Background: The drug-resistant mechanisms of the first-generation Bruton’s tyrosine kinase (BTK) inhibitor, ibrutinib, has been extensively explored in Chronic Lymphocytic Leukemia (CLL) patients. However, the resistant mechanisms of the second-generation BTK inhibitor such as zanubrutinib remained largely unexplored.
Aims: To assess the clonal characteristics in CLL patients with zanubrutinib resistance.
Methods: We retrospectively identified 8 CLL patients with zanubrutinib resistance. Deep targeted-gene NGS and ddPCR detecting BTK and PLCG2 mutation were assessed in serial samples. scRNA-seq of matching peripheral blood (PB) and lymph node (LN) were performed in 3 zanubrutinib-resistant patients showing progressive lymphadenopathy.
Results: Median time from zanubrutinib initiation to progression was 30.5 months, including 2 Richter Transformation (RT) patients after 13 months’ treatment. NGS showed that BTK Cys481 mutation was detected in 5 of 8 patients (1 patients also harbored BTK Leu528Trp with low frequency) and PLCG2 mutation in one patient without BTK Cys481 mutation. For 3 patients with undetectable BTK mutation by NGS, ddPCR were performed and BTK Cys481Ser was identified in one patient, suggesting spatial heterogeneity. Clonal evolution model were depicted in patients available for longitudinal NGS. TP53, EGR2, NOTCH1 and SF3B1 were main driving clones during zanubrutinib resistance.
scRNA-seq analysis showed that RT showed significantly higher expression of MCL-1 while progressive CLL showed relatively higher expression of BCL-2 (p<0.0001), indicating the shift of BCL-2 family dependence among different disease characteristics.
Summary/Conclusion: NGS and ddPCR should be used comprehensively to evaluate the emergence of resistant clones. BTK Cys481 and Leu528 were two main BTK mutations leading to zanubrutinib resistance. The shift of BCL2 anti-apoptotic family gene expression indicates that higher MCL-1 expression of RT might lead to insensitivity to venetoclax treatment following zanubrutinib resistance.
Keywords: Drug resistance, Bruton’s tyrosine kinase inhibitor (BTKi), B cell chronic lymphocytic leukemia