MYD88L265P Detection in IgM Monoclonal Gammopathies: Methodological Considerations for Routine Implementation

Abstract

In IgM monoclonal gammopathies MYD88L265P is a prognostic and predictive biomarker of therapy response. MYD88L265P detection is mainly performed by allele-specific quantitative PCR (ASqPCR), however recently, droplet digital PCR (ddPCR) has been proved to be suitable for MYD88L265P screening and minimal residual disease monitoring (MRD). This study compared ASqPCR and ddPCR to define the most sensitive method for MYD88L265P detection in bone marrow (BM), peripheral blood (PB) sorted or unsorted CD19+ cells, and in plasma cell-free DNA (cfDNA). Overall, the analysis showed a good concordance rate (74%) between the two methods, especially in BM samples, while discordances (26%) were mostly in favor of ddPCR (ddPCR+ vs. ASqPCR-) and were particularly evident in samples with low mutational burden, such as PB and cfDNA. This study highlights ddPCR as a feasible approach for MYD88L265P detection across different specimen types (including cfDNA). Interestingly, its high sensitivity makes CD19+ selection dispensable. On the other hand, our results showed that MYD88L265P detection on PB samples, especially with ASqPCR, is suboptimal for screening and MRD analysis. Finally, significantly different MYD88L265P mutational levels observed between Waldenström Macroglobulinemia and IgM monoclonal gammopathy of undetermined significance patients suggest the need for further studies in order to identify possible correlations between mutational levels and risk of progression to Waldenström.

Digital Droplet PCR in Hematologic Malignancies: A New Useful Molecular Tool

“…In 2018, our group contributed to set ddPCR for the identification of the MYD88 mutation; this technique detected mutation in 96% of MW and 87% of IgM-MGUS cases (vs 81% and 58% by QT-PCR, respectively); the concordance rate with QT-PCR amounted to 78% on bone marrow and 68% on peripheral blood samples; in the remaining cases, ddPCR confirmed its advantage. The most interesting finding of this work was the possible application of this molecular tool to the circulating tumor DNA (ctDNA) harvested from plasma [ 88 ] or in the cerebral spinal fluid [ 89 ]. In 2019, another group employed ddPCR for detecting MYD88L265P mutation in a cohort of 39 patients; with a sensitivity of 1 × 10 −3 , the authors identified the mutation in 90% of MW cases, in 44% of patients affected by LPL, in 5% of IgM MM, and no in CLL or mantle cell lymphoma (MCL) cases [ 90 ].…”

Nucleic Acid Biomarkers in Waldenström Macroglobulinemia and IgM-MGUS: Current Insights and Clinical Relevance

“…Therefore, the MYD88 L265P /MYD88 WT ratio might also be proposed as a quantitative marker and useful diagnostic tool for MRD analysis. Recently, digital PCR (dPCR) has been described as more sensitive than AS-qPCR across different specimen types (including plasma-cfDNA), for MYD88 L265P screening and MRD analysis, suggesting that the implementation of dPCR assay in routine diagnostic laboratories might avoid the need for CD19+ selection [62,99].…”

“…Cast-PCR plasma 92 88% 51 80% ND [192] Up to now, there is a lack of consensus regarding the optimal specimen and analytical method for mutational detection in WM, in terms of operating procedures, test sensitivity and result interpretation [99,192]. Researchers must be aware that differences in method sensitivity may lead to both a misclassification of disease status and an overestimation of the efficacy of novel treatments.…”

“…In a recent publication, different PCR methods (qPCR vs. dPCR) have been compared in BM, PB, CD19+ and cfDNA samples: dPCR appeared to be the most sensitive approach for MYD88 detection. Moreover, an algorithm was provided to suggest the most convenient PCR method based on available specimens and laboratory equipment [99]. Although highly relevant and promising, we are aware that the available data are premature to establish cfDNA as a single approach for disease screening and monitoring.…”

Use of BTK inhibitors with special focus on ibrutinib in Waldenström macroglobulinemia: An expert panel opinion statement

“…A sub analysis showed a 50% major response rate with zanubrutinib in 26 MYD88 WT patients 52 . However, it should be noted that different methods were used in the detection of MYD88L265P mutation, including Sanger sequencing.1, allele specific polymerase chain reaction (PCR) 53 and more recently droplet digital PCR and NGS‐based techniques, making comparison across studies and standardized sensitive detection of MYD88 mutations problematic 54‐57 …”