Clinical Validation of a CXCR4 Mutation Screening Assay for Waldenstrom Macroglobulinemia

Abstract Objectives Waldenstrom macroglobulinemia (WM) is a B-cell lymphoma characterized by the accumulation of lymphocytes and plasmacytic cells in the bone marrow and by excess production of immunoglobulin M in serum. WM has been closely linked with the MYD88 L265P mutation. Whole genome sequencing has identified somatic mutations in the CXCR4 gene in ∼29% of WM cases with MYD88 L265P . CXCR4 mutations may interfere with treatment response to ibrutinib. The goal of this study was to design and validate a clinical assay to detect CXCR4 mutations. Methods Thirty-three low-grade B-cell lymphomas with plasmacytic differentiation (23 MYD88 L265P and 10 MYD88 WT ) involving various samples types (fresh and formalin-fixed tissues) formed the study group. We designed and validated Sanger sequencing and pyrosequencing assays to detect mutations in CXCR4 in a Clinical Laboratory Improvement Amendments-approved clinical laboratory. Results We identified 8 cases with CXCR4 mutations, including 5 single nucleotide substitutions (3 resulting in p.S338* and 1 in p.R334*), and 3 insertion/deletions. Seven of 8 CXCR4 mutated cases were also MYD88 L265P mutant. Among the single nucleotide substitutions, we identified a novel missense variant (p.L326P) and a previously reported variant (G335S) of uncertain clinical significance. Conclusions We successfully validated a set of clinical assays to detect mutations in CXCR4 mutations in a clinical laboratory.