Abstract
Patients with chronic lymphocytic leukemia (CLL) that develop resistance to Bruton tyrosine kinase (BTK) inhibitors are typically positive for mutations in BTK or phospholipase c gamma 2 (PLCγ2). We developed a high sensitivity (HS) assay utilizing wild-type blocking polymerase chain reaction achieved via bridged and locked nucleic acids. 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 treated with the BTK inhibitor, ibrutinib. We also tested ibrutinib-naïve patients with CLL. HS testing achieved 100x greater sensitivity than Sanger. HS Sanger sequencing was capable of detecting < 1 mutant allele in background of 1000 wild-type alleles (1:1000). Similar sensitivity was achieved with HS NGS. No BTK or PLCγ2 mutations were detected in any of the 44 ibrutinib-naïve CLL patients. We demonstrate that without the HS testing 56% of positive samples would have been missed for BTK and 85% of PLCγ2 would have been missed. With the use of HS, we were able to detect multiple mutant clones in the same sample in 37.5% of patients; most would have been missed without HS testing. We also demonstrate that with HS sequencing, plasma cfDNA is more reliable than cellular DNA in detecting mutations. Our studies indicate that wild-type blocking and HS sequencing is necessary for proper and early detection of BTK or PLCγ2 mutations in monitoring patients treated with BTK inhibitors. Furthermore, cfDNA from plasma is very reliable sample-type for testing.
Highlights
Bruton tyrosine kinase (BTK) inhibitors like ibrutinib have demonstrated high clinical response rates and durable remissions in patients with chronic lymphocytic leukemia (CLL) including refractory patients to conventional therapy or patients with tumor protein p53 (TP53) mutations [1,2,3,4,5]
We used this high sensitivity assay in combination with Sanger sequencing and generation sequencing (NGS) and tested cellular DNA and cell-free DNA from patients with CLL treated with the BTK inhibitor, ibrutinib
Our studies indicate that wild-type blocking and high sensitivity (HS) sequencing is necessary for proper and early detection of BTK or phospholipase c γ 2 (PLCγ2) mutations in monitoring patients treated with BTK inhibitors
Summary
Bruton tyrosine kinase (BTK) inhibitors like ibrutinib have demonstrated high clinical response rates and durable remissions in patients with chronic lymphocytic leukemia (CLL) including refractory patients to conventional therapy or patients with tumor protein p53 (TP53) mutations [1,2,3,4,5]. Patients who develop resistance to ibrutinib therapy typically have mutations in either BTK or phospholipase c γ 2 (PLCγ2) [1, 6]. In clinical trials of CLL patients on BTK inhibitor (BTKi) therapy, whole exome sequencing with next-generation sequencing (NGS) has typically been used to detect specific mutations in BTK or PLCγ2 genes [1, 6]. Accurate, high-sensitivity assays that can be run in large volumes in a clinical setting are a necessity to further understand the relationship between the appearance of a mutation and the development of resistance to therapy and clinical progression
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