Multiplex digital PCR enables sensitive detection of resistance to BTK inhibitors.

The advent of BTK inhibitors has been transformative in the management of patients with chronic lymphocytic leukemia or other B-cell lymphoproliferative disorders. However, emergence of BTK or PLCG2 mutations lead to resistance to these compounds and are now a growing concern in clinical practice. Assessing BTK mutations is now becoming a priority to guide the therapeutic decision at further relapse. To this end, targeted next-generation sequencing (NGS) is a valid tool, but lack of sensitivity and the required time for delivering results remain major challenges. Digital PCR could be more sensitive but is also limited by the number of mutations that can be screened. We here overcame these challenges by multiplexing digital PCR (mdPCR) in three assays that can cover 96% of ibrutinib-resistant cases. We investigated a cohort of 28 patients progressing on ibrutinib and for whom NGS revealed BTK mutations (C481S, C481F and C481R) and/or PLCG2 R665W mutation. Overall, 49 mutations were detected by NGS and 68 by mdPCR. We found mdPCR to bemore sensitive than NGS, particularly at low allelic frequencies, making it more suitable for the detection and quantification of small mutated clones. Thus, mdPCR offers high sensitivity, is expected to be more rapid and cost-effective than NGS in detecting resistance mutations to improve the therapeutic choice at relapse after exposure to BTK inhibitors.