HemaSphere | 2021
Diagnostic Next-generation Sequencing Frequently Fails to Detect MYD88L265P in Waldenström Macroglobulinemia
Abstract
Mutations in MYD88 (MYD88 MUT) are present in approximately 93%–97% of patients with Waldenström macroglobulinemia (WM), nearly all of which correspond to the c.978T>C transversion resulting in a p.Leu265Pro (L265P) substitution at the protein level.1,2 MYD88MUT helps support the diagnosis of WM and differentiate from other IgM-secreting B-cell malignancies, such as marginal zone lymphoma and IgM myeloma, where it is absent or rarely expressed.2 The presence of MYD88MUT is also associated with a better prognosis, lower risk of histological transformation, and predicts response to the BTK inhibitor ibrutinib in WM patients.3–7 These findings prompted the World Health Organization, National Comprehensive Cancer Network, and WM Workshop guidelines to recommend MYD88MUT testing for all suspected WM cases.8 Despite the importance of MYD88MUT, a uniform means for identifying them is currently lacking. The original studies that established the incidence of MYD88L265P used an allele-specific polymerase chain reaction (AS-PCR) with CD19-selected bone marrow (BM) aspirates to maximize sensitivity.2 However, presorting B-cells before AS-PCR is not feasible for most clinical laboratories; hence, unselected BM aspirates are routinely used for the clinical detection of MYD88L265P.9 Additional testing with Sanger sequencing is recommended in patients with wildtype (WT) MYD88 by AS-PCR to evaluate for rare non-L265P MYD88MUT.9 Targeted next-generation sequencing (NGS) has emerged as an alternative to AS-PCR to identify MYD88L265P, but the sensitivity of NGS for MYD88L265P detection in WM patients is unknown. This prompted us to compare the results for MYD88L265P detection by NGS against AS-PCR in 414 consecutive WM patients who had both assays performed synchronously. We used CD19-selected BM aspirate to detect MYD88L265P by quantitative AS-PCR, followed by Sanger sequencing to evaluate for non-L265P MYD88MUT in patients with MYD88WT by AS-PCR as previously described.2,6,7 Qualitative AS-PCR (lower limit of detection ~1%) for MYD88L265P was also performed on unselected BM aspirate in the Molecular Diagnostics Laboratory, Brigham & Women’s Hospital (Boston, MA). The findings for MYD88L265P were compared against a clinically validated and targeted NGS assay (Rapid Heme Panel) using unselected BM aspirate from the same patients.10 The NGS assay has an average coverage of 1500X with <5% of the amplicons with 50X coverage, and reproducibly could detect single nucleotide variants at allele frequencies of ≥5%.10 The median coverage of the MYD88 amplicon was 1521X (range 305–3707X). Calculations were performed with R (R Foundation for Statistical Computing, Vienna, Austria). The Dana-Farber/Harvard Cancer Center IRB approved this study, and all patients provided written consent for the use of their samples. Clinical characteristics at the time of MYD88MUT testing are shown in Supplemental Digital Table 1, http://links.lww. com/HS/A180. Using AS-PCR with CD19-selected BM samples, 391 patients (94.4%) had MYD88L265P identified. Sanger sequencing of the 23 patients with MYD88WT by AS-PCR revealed that one patient had a dinucleotide substitution that resulted in MYD88L265P, while another had a MYD88S243N mutation. Overall, the prevalence of MYD88MUT in this cohort was 393/414 (95%), and did not differ between treatment-naïve and previously treated WM patients (94% versus 96%, respectively; P = 0.37). We compared the results for MYD88L265P by NGS with unselected BM aspirate against AS-PCR with CD19-selected BM. Among the 391 patients with MYD88L265P identified by AS-PCR, only 259 patients (66%) had MYD88L265P identified by the NGS method; the median variant allele fraction for MYD88L265P was 5.95% (range 0.5%–86.5%). No patient had MYD88L265P identified by NGS that AS-PCR did not also identify. The test performance statistics comparing the 2 methods are summarized in Table 1. We then evaluated factors that impacted the sensitivity of MYD88L265P detection by NGS. Modeling the false negative results by age, sex, hemoglobin level, platelet count, serum IgM 1Bing Center for Waldenström’s Macroglobulinemia, Dana-Farber Cancer Institute, Boston, Massachusetts, USA 2Department of Medicine, Harvard Medical School, Boston, Massachusetts, USA 3Division of Medical Oncology, Massachusetts General Hospital, Boston, Massachusetts, USA 4Boston University School of Medicine, Massachusetts, USA Supplemental digital content is available for this article. Copyright © 2021 the Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the European Hematology Association. This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial License 4.0 (CCBY-NC), where it is permissible to download, share, remix, transform, and buildup the work provided it is properly cited. The work cannot be used commercially without permission from the journal. HemaSphere (2021) 5:8(e624). http://dx.doi.org/10.1097/HS9.0000000000000624. Received: 22 March 2021 / Accepted: 4 June 2021