Acquired mutations associated with ibrutinib resistance in Waldenström macroglobulinemia

Abstract

Ibrutinib produces high response rates and durable remissions in Waldenström macroglobulinemia (WM) that are impacted by MYD88 and CXCR4^WHIM mutations. Disease progression can develop on ibrutinib, although the molecular basis remains to be clarified. We sequenced sorted CD19⁺ lymphoplasmacytic cells from 6 WM patients who progressed after achieving major responses on ibrutinib using Sanger, TA cloning and sequencing, and highly sensitive and allele-specific polymerase chain reaction (AS-PCR) assays that we developed for Bruton tyrosine kinase (<i>BTK</i>) mutations. AS-PCR assays were used to screen patients with and without progressive disease on ibrutinib, and ibrutinib-naïve disease. Targeted next-generation sequencing was used to validate AS-PCR findings, assess for other <i>BTK</i> mutations, and other targets in B-cell receptor and MYD88 signaling. Among the 6 progressing patients, 3 had <i>BTK</i>^{<i>Cys481</i>} variants that included <i>BTK</i>^{<i>Cys481Ser(c.1635G>C</i> and <i>c.1634T>A)</i>} and <i>BTK</i>^{<i>Cys481Arg(c.1634T>C)</i>} Two of these patients had multiple <i>BTK</i> mutations. Screening of 38 additional patients on ibrutinib without clinical progression identified <i>BTK</i>^{<i>Cys481</i>} mutations in 2 (5.1%) individuals, both of whom subsequently progressed. <i>BTK</i>^{<i>Cys481</i>} mutations were not detected in baseline samples or in 100 ibrutinib-naive WM patients. Using mutated <i>MYD88</i> as a tumor marker, <i>BTK</i>^{<i>Cys481</i>} mutations were subclonal, with a highly variable clonal distribution. Targeted deep-sequencing confirmed AS-PCR findings, and identified an additional <i>BTK</i>^{<i>Cys481Tyr(c.1634G>A)</i>} mutation in the 2 patients with multiple other <i>BTK</i>^{<i>Cys481</i>} mutations, as well as <i>CARD11</i>^{<i>Leu878Phe(c.2632C>T)</i>} and <i>PLCγ2</i>^{<i>Tyr495His(c.1483T>C)</i>} mutations. Four of the 5 patients with <i>BTK</i>^<i>C481</i> variants were CXCR4 mutated. <i>BTK</i>^{<i>Cys481</i>} mutations are common in WM patients with clinical progression on ibrutinib, and are associated with mutated <i>CXCR4</i>.