Using high-sensitivity sequencing for the detection of mutations in BTK and PLCγ2 genes in cellular and cell-free DNA and correlation with progression in patients treated with BTK inhibitors
Metrics: PDF 1473 views | HTML 1765 views | ?
Adam Albitar1, Wanlong Ma1, Ivan DeDios1, Jeffrey Estella1, Inhye Ahn2, Mohammed Farooqui3, Adrian Wiestner3, Maher Albitar1
1NeoGenomics Laboratories, Irvine, CA, USA
2Medical Oncology Service, National Cancer Institute, Bethesda, MD, USA
3Hematology Branch, National Heart, Lung, and Blood Institute, Bethesda, MD, USA
Maher Albitar, email: firstname.lastname@example.org
Keywords: ibrutinib, CLL, resistance, PLCγ2, BTK
Received: August 23, 2016 Accepted: January 27, 2017 Published: February 13, 2017
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.
All site content, except where otherwise noted, is licensed under a Creative Commons Attribution 3.0 License.