ORIGINAL ARTICLE MYD88 L265P is a marker highly characteristic of, but not restricted to, Waldenstro ¨m’s macroglobulinemia C Jime ´ nez 1 , E Sebastia ´n 1 , MC Chillo ´n 1,2 , P Giraldo 3 , J Mariano Herna ´ ndez 4 , F Escalante 5 , TJ Gonza ´ lez-Lo ´ pez 6 , C Aguilera 7 , AG de Coca 8 , I Murillo 3 , M Alcoceba 1 , A Balanzategui 1 , ME Sarasquete 1,2 , R Corral 1 , LA Marı ´n 1 , B Paiva 1,2 , EM Ocio 1,2 , NC Gutie ´ rrez 1,2 , M Gonza ´lez 1,2 , JF San Miguel 1,2 and R Garcı ´a-Sanz 1,2 We evaluated the MYD88 L265P mutation in Waldenstro ¨ m’s macroglobulinemia (WM) and B-cell lymphoproliferative disorders by specific polymerase chain reaction (PCR) (sensitivity B10 À 3 ). No mutation was seen in normal donors, while it was present in 101/117 (86%) WM patients, 27/31 (87%) IgM monoclonal gammapathies of uncertain significance (MGUS), 3/14 (21%) splenic marginal zone lymphomas and 9/48 (19%) non-germinal center (GC) diffuse large B-cell lymphomas (DLBCLs). The mutation was absent in all 28 GC-DLBCLs, 13 DLBCLs not subclassified, 35 hairy cell leukemias, 39 chronic lymphocytic leukemias (16 with M-component), 25 IgA or IgG-MGUS, 24 multiple myeloma (3 with an IgM isotype), 6 amyloidosis, 9 lymphoplasmacytic lymphomas and 1 IgM-related neuropathy. Among WM and IgM- MGUS, MYD88 L265P mutation was associated with some differences in clinical and biological characteristics, although usually minor; wild-type MYD88 cases had smaller M-component (1.77 vs 2.72 g/dl, P ¼ 0.022), more lymphocytosis (24 vs 5%, P ¼ 0.006), higher lactate dehydrogenase level (371 vs 265 UI/L, P ¼ 0.002), atypical immunophenotype (CD23 À CD27 þþ FMC7 þþ ), less Immunoglobulin Heavy Chain Variable gene (IGHV) somatic hypermutation (57 vs 97%, P ¼ 0.012) and less IGHV3–23 gene selection (9 vs 27%, P ¼ 0.014). These small differences did not lead to different time to first therapy, response to treatment or progression-free or overall survival. Leukemia (2013) 27, 1722–1728; doi:10.1038/leu.2013.62 Keywords: MYD88 mutations; Waldenstro ¨ m’s macroglobulinemia; IgM monoclonal gammapathies; diagnosis; prognosis; immunophenotyping INTRODUCTION Waldenstro ¨ m’s macroglobulinemia (WM) is a rare hematological malignancy with an incidence of 3.6–5.5 cases per million person– years in the EU and US. 1–4 Despite its low frequency, WM is particularly interesting due to its singular pathogenic features, which represent the most genuine intermediate stage between lymphoproliferative disorders (LPDs) and plasma cell dyscrasias, in which clinical and biological features mimic the two ends of the spectrum of such disorders. Moreover, the study of WM may be relevant to better understand the genetic mechanisms involved in those LPDs derived from abnormalities in the terminal B-cell differentiation process. Although most WM patients have been reported to have genetic aberrancies, few recurrent chromosomal changes have been described in this disease, probably due to the difficulty of obtaining tumor metaphases for karyotype studies. 5 Whole- genome sequencing has confirmed that virtually all WM patients have molecular DNA alterations. 6 In particular, the L265P mutation at the MYD88 gene (38182641 in chromosome 3p22.2), which results in a leucine to proline change at the L265P amino acid, was reported in 26 of the 30 WM patients initially evaluated with whole-genome sequencing. 6 An extended evaluation of this mutation in a larger series of patients showed it to be present in 91% (49/54) of WM patients, and it was suggested that in IgM monoclonal gammapathies it looked to be almost exclusive of lymphoplasmacytic bone marrow (BM) infiltrative forms of the disorder. This was based on the fact that the mutation was very infrequent in IgM monoclonal gammopathy of uncertain significance (MGUS, 10% of cases) and in marginal zone lymphomas (MZLs, 7%), and it was completely absent from multiple myeloma patients (0/10) and healthy donors. 6 Apart from this, MYD88 L265P mutation is also present in a fraction of diffuse large B cell lymphomas (DLBCLs) of the activated B-cell type (14–29%) 7–9 and leg type (69%), 10 primary central nervous system lymphoma (36–38%) 11,12 and mucosa-associated lymphoid tissue lymphoma (9%). 7 In addition, most of these studies have been carried out with conventional sequencing techniques, such as Sanger sequencing, which has a relatively low sensitivity and requires at least 20–30% of cells to carry the mutation to be able to detect it among the vast majority of normal alleles. As many of the aforementioned tumors can have low infiltration in the tissues available for analysis, this technical limitation could lead to underestimate the mutation frequency. Such an effect is especially important in some entities, such as IgM-MGUS, where the percentage of true monoclonal cells in the BM aspiration could be as low as o1%. 13 Polymerase chain reaction with allele-specific oligonucleotides (ASO-PCR) is a technique that can discriminate low levels of the mutant sequence against a background high in wild-type DNA. 14 In ASO-PCR, the primer pair is designed so that one of the 3 0 ends 1 Department of Hematology, University Hospital of Salamanca, Salamanca, Spain; 2 Center of Investigation in Cancer (CIC), Instituto Biosanitario de Salamanca (IBSAL), Salamanca, Spain; 3 Department of Hematology, Hospital Miguel Servet, Zaragoza, Spain; 4 Department of Hematology, Hospital General, Segovia, Spain; 5 Department of Hematology, Hospital Virgen Blanca, Leo ´ n, Spain; 6 Department of Hematology, Hospital General de Burgos, Burgos, Spain; 7 Department of Hematology, Hospital Comarcal del Bierzo, Leo ´ n, Spain and 8 Department of Hematology, Hospital Clı ´nico, Valladolid, Spain. Correspondence: Dr R Garcı ´a-Sanz, Department of Hematology, University Hospital of Salamanca, Paseo de San Vicente, 58-182, Salamanca 37007, Spain. E-mail: rgarcias@usal.es Received 29 December 2012; revised 9 February 2013; accepted 13 February 2013; accepted article preview online 28 February 2013; advance online publication, 22 March 2013 Leukemia (2013) 27, 1722–1728 & 2013 Macmillan Publishers Limited All rights reserved 0887-6924/13 www.nature.com/leu