Pyrosequencing as a Fast and Reliable Method in Detecting the MYD88 p.L265P Mutation in Decalcified Formalin-Fixed and Paraffin-Embedded Tissues

Dear Editor, MYD88 p.L265P has recently been identified as a considerably recurrent mutation in lymphoplasmacytic lymphomas with an IgM monoclonal protein (Waldenström’s macroglobulinemia [WM]). The mutation was also described as a common event in several other lymphoproliferative disorders, including diffuse large B-cell lymphoma and primary central nervous system (CNS) lymphoma [1]. Detecting MYD88 p.L265P mutations was subsequently proposed as critical to the differential diagnosis of WM, multiple myeloma, and marginal zone lymphoma [2, 3]. A more recent study indicated that MYD88 mutation frequency in WM was slightly lower than was previously assumed. Patients with wild-type MYD88, however, often show amplifications on chromosome 3 at the 3p22 locus, which includes the MYD88 gene [4]. Because MYD88 plays a role in activating nuclear factor (NF)-κB signaling, its mutation will likely be relevant to targeted therapeutics [5]. Previously published methods for the detection of MYD88 p.L265P include high-resolution melting analysis (HRMA), allele specific polymerase chain reaction (AL-PCR), and direct DNA sequencing [2, 6]. The purpose of this study was to establish a pyrosequencing assay using decalcified formalin-fixed and paraffin-embedded (dFFPE) bone marrow trephine biopsies from 14 patients with WM and 10 patients with multiple myeloma. To extend the application of the technique, we used the assay to evaluate fresh bone marrow mononuclear cell samples (n=5) and peripheral blood samples (n=5) collected from five of the 14 WM patients (Cases 1, 4-7). All samples were collected as part of standard clinical care and diagnosed at the Reference Center for Lymph Node Pathology and Hematopathology, University Hospital of SchleswigHolstein, Campus Luebeck, Germany. All studies were approved by the Ethics Committee at the University of Luebeck and were in accordance with the Declaration of Helsinki. Pyrosequencing was performed as described previously [7]. DNA was extracted with the QiaAmp Mini Kit 250 (Qiagen, Hilden, Germany), according to the manufacturer’s instructions. A short sequence of DNA encompassing the mutation site was amplified by using a specific pair of primers, one of which was biotinylated (in this case the reverse primer). Next, a single strand of the amplified mutation region was prepared by using streptavidin-coated Sepharose beads to specifically bind the biotin tag on the reverse primer. Sequencing was subsequently performed on a PyroMark Q24 platform (Qiagen) following incubation with a forward sequencing primer. Allele frequency was quantified utilizing the PyroMark Software (Qiagen). Primers