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Multiplex digital PCR enables sensitive detection of resistance to BTK inhibitors
Manon Garcia1,2, Carolyne Croizier1,2, Grégory Lazarian3,4
1Service de Thérapie Cellulaire et d'Hématologie Clinique, CHU Estaing, Clermont-Ferrand, France.
Abstract:
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.
Insights
Multiplexing digital PCR (mdPCR) offers a more sensitive and rapid method for detecting resistance mutations in patients with B-cell lymphoproliferative disorders treated with BTK inhibitors. This approach aids in guiding therapeutic decisions at relapse.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- Bruton's tyrosine kinase (BTK) inhibitors have revolutionized chronic lymphocytic leukemia (CLL) and B-cell lymphoproliferative disorder treatment.
- Emergence of BTK or PLCG2 mutations causes resistance to BTK inhibitors, posing a clinical challenge.
- Accurate assessment of these mutations is crucial for guiding subsequent therapeutic strategies.
Purpose of the Study:
- To develop and validate a highly sensitive method for detecting BTK and PLCG2 resistance mutations.
- To overcome the limitations of sensitivity and turnaround time associated with targeted next-generation sequencing (NGS).
- To offer a more rapid and cost-effective alternative for mutation detection.
Main Methods:
- Development of multiplexing digital PCR (mdPCR) assays targeting common resistance mutations.
- Analysis of 28 patients with BTK inhibitor-resistant disease using both NGS and mdPCR.
- Comparison of sensitivity and mutation detection rates between NGS and mdPCR.
Main Results:
- mdPCR detected 68 mutations across 28 patients, compared to 49 mutations detected by NGS.
- mdPCR demonstrated superior sensitivity, especially for detecting low allelic frequency mutations.
- The developed mdPCR assays covered 96% of known ibrutinib-resistant cases.
Conclusions:
- mdPCR is a highly sensitive and potentially faster, more cost-effective method for detecting BTK and PLCG2 resistance mutations.
- This technique can accurately detect and quantify small mutated clones, improving patient management.
- mdPCR facilitates informed therapeutic choices for patients relapsing after BTK inhibitor therapy.
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