The mutational landscape of resistance to BTK and BCL2 inhibitors in CLL: a real-world multicentric study

Resistance mechanism to BTK and BCL2 inhibitors in CLL remains poorly characterized, particularly regarding clonal dynamics and compartment-specific evolution. We analyzed 114 samples from 101 patients discontinuing therapy due to progression, toxicity, or Richter transformation using a 33-gene NGS panel. Notably, 34% of relapsed/refractory patients lacked BTK/PLCG2 mutations, a proportion that increased to 71% when considering only clonal alterations (VAF > 25%). While the BTK C481 variant predominated under covalent BTKi (94.4%), non-covalent resistance mutations (T474, L528) were also detected, suggesting potential cross-resistance. Distinct genomic profiles emerged based on treatment duration: late progressors (>24 months) exhibited a significantly higher frequency of BTK / PLCG2 mutations compared to early progressors (82% vs 11%; p < 0.001), suggesting heterogeneous resistance mechanisms. In the BCL2i cohort, BCL2 mutations were detected in 38% of cases. Compartment-specific analysis revealed that lymph nodes consistently harbored higher VAFs than paired bone marrow or peripheral blood. Furthermore, the persistence of BTK mutations after subsequent BCL2i therapy correlated with high pre-treatment VAFs. This study provides a descriptive map of the highly heterogeneous mutational landscape at targeted therapy discontinuation. The data demonstrate that this landscape is defined by distinct subclonal architectures, significant anatomical compartment variations in VAF, and divergent genomic profiles based on the treatment duration.