High Sensitivity Testing Shows Multiclonal Mutations in Patients with CLL Treated with BTK Inhibitor and Lack of Mutations in Ibrutinib-Naive Patients

Background: Patients with chronic lymphocytic leukemia (CLL) that develop resistance to Bruton’s tyrosine kinase (BTK) inhibitors are typically positive for mutations in BTK or phospholipase c gamma 2 (PLCγ2). Mutations in BTK at the C481S hotspot alter the active site of the mutant BTK to the effect that Ibrutinib is reversibly bound. PLCγ2 is downstream of BTK in the B-cell signaling pathway; mutations in PLCγ2 at either of the R665W, L845F, or S707Y hotspots result in a constitutively activated PLCγ2. In order to better understand the development of these resistance mechanisms in patients with CLL, we developed a high sensitivity (HS) assay utilizing branched and locked nucleic acids (BNA and LNA, respectively). We used this high sensitivity assay in combination with Sanger sequencing and next generation sequencing (NGS) and tested cellular DNA and cell free DNA (cfDNA) from patients with CLL.