BACKGROUND
Ibrutinib is an irreversible inhibitor of Bruton’s tyrosine kinase (BTK) and is effective in chronic lymphocytic leukemia (CLL). Resistance to irreversible kinase inhibitors and resistance associated with BTK inhibition have not been characterized. Although only a small proportion of patients have had a relapse during ibrutinib therapy, an understanding of resistance mechanisms is important. We evaluated patients with relapsed disease to identify mutations that may mediate ibrutinib resistance.
METHODS
We performed whole-exome sequencing at baseline and the time of relapse on samples from six patients with acquired resistance to ibrutinib therapy. We then performed functional analysis of identified mutations. In addition, we performed Ion Torrent sequencing for identified resistance mutations on samples from nine patients with prolonged lymphocytosis.
RESULTS
We identified a cysteine-to-serine mutation in BTK at the binding site of ibrutinib in five patients and identified three distinct mutations in PLCγ2 in two patients. Functional analysis showed that the C481S mutation of BTK results in a protein that is only reversibly inhibited by ibrutinib. The R665W and L845F mutations in PLCγ2 are both potentially gain-of-function mutations that lead to autonomous B-cell–receptor activity. These mutations were not found in any of the patients with prolonged lymphocytosis who were taking ibrutinib.
CONCLUSIONS
Resistance to the irreversible BTK 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 BTK, underscores the importance of the B-cell–receptor pathway in the mechanism of action of ibrutinib in CLL. (Funded by the National Cancer Institute and others.)
B-cell receptor (BCR) signaling is aberrantly activated in chronic lymphocytic leukemia (CLL). Bruton tyrosine kinase (BTK) is essential to BCR signaling and in
IntroductionChronic lymphocytic leukemia (CLL) is the most prevalent adult leukemia with an immunophenotype expressing the T-cell marker CD5 together with CD19, CD20, CD23, and dim-surface immunoglobulin. 1 Although immunophenotypically similar to the normal B1 lymphocytes, CLL cells have a distinct mRNA gene expression profile that most approximates a postgerminal memory B cell. 2 For many years CLL has been viewed as a nonproliferating leukemia based on the nonproliferating blood compartment; however, as with normal B cells, it has come to be recognized that CLL cell proliferation probably occurs in sites where microenvironmental stimulation occurs such as the lymph nodes and spleen. In such sites, proliferation centers are observed with a high proportion of dividing CLL cells expressing survivin that are often surrounded by either T cells or accessory stromal cells capable of providing cytokine costimulation. 3,4 Studies administering heavy water allow accurate measurement of all body compartments of CLL and assess the birth rate of CLL tumor cells in vivo. 5 These studies have demonstrated a broad range of proliferation of CLL cells that varies based on disease state and also immunoglobulin heavy chain variable (IVGH) mutational status. 5,6 In particular, a higher tumor birth rate is noted in CLL patients with IVGH unmutated disease and ZAP-70 expression. Multiple studies have documented evidence of enhanced B-cell receptor (BCR) signaling in patients with IVGH unmutated disease or those with increased ZAP-70 expression. [7][8][9] Thus, accessory cytokines, cell-cell contact in the microenvironment, and also BCR-signaling coupled to B-cell proliferation appear sentinel to CLL progression and pathogenesis.While understanding of CLL biology has improved dramatically, until very recently integration of these findings to treatment interventions has been lacking. Specifically, treatment has included alkylators, nucleoside analogs, and their combination where small advances in improved response and progression-free survival (PFS) have been noted. 10,11 However, these therapies have had very little impact on overall survival of CLL. The addition of the chimeric CD20 antibody, rituximab, perhaps represents the most significant advance in CLL therapy. Rituximab single agent activity 12 and phase 2 studies combining it with fludarabine (FR) or fludarabine and cyclophosphamide (FCR) have demonstrated improved overall survival (OS) over historical controls. 13,14 A randomized trial of FCR versus fludarabine or cyclophosphamide alone 15 demonstrated significant improvement in response; PFS and OS. While the presumptive mechanism of rituximab in CLL has been assumed to be immunologic (reviewed in Jaglowski and Byrd 16 ), a recent study demonstrated a direct effect on BCR-signaling in both normal and malignant B cells via perturbation of membrane rafts by CD20 anti...
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