Abstract 3150: PLCG2 C2-domain mutations co-occur with BTK and PLCG2 resistance mutations in chronic lymphocytic leukemia undergoing treatment with the BTK inhibitor ibrutinib
Authors: Jones Dan , Woyach Jennifer A. , Zhao Weiqiang , Caruthers Sean , Tu Huolin , Coleman Joshua , Byrd John C. , Johnson Amy J. , Lozanski Gerard 
Published: July 2017

Abstract
Background: The Bruton agammaglobulinemia tyrosine kinase (BTK) activates B-cell receptor signaling through activation of phospholipase C gamma 2 (PLCG2). Clinical resistance to the Bruton tyrosine kinase (BTK) inhibitor ibrutinib in chronic lymphocytic leukemia (CLL) is highly associated with emergence of the BTK C481 mutations that prevent ibrutinib covalent binding. PLCG2 mutations also occur in these ibrutinib-resistant samples but the spectrum of mutations and their occurrence with BTK changes have not been fully delineated.
Materials and Methods: All peripheral blood samples with adequate depth of sequencing coverage were included from CLL patients receiving ibrutinib (with or without other therapies) that were submitted from Ohio State University (OSU) to the OSU James Polaris Molecular Laboratory. Genomic DNA was extracted from negatively selected B cells and deep sequencing of the entire coding regions of BTK and PLCG2 performed using a custom Ion Torrent Ampliseq panel. A mean depth of greater than 1000X was obtained with hotspot mutations validated down to 1% variant allele fraction (VAF) in the B cell preparations using orthogonal mutation-specific detection methods.
Results: Among 1063 CLL samples from 380 patients who received ibrutinib, BTK C481 resistance mutations were identified in 79 (20.8%) patients including 20 patients that also had co-occurring PLCG2 mutations. 11 patients (2.9%) had PLCG2 mutations without accompanying BTK C481 alterations for a cumulative incidence of PLCG2 mutations in 8.2% of ibrutinib-treated patients. These included previously described mutations in the SH2 and SH3 domain of PLCG2 (R665W, S707F, A708P and L845F) but also previously uncharacterized mutations in the PLCG2 C2 domain that were seen in 12 patients (3.2%). C2 domain mutations, always seen in association with another PLCG2 and/or BTK resistance mutation, affected codons 1140-1144 that include the highly conserved aspartic acid residues that bind calcium and mediate membrane localization in other C2-domain containing proteins. In sequential samples, PLCG2 C2-domain mutations tracked at similar levels to the co-occurring BTK and PLCG2 resistance mutations indicating their presence in the same population of CLL cells.
Conclusions: Mutations in three different PLCG2 structural domains commonly co-occur with BTK C481 mutations. The identification of PLCG2 mutations in the calcium-regulated C2 domain expands the possible mechanisms that can produce PLCG2 activation following ibrutinib treatment. The diversity of recurrent mutations observed supports the need for complete PLCG2 sequencing for full characterization of ibrutinib-treated CLL samples.
Citation Format: Dan Jones, Jennifer A. Woyach, Weiqiang Zhao, Sean Caruthers, Huolin Tu, Joshua Coleman, John C. Byrd, Amy J. Johnson, Gerard Lozanski. PLCG2 C2-domain mutations co-occur with BTK and PLCG2 resistance mutations in chronic lymphocytic leukemia undergoing treatment with the BTK inhibitor ibrutinib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3150. doi:10.1158/1538-7445.AM2017-3150