genes involved in apoptotic regulation—at late ibrutinib response (D). Bulk RNA sequencing confirming upregulation trends in late follow-up samples for markers involved in B-cell signaling and survival (E). Abbreviations *: p < 0.05, ns: not significant. Dotted lines in (C) separate early and late response profiles. Dots in (E) show individual expression levels for the bulk RNA sequencing samples when expressed.