Ibrutinib Resistance Mutations Identified in CLL Patients


Key Points

  • The C481S mutation in BTK confers relative resistance to ibrutinib by preventing irreversible binding.
  • R665W and L845F mutations in PLCγ2 are potential gain-of-function mutations that may lead to autonomous B-cell-receptor activity.

Ibrutinib is an irreversible inhibitor of Bruton’s tyrosine kinase that was recently approved for treatment of chronic lymphocytic leukemia (CLL) patients who have received at least one prior therapy. A small proportion of CLL patients have been observed to relapse during ibrutinib treatment, but mechanisms of resistance to irreversible kinase inhibitors and resistance associated with BTK inhibition have not been elucidated. As reported in The New England Journal of Medicine, Woyach et al have identified mutations conferring resistance to ibrutinib in CLL.

The study involved whole-exome sequencing of samples at baseline and time of relapse in six CLL patients with acquired resistance to ibrutinib therapy, manifested as progressive CLL, and functional analysis of identified mutations.

Identified Mutations

In five patients, sequencing of the relapse sample revealed a cysteine-to-serine mutation in BTK at position 481 (C481S). In the remaining patient, the relapse sample had an arginine-to-tryptophan mutation in PLCγ2 at position 665 (R665W). In one patient with a low-frequency C481S mutation, three PLCγ2 mutations were found, consisting of a R665W mutation, a leucine-to-phenylalanine mutation at position 845 (L845F), and a serine-to-tyrosine mutation at position 707. At baseline, no patient had evidence of mutations in BTK or PLCγ2 on whole-exome sequencing, and Ion Torrent sequencing in three patients identified mutations in no more than 0.2% of reads. At relapse, none of the patients had mutations in other kinases containing a residue homologous to BTK C481 that are irreversibly inhibited by ibrutinib (ITK, BMX, TEC, EGFR, JAK3, HER2, HER4, and BLK), and no other recurrent mutation was identified in any of the patients at relapse.

Functional Analysis

Functional analysis showed that the C481S mutation conferred relative resistance to ibrutinib by preventing irreversible binding, confirmation that it is a functionally relevant resistance mutation. The R665W and L845F mutations in PLCγ2 were found to be potential gain-of-function mutations in the presence of B-cell receptor stimulation that could lead to autonomous B-cell receptor activity.

No Mutations in Prolonged Lymphocytosis

Ion Torrent sequencing for the identified resistance mutations was also performed in samples from nine CLL patients with prolonged lymphocytosis on ibrutinib to determine whether presence of the mutations might portend relapse in this setting. No patient had evidence of any mutation in BTK or PLCγ2, suggesting that persistent lymphocytosis is not associated with the identified resistance mutations.

The investigators concluded, “Resistance to the irreversible [Bruton’s tyrosine kinase] inhibitor ibrutinib often involves mutation of a cysteine residue where ibrutinib binding occurs. This finding, combined with two additional mutations in PLCγ2 that are immediately downstream of [Bruton’s tyrosine kinase], underscores the importance of the B-cell–receptor pathway in the mechanism of action of ibrutinib in CLL.”

John C. Byrd, MD, of Ohio State University, is the corresponding author for The New England Journal of Medicine article.

The study was funded by the National Cancer Institute, Four Winds Foundation, D. Warren Brown Foundation, Mr. and Mrs. Michael Thomas, Mr. and Mrs. Al Lipkin, Harry T. Mangurian Jr. Foundation, Leukemia and Lymphoma Society, Conquer Cancer Foundation, an American Society of Hematology Scholar Award, Else Kroner-Fresenius-Stiftung, Helmholtz Virtual Institute, German Research Foundation, and Pharmacyclics. For full disclosures of the study authors, visit

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