Fast Detection Of MYD88-L265P Mutation By PCR-RFLP In Chronic Lymphoproliferative Disorders

Waldenstrom’s macroglobulinemia (WΜ) is an incurable disorder characterized by bone marrow infiltration from neoplastic lymphoplasmacytic B-lymphocytes and monoclonal IgM production. Recent data suggest a possible causal relationship of MYD88-L265P mutation with the pathogenesis of the disease (Treon Sp et al. N Eng J Med 2012;367:826-33). Additional studies have conformed that MYD88-L265P is characteristic of WΜ, but it is also rarely present in other chronic lymphoproliferative disorders (LPDs) (Jiménez C et al. Leukemia 2013,doi: 10.1038/leu.2013.62; Varettoni M et al. Blood 2013;121:2522-8). The purpose of this study was to analyse the prevalence of the aforementioned mutation in patients with WM and other LPDs, using a fast and reliable polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) protocol.

Genomic DNA was extracted from the bone marrow of 11 patients with WΜ, 12 patients with B-cell chronic lymphocytic leukemia (CLL), 8 with splenic lymphoma with villous lymphocytes (SLVL). Furthermore, consecutive samples of peripheral blood and bone marrow isolated CD19⁺ cells, derived from a patient with monoclonal IgM gammopathy of undetermined significance (MGUS-IgM), were also retrospectively analysed for the presence of MYD88-L265P defect. The detection of the mutation was performed by PCR amplification of the MYD88 exon 5 region, followed by RFLP analysis, since the presence of the mutation results in the generation of BsiEI restriction enzyme site. PCR-RFLP results were also confirmed by direct sequencing of purified CD19⁺cells.

The MYD88-L265P mutation was detected in 10 patients with WΜ (90.9%), in 1 patient with SLVL with markers of lymphoplasmatocytoid differentiation (12.5%) and was absent in all CLL patients. Interestingly, our PCR-RFLP protocol exhibited a greater sensitivity than direct sequencing, when applied to total bone marrow cells (12.5% vs 25%). Moreover, the patient with MGUS-IgM displayed the MYD88-L265P mutation in isolated CD19⁺cells of both bone marrow and peripheral blood in all consecutive samples and remains in a stable condition for the last 7 years.

PCR-RFLP is a rapid, sensitive and reliable technique for the detection of MYD88-L265P mutation in patients with WM and lymphomas with lymphoplasmatocytoid differentiation. Additionally, the presence of MYD88-L265P mutation in MGUS-IgM in the absence of disease progression for many years, suggests that this genetic defect alone is not sufficient to lead to overt neoplastic disease.

No relevant conflicts of interest to declare.