MYD88 L265P in Waldenström macroglobulinemia, immunoglobulin M monoclonal gammopathy, and other B-cell lymphoproliferative disorders using conventional and quantitative allele-specific polymerase chain reaction

Abstract

By whole-genome and/or Sanger sequencing, we recently identified a somatic mutation (MYD88 L265P) that stimulates nuclear factor κB activity and is present in >90% of Waldenström macroglobulinemia (WM) patients. MYD88 L265P was absent in 90% of immunoglobulin M (IgM) monoclonal gammopathy of undetermined significance (MGUS) patients. We therefore developed conventional and real-time allele-specific polymerase chain reaction (AS-PCR) assays for more sensitive detection and quantification of MYD88 L265P. Using either assay, MYD88 L265P was detected in 97 of 104 (93%) WM and 13 of 24 (54%) IgM MGUS patients and was either absent or rarely expressed in samples from splenic marginal zone lymphoma (2/20; 10%), CLL (1/26; 4%), multiple myeloma (including IgM cases, 0/14), and immunoglobulin G MGUS (0/9) patients as well as healthy donors (0/40; P < 1.5 × 10(-5) for WM vs other cohorts). Real-time AS-PCR identified IgM MGUS patients progressing to WM and showed a high rate of concordance between MYD88 L265P ΔCT and BM disease involvement (r = 0.89, P = .008) in WM patients undergoing treatment. These studies identify MYD88 L265P as a widely present mutation in WM and IgM MGUS patients using highly sensitive and specific AS-PCR assays with potential use in diagnostic discrimination and/or response assessment. The finding of this mutation in many IgM MGUS patients suggests that MYD88 L265P may be an early oncogenic event in WM pathogenesis.

Figure 1

Agarose gel-based conventional AS-PCR assay for detection of MYD88 L265P. (A) MYD88 L265P AS-PCR primer design. The reverse primers with an internal mismatch in the third position from the 3′-end and the common forward primer are indicated by arrows. (B) Sensitivity of the conventional AS-PCR assay was established by serial dilutions of DNA from OCI-LY3 against OCI-LY19 cells. The PCR products (159 bp) for this assay were separated on a 2% agarose gel as indicated by arrows. The mutant MYD88 L265P allele was detected to a dilution of 0.1%. MW, molecular weight.

Figure 2

MYD88 L265P in DLBCL and WM cell lines and primary WM patient cells using conventional AS-PCR assay. Detection of MYD88 L265P in OCI-LY3 and OCI-LY19 DLBCL, BCWM.1, and MWCL-1 WM cell lines (A) and primary WM patient tumor cells isolated from BM aspirates (B) by conventional AS-PCR assay. The depicted zygosity of MYD88 L265P in primary WM patient cells shown was confirmed by WGS. The position of MYD88 L265P locus is indicated by arrows. Complementary strand sequences are shown.

Figure 3

Sensitivity and specificity plots for real-time AS-PCR assay. (A) Δ reaction curve for real time AS-PCR assay. The MYD88 L265P mutant DNA (OCI-LY3) was diluted with the wild-type DNA (OCI-LY19) at the concentrations indicated in the amplification plot, with the MYD88 L265P allele detectable to a dilution of 0.08%. (B) Standard curve for real time AS-PCR assay. The correlation coefficient was 0.998 with a slope value of −3.48. (C) Dissociation curve for real time AS-PCR assay. Melting curve analysis revealed that the MYD88 L265P mutant-specific amplicon melted at 84°C. A minor nonspecific amplification was only found in the dilution of 0.4% or lower with a melting peak at 80°C. WT, wild-type.

Figure 4

Real-time AS-PCR results for MYD88 L265P in samples from patients with WM, IgM MGUS, and other B-cell lymphoproliferative disorders. Violin plot representing AS-PCR differences in cycle threshold (ΔC_T). The span of grey area for each cohort represents the kernel density estimation of the sample distribution, and highlights the bimodal nature of the data. Box plots with interquartile ranges are shown in black with an overlay of the individual data points. Samples evaluated were from healthy donors (HD, n = 40); along with patients with IgG (n = 9) and IgM (n = 24) MGUS; CLL (n = 26); MM including 3 patients with IgM myeloma (n = 14); MZL (n = 20), and WM (n = 104). The light grey bar represents the distance between the highest positive (7.3), and lowest negative (9.6) sample ΔC_T values. Circled area depicts results for 3 IgM MGUS patients who progressed to WM.