A Simple and Robust Real-Time Quantitative PCR Method for the Detection of MYD88 L265P Mutation in Waldenström'smacroglobulinemia.

Introduction

Currently used methods in the detection MYD88^L265P mutation often involve allele-specific polymerase chain reaction (AS-PCR) and Sanger sequencing in the BM biopsy of patients with IgM monoclonal gammopathies, assays which are rather qualitative than quantitative. We have recently evaluated the role of peripheral blood (PB) cell-free DNA (cfDNA) in characterizing the mutational status of MYD88as a tool for a non-invasive screening of patients with IgM monoclonal gammopathies. In this study we sought to develop a highly sensitive, allele specific quantitative real time PCR (ASqPCR) assay for the identification of MYD88 L265P mutation in patients with IgM monoclonal gammopathies, that can be easily applied in routine clinical screening in the cfDNA of these patients.

Method design

Our quantitative allele specific PCR approach is based on CAST PCR technology which is a competitive allele-specific TaqMAN PCR that utilizes an allele-specific primer for mutant allele detection that competes with an MGB blocker oligonucleotide that suppress the wild-type background by minimizing the amplification of mismatched products derived from the wild-type allele. This technology is highly specific and sensitive, as it can detect rare amounts of mutated DNA in a sample that contains large amounts of normal, wild-type DNA. This new technology is capable of detecting one mutant allele molecule in 10,000,000 wild-type molecules. Each assay also included the amplification of the MYD88 wild type as an internal control sequence in a separate reaction.

Results

Our patients cohort included 37/94 (39%) with IgM-MGUS, 34/94(36%) with asymptomatic WM, 18/94 (19%) with symptomatic WM, 2/94 (2%) in relapse and 2/94 (2%) in remission.

Overall 83/94 (88%) patients were identified as having the MYD88^L265P mutation. Among the 11 patients who carried the MYD88^WT, 4 patients were IgM-MGUS, meaning that the WM patients who carried the MYD88^L265P mutation were 92%. Forty four patients in this series had paired BM and PB samples and the rate of MYD88^L265P mutation detection was 94% in BM and 78% in PB. Our analysis included 51 CD19⁺ selected samples from BM aspirates and 87 cfDNA from PB samples. 10 patients with multiple myeloma (MM), 3 MM cell lines, 3 ovarian cancer cell lines and 5 healthy donors peripheral blood were used as negative controls. For positive control, DNA from WM cell lines MWCL1 and BCWM1 were used. The mutation cut-off ratio was determined based on the analysis of the positive and negative controls where a ΔCt value>9.03 was defined as 100% wild-type while a ΔCt value equal to 0 was defined as 100% mutant.

Our results from the mutation detector analysis software showed that 22/37 MYD88^L265PIgM-MGUS patients with available CD19⁺ samples had an average mutation fraction ratio 53.43% (range 0.2-100%) while 26/37 patients with available cfDNA had a mutation fraction ratio 6.18% (range 0.2-30%). In MYD88^L265P asymptomatic WM patients, 17/34 with available CD19⁺ samples had a mutation fraction ratio 79.24% (range 1.2-100%) while 22/34 patients with available cfDNA had a mutation fraction ratio 9.31% (range 0.5-33.8%). Finally, in MYD88^L265P symptomatic WM patients, 10/18 with available CD19⁺ samples had a mutation fraction ratio 86.7% (range 6.9-100%) while 15/18 patients with available cfDNA had a mutation fraction ratio 27.8% (range 0.9-100%). Overall our study shows that the mutation fraction rates are higher among patients with symptomatic WM compared to patients with asymptomatic WM, and higher in patients with asymptomatic WM compared to IgM-MGUS patients highlighting the possible impact MYD88^L265P mutation presence and burden ratio on disease progression of these patients.

Conclusion

In conclusion our study shows that CAST PCR technology by a simple real-time PCR is a feasible and highly sensitive assay for MYD88^L265P mutational screening and mutation fraction monitoring in patients with IgM monoclonal gammopathies. This technology is particularly interesting as it can be applied in the plasma cfDNA from the peripheral blood of patients where the amounts of DNA are especially low. Furthermore it represents a cost-effective sensitive tool avoiding the use of costly instruments such as digital droplet PCR accompanied with costly consumables which makes it hard to use for everyday screening.