Targeted sequencing in ibrutinib treated CLL. BTK/PLCG2 mutations in CLL

Patients with chronic lymphocytic leukemia (CLL) who relapse and progress on ibrutinib constitute an unmet clinical need. BTK/PLCG2 mutations have been associated to resistance in patients with CLL relapsing on ibrutinib, though their frequency, clonality and overall relevance to progression are not fully understood. In this multicenter retrospective observational study, we analyzed 98 patients with CLL treated with ibrutinib (49 relapsing after an initial response and 49 still responding after ≥1 year of continuous treatment) using a targeted next-generation sequencing panel (1% sensitivity) comprising 13 genes relevant to CLL pathogenesis, including BTK and PLCG2. BTK hotspot mutations were validated by digital droplet PCR (ddPCR) (0.1% sensitivity). By integrating both NGS and ddPCR analysis, 32 of 49 relapsing cases (65%) carried at least 1 hotspot BTK mutation and/or PLCG2 mutation(s); in 6 of these, mutations were only detected by ddPCR [variant allele frequency (VAF) 0.1-1.2%]. BTK/PLCG2 mutations were identified in only 6/49 responding patients (12%; 5/6 VAF <10%), of whom 2 progressed at later time points. Among the relapsing patients, the BTK-mutated (BTKmut) group was enriched for EGR2 mutations, while BTK-wildtype (BTKwt) cases were enriched for BIRC3/NFKBIE mutations. Finally, no difference in TP53 mutation burden was observed between BTKmut versus BTKwt relapsing cases, and ibrutinib treatment did not appear to favor selection of TP53-aberrant clones. In conclusion, we show that BTK/PLCG2 mutations were absent in a substantial fraction (35%) of patients in a real-world cohort failing ibrutinib, alluding to additional mechanisms that may be cooperating in determining progression on ibrutinib.