Detection of MYD88 L265P mutation by next-generation deep sequencing in peripheral blood mononuclear cells of Waldenström's macroglobulinemia and IgM monoclonal gammopathy of undetermined significance.

Ayako Nakamura,Osamu Ohara,Kosei Matsue,Yasumasa Sugita,Chiaki Nakaseko,Chikako Ohwada,Yusuke Takeda,Shokichi Tsukamoto,Hisashi Wakita,Koutaro Yokote,Hiroaki Tanaka,Nobuyuki Aotsuka,Naoya Mimura,Oshima-Hasegawa Nagisa,Emiko Sakaida,Masahiro Takeuchi,Francesco Bertolini

doi:10.1371/journal.pone.0221941

Abstract

We investigated the feasibility of using next-generation sequencing (NGS) technique using molecular barcoding technology to detect MYD88 L265P mutation in unselected peripheral blood mononuclear cells (PBMCs) in 52 patients with Waldenström’s macroglobulinemia [1] and 21 patients with IgM-monoclonal gammopathy of undetermined significance (MGUS). The NGS technique successfully detected the MYD88 L265P in unselected PBMCs at a sensitivity of 0.02%, which was ×5 higher than that of AS-PCR. All the results between paired BM and PB samples from 2 IgM MGUS and 4 untreated WM patients matched completely. MYD88 L265P mutation was detected in 14/21 (66.7%), 14/19 (73.7%), and 10/33 (30.3%) with the median mutant allele burden of 0.36% (range, 0.06–2.85%), 0.48% (range, 0.02–32.3%), and 0.16% (range, 0.02–33.8%), in IgM-MGUS, untreated WM, and previously treated WM, respectively. Multiple linear regression analysis identified an absolute peripheral lymphocyte count as the positive predictor of PB mutant allele burden (R2 = 0,72, P<0.0001). Our non-invasive, simple NGS method has the potential to detect MYD88 L265P mutations in PBMCs of IgM MGUS and WM patients, which may especially utilized for monitoring minimal residual tumor burden after treatment.

Highlights

Waldenstrom’s Macroglobulinemia [1] is a B-cell malignancy characterized by lymphoplasmacytic cells in bone marrow (BM), lymph nodes and spleen, as well as the abnormal increase in serum immunoglobulin-M (IgM), resulting in many problematic clinical symptoms [2]
Recently developed allele-specific polymerase chain reaction (AS-PCR) is highly sensitive in determining the MYD88 L265P status and its quantitative assessment may be utilized in monitoring tumor burden [9], CD19-selection technique is required to achieve enough sensitivity when peripheral blood (PB) is used [7], which may not be suitable for clinical use
To provide a more simple, non-invasive, inclusive, as well as sensitive method, we investigated the feasibility of using next-generation sequencing (NGS) technique to detect MYD88 L265P from unselected PB mononuclear cells (PBMCs) in WM and IgM-monoclonal gammopathy of undetermined significance (MGUS)

Summary

Introduction

Waldenstrom’s Macroglobulinemia [1] is a B-cell malignancy characterized by lymphoplasmacytic cells in bone marrow (BM), lymph nodes and spleen, as well as the abnormal increase in serum immunoglobulin-M (IgM), resulting in many problematic clinical symptoms [2]. Since the majority of IgM monoclonal gammopathy of undetermined significance (MGUS) and a good portion of WM patients may relish asymptomatic phase of the disease [8], a non-invasive method to accurately assess tumor progression is eagerly awaited. Recently developed allele-specific polymerase chain reaction (AS-PCR) is highly sensitive in determining the MYD88 L265P status and its quantitative assessment may be utilized in monitoring tumor burden [9], CD19-selection technique is required to achieve enough sensitivity when peripheral blood (PB) is used [7], which may not be suitable for clinical use. To provide a more simple, non-invasive, inclusive, as well as sensitive method, we investigated the feasibility of using next-generation sequencing (NGS) technique to detect MYD88 L265P from unselected PB mononuclear cells (PBMCs) in WM and IgM-MGUS

Methods

Results

Conclusion