MYD88 L265P Mutation Detection: Analysis of Flow Cytometry Sorted Plasma and Lymphoid Cell Clones Improves Sensitivity and Specificity for WM/LPL Diagnosis
Authors: Wang Zhixing , Fritschle Wayne , Bennington Richard , Burnworth Bettina , Bennington Angela , Wentzel Collette , Verkamp Stephanie , Nguyen Phung , Ghirardelli Keely , Broderson Lisa , Wells Denise A. , Loken Michael R. , Zehentner Barbara Katharina 
Published: October 2019

Abstract
Background:
Waldenström's macroglobulinemia (WM) and lymphoplasmacytic lymphoma (LPL) are lymphoproliferative disorders with bone marrow infiltration by clonal lymphoplasmacytic cells (Treon et al., 2003, 2005). The somatic point mutation L265P in the myeloid differentiation primary response gene 88 (MYD88) has been reported in more than 90% of WM patients (Treon et al., 2012). Therefore MYD88 mutation analysis has been implemented in clinical practice to support the diagnosis of LPL/WM.
After the implementation of MYD88 L265P assays with increased detection sensitivity, a substantial portion of patients with IgM monoclonal gammopathy of undetermined significance (IgM-MGUS) was also reported MYD88 L265P positive. Splenic marginal zone lymphoma (SMZL), B-cell chronic lymphoproliferative disorders (B-CLPD) and diffused large B-cell lymphoma (DLBCL) have been found positive with much lower incidence rates (Varettoni et al., 2013; Xu et al., 2013).
Hence the remaining need to differentiate WM/LPL from other lymphoproliferative disorders with co-occurring plasma cells with high confidence.
Patients and Methods:
In this study, flow cytometric cell sorting was utilized to isolate clonal plasma and B lymphoid cell fractions as previously described (Zehentner et al., 2011). 69 patient specimen fractions with a clinical history of WM /LPL, multiple myeloma, CLL and lymphoma were analyzed for MYD88 L265P mutation using Sanger sequencing. Furthermore, the Biomed-2 primer sets for the immunoglobulin heavy (IgH) and/or the immunoglobulin kappa light chain region (IgK) were used to compare B cell clonality profiles in the lymphoid versus plasma cell compartments.
Results:
MYD88 L265P mutation was detected in all specimens with confirmed Waldenström's macroglobulinemia (17/17, 100%). Of these 16/17 (94%) revealed MYD88 L265P as well as identical monoclonality profile by gene rearrangement analysis in both the plasma and the B lymphoid cell collections. In 47% (8/17), the mutation was only detected in the plasma and B cell fractions, but not in the whole bone marrow specimens.
21 patient specimens with a known clinical history of LPL and co-occurring clonal plasma cells were tested. 9 of 21 (43%) were categorized with identical B cell clonality profile when comparing plasma and B lymphoid cells; whereas 12 (57%) had unrelated clonality profiles. 7 of the 9 (78%) specimens in the identical clonality group tested positive for MYD88 L265P in both the plasma and B lymphoid clone. None of the unrelated clonality group specimens carried the mutation in both cell fractions; for 7/12 (58%) MYD88 L265P was found in either the plasma (2) or the B-cell fraction (5) whereas 5/12 (42%) tested negative.
11 bone marrow aspirate specimen with known presence of lymphoma (including splenic marginal zone (SMZL), mantle cell and marginal zone) were analyzed. 10/11 (90%) tested negative for MYD88 L265P, with the exception of one SMZL specimen. Furthermore, 12 known myeloma, 5 CLL and 3 healthy donor specimens were analyzed, all tested negative.
Conclusions:
In this study, we developed and tested a novel approach to assess MYD88 L265P mutation status in order to assist WM/LPL diagnosis. Flow cytometric cell sorting for clonal plasma and B cell populations was combined with molecular analysis. Subsequently, MYD88 L265P mutation as well as B-cell clonality profile was compared in both cell fractions.
Our study postulates a significant improvement in sensitivity and most importantly specificity when applying MYD88 L265P mutation status in combination with cell sorting for WM/LPL diagnostic decisions. 47% WM patients (8/17) and 44% LPL patients (4/9) were positive for the MYD88 mutation exclusively in both flow cytometry sorted cell fractions but not in whole bone marrow specimens. 94% (16/17) WM as well as 78% (7/9) LPL specimens with identical plasma and B-cell clonality profile revealed the presence of the MYD88 L265P mutation in both the plasma cell and the B lymphoid cell clones. Whereas LPL specimens with unrelated clonality profile of the plasma and lymphoid cell fractions as well as other control specimens (lymphoma, myeloma, CLL and healthy) either tested negative or positive only in one of the sorted cell fractions.
We therefore conclude that confirming the presence of MYD88 L265P in both B-lymphoid and plasma cell fraction is an important prerequisite to distinguish LPL/WM from related disorders with high confidence.

Disclosures
Wang: HematoLogics Inc.: Employment. Fritschle:HematoLogics Inc.: Employment. Bennington:HematoLogics Inc.: Employment. Burnworth:HematoLogics Inc.: Employment. Bennington:HematoLogics Inc.: Employment. Wentzel:HematoLogics Inc.: Employment. Verkamp:HematoLogics Inc.: Employment. Nguyen:HematoLogics Inc.: Employment. Ghirardelli:HematoLogics Inc.: Employment. Broderson:HematoLogics Inc.: Employment. Wells:HematoLogics Inc.: Employment, Equity Ownership. Loken:HematoLogics Inc.: Employment, Equity Ownership. Zehentner:HematoLogics Inc.: Employment, Equity Ownership.