Detection Of MYD88 L265P In Peripheral Blood Of Patients With Waldenström's Macroglobulinemia and IgM Monoclonal Gammopathy Of Undetermined Significance

Waldenström's macroglobulinemia (WM) is characterized by bone marrow (BM) infiltration with lymphoplasmacytic cells and production of an IgM paraprotein. Lymphocytosis is uncommon in WM, and many patients exhibit peripheral B-cell lymphopenia. Recently, by whole genome sequencing, we identified a highly recurrent somatic mutation (L265P) in MYD88. Using real-time allele-specific polymerase chain reaction (AS-PCR), we demonstrated the presence of MYD88 L265P in 93% and 54% of patients with WM and IgM monoclonal gammopathy of undetermined significance (MGUS), respectively (Xu et al, Blood 2013).

To clarify the feasibility of using real-time AS-PCR assay to detect MYD88 L265P in peripheral blood, we first examined unselected mononuclear cells from peripheral blood (PB) samples of 88 patients with untreated WM. 81/88 (92%) of these patients were MYD88 L265P positive by bone marrow (BM) examination of CD19 selected B-cells; Of the 81 BM-MYD88 L265P positive patients, 32 (40%) demonstrated MYD88 L265P by AS-PCR in unselected PB mononuclear cells (PBMC) obtained at the time of BM examination. To enhance the ability to detect MYD88 L265P in PB samples, we next used AS-PCR in CD19 selected PBMC obtained from 198 WM patients which included untreated and previously treated patients. Among untreated patients, 106/118 (89.8%) were positive for MYD88 L265P in CD19 selected PB, whereas among previously treated patients, 55/80 (68.8%) were positive for MYD88 L265P (p=0.0002 vs. untreated). Simultaneously analyzed PB and BM samples from 65 untreated WM patients demonstrated positivity for MYD88 L265P in 58/65 (89.2%) and 55/65 (84.6%) of CD19 selected BM and PB samples, respectively. These findings yielded a sensitivity of 94.8%, specificity of 100%, positive and negative predictive values of 100% and 70%, respectively for AS-PCR testing of PB CD19⁺ cells for MYD88 L265P in untreated WM patients. In addition, we analyzed 12 untreated IgM MGUS patients with paired sampling of CD19 selected PB and BM cells. 6/12 (50%) IgM MGUS patients were positive for MYD88 L265P in BM CD19⁺ cells, with 5/6 (83%) of these positive patients demonstrating MYD88 L265P by AS-PCR in CD19 selected PB samples.

The overall results demonstrate a high concordance of MYD88 L265P status between PB and BM CD19 selected samples, particularly in untreated WM patients. The detection of MYD88 L265P by CD19 selected PB AS-PCR examination provides a convenient and less invasive method to support the diagnosis of WM and IgM MGUS.