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

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.

Functional characterization of phospholipase C-γ2 mutant protein causing both somatic ibrutinib resistance and a germline monogenic autoinflammatory disorder

“…Very recently, two CLL patients progressing on ibrutinib have been described carrying a 6-nucleotide deletion in exon 20 of PLCG2 (c.2120-2125del), leading to the deletion of both S707 and A708 [ 8 , 9 ]. In addition, two patients were found with a A708P point mutation and one with a S707F|A708P double mutation [ 7 ].…”

“…8 and 9 , if the mutations occur in a single gene, they clearly have the potential to synergize in regard to enhancing PLCγ 2 activity in intact cells. This view is strongly supported by the experiment investigating the combinatorial functional effects of deletions in positions S707 and A708, both encoded by exon 20, although thus far only a deletion of both residues has been reported for ibrutinib-resistant patients [ 8 , 9 ].…”

“…Deletions of exons 19 or 20–22 cause cold urticaria and PLCγ 2 –associated antibody deficiency and immune dysregulation, PLAID [ 1 , 2 ], while a point mutation (S707Y) is the basis of autoinflammation and PLCγ 2 -associated antibody deficiency and immune dysregulation, APLAID [ 3 ]. In addition, several point mutations as well as small deletions have been found to mediate resistance of chronic lymphocytic leukemia (CLL) cells to the Btk inhibitor ibrutinib [ 4 – 9 ]. PLCG2 point mutations have also been identified in association with childhood-onset steroid-sensitive nephrotic syndrome [ 10 ], Burkitt lymphoma [ 11 ], and inflammatory bowel disease [ 12 ].…”

Noncatalytic Bruton's tyrosine kinase activates PLCγ2 variants mediating ibrutinib resistance in human chronic lymphocytic leukemia cells

“…Thus, wholeexome sequencing of CLL cells causing late relapses revealed (i) C481S and a small number of other mutations in the same and in other positions in BTK and (ii) a higher variety of mutations in PLCG2. To date, some 25 point mutations of one or two adjacent residues are known in PLCG2, some of which cluster in specific regions of the encoded PLC 2 protein, such as one of the two SH2 domains and the C2 domain (34)(35)(36). Collectively, the BTK and PLCG2 mutations are associated with about 85 % of CLL cases with acquired resistance to BTK inhibitors (37).…”

Potent anti-leukemic activity of a specific cyclin-dependent kinase 9 inhibitor in mouse models of chronic lymphocytic leukemia

“…Patients treated with these novel agents still progress after treatment or may not tolerate them. Treatment failure with the novel agents may occur due to acquired resistance, which in the case of the BTK inhibitor ibrutinib was shown to be mediated by mutations in the ibrutinib-binding site of BTK [ 9 ]. Furthermore, patients also progress under treatment with the BCL-2 inhibitor venetoclax.…”