Introduction: Covalent Bruton tyrosine kinase inhibitors (cBTKi) have revolutionized the treatment of Chronic Lymphocytic Leukemia (CLL). They target Bruton tyrosine kinase (BTK), which mediates BCR signaling, and triggers pathways involved in cell survival, proliferation and migration. BTK inhibition in neoplastic lymphocytes results in their demarginalization from lymph nodes into peripheral blood, leading to redistribution lymphocytosis, characterized by a transient increase in absolute lymphocyte count (ALC) after therapy initiation. However, cases of absent or attenuated lymphocytosis after treatment initiation have been reported in literature, particularly in patients with trisomy 12 (+12), the clinical impact of this phenomenon remains unclear. Trisomy 12 cells show increased expression of adhesion integrins LFA-1, Mac-1 and VLA4-4, promoting retention of +12 CLL cells within tissues. The aim of this study was to describe CLL patients treated with cBTKi who did not show any lymphocytosis after treatment initiation and its impact on Progression-free survival (PFS), Time to next treatment (TTNT) and Overall survival (OS).

Material and Methods: We retrospectively analyzed a cohort of 346 treatment-naïve CLL patients from 16 Italian centers, all treated with target-dose ibrutinib, acalabrutinib, or zanubrutinib. We assessed the median ALC at baseline and on days +15, +30, +60, +90, +120, +180, +270 and +360. Then we conducted a descriptive statistical analysis based on clinical and biological characteristics. Additionally, clinical outcomes as PFS, TTNT and OS were assessed in 284 patients with sufficient follow-up. The study was carried out according to the Helsinki Declaration, Good Clinical Practice, and the applicable national regulations and was approved by the local ethic committee.

Results: We identified 51/346 patients (15%) who did not develop lymphocytosis following cBTKi initiation. These patients demonstrated a persistent reduction in ALC from day 15 onward. No significant differences in lymphocyte kinetics or frequency of non-lymphocytosis were found between the three cBTKi. According to the cytogenetic profile, 45% had CLL with +12, 18% del(17p), 25% del(11q), 35% del(13q) and 12% normal karyotype. Only for 22/51 patients we had data about CD49d (VLA-4 integrin alfa subunit) expression before starting treatment: 16/22 (73%) patients CD49d+ and 6/22 (27%) CD49d-. Regarding immunoglobulin heavy chain variable region (IGHV) gene mutational status: 71% had unmutated IGHV and 29% had mutated IGHV. No statistical differences in lymphocyte kinetics were observed between mutated and unmutated IGHV. We noticed that the +12 patients (23 patients) had a smaller increase in blood lymphocytosis from the first to the sixth month. In +12 CLL patients, we did not study the specific role of CD49d+ due to its widespread expression (89%) in this group of patients. We further analyzed the role of CD49d in patients without +12 (28 patients) to detect the independent role of CD49d. All the 37 CD49d+ CLL patients showed a more pronounced reduction in lymphocyte counts during the first 3 months of therapy. Clinical outcomes were assessed in 284 patients, comparing those with (n=244) and without (n=40) cBTKi-induced lymphocytosis. No significant differences were observed in PFS, TTNT and OS, regardless of presence or absence of lymphocytosis (PFS p=0.9651; TTNT p=0.5346; OS p=0.6848).

Conclusion: The absence of lymphocytosis during cBTKi therapy in CLL is associated with specific biological features, notably CD49d expression and trisomy 12. CD49d appears to be the key driver of the no-lymphocytosis phenotype, while +12 may have an indirect role due to its strong association with CD49d. Importantly, lack of lymphocytosis after cBTKi treatment does not correlate with inferior outcomes (PFS, TTNT, OS) comparing patients with or without lymphocytosis, independently of CD49d expression. Future studies with larger cohorts and extended follow-up are warranted to clarify the prognostic impact of CD49d on long-term outcomes in this setting.

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2025
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