PS1319 MYD88L265P MUTATION IN WM AND IGM‐MGUS: COMPARISON OF FEASIBILITY AND BENEFIT BETWEEN DDPCR AND STANDARD ASQPCR IN CD19+ SELECTED AND UNSELECTED SAMPLES

Background: In Waldenström Macroglobulinemia (WM) whole genome sequencing has revealed recurring somatic mutations, affecting the NFK‐B and Bruton tyrosine kinase pathways, which are responsible for drug response prediction. MYD88 L265P is the highly prevalent mutation, detected in up to 95% of patients, and represents a suitable molecular marker. Mutational analysis currently performed by ASqPCR has some technical limitations, in particular: marker identification failure at low tumor level, especially after chemo‐immunotherapy, and requirement of CD19+ cells selection in routine diagnostic laboratories. Furthermore, 1.5–2% of monoclonal gammopathy of undetermined significance (IgM‐MGUS) patients, per year, progress to WM and there are not reliable biological markers able to distinguish between these two conditions. Recently, droplet digital PCR (ddPCR) demonstrated to be highly suitable for WM screening and MRD monitoring. Aims: In this study we aim to compare ASqPCR and ddPCR in CD19+ selected and unselected samples from WM and IgM‐MGUS patients, to assess whether: 1) cell selection could be avoided in diagnostic routine, for MYD88 L265P detection; 2) the MYD88 L265P quantitative level might discriminate WM from IgM‐MGUS. Methods: We analyzed two retrospective samples series from WM and IgM‐MGUS patients collected at the Hematology Units of Torino and Varese. All patients provided written informed consent in accordance with Helsinki's declaration. Both ddPCR and ASqPCR were performed on CD19+ selected cells (Whole Blood CD19 MicroBeads‐MACS, Miltenyi Biotec) collected in Varese and on unselected mononuclear cells (MNC) collected in Torino. The experiments were performed in parallel and in blind on the same series, by Varese (ASqPCR) and Torino (ddPCR), as previously described (Hunter Z et al, Drandi D et al). Results: Method comparison was performed on 130 samples from 110 patients (66 WM and 34 IgM‐MGUS), 75 CD19+ selected samples (53 BM, 22 PB at baseline) and 55 unselected MNC (30 BM, 25 PB, both at baseline and follow‐up). As reported in literature, ddPCR detected MYD88 L265P in 96% (23/24) of baseline CD19+ BM WM samples while ASqPCR in 83% (20/24). If considering the whole series, 31% (41/130) of samples were discordant between the two techniques mostly (39/41) mutated by ddPCR and WT by ASqPCR and regardless of disease status. Focusing on CD19+ selected samples, 36% (27/75) were discordant, mostly (26/27) mutated by ddPCR and WT by ASqPCR, with a median MYD88 L265P MUT/WT ratio of 2.36E‐03 (range:3.62E‐04–4.78E‐02). Concordantly, 25% (14/55) of MNC samples were discordant, mostly (13/14) mutated by ddPCR and WT by ASqPCR, with a median MYD88 L265P MUT/WT ratio of 5.53E‐04 (range:4.33E‐04–9.00E‐04) (Figure1). Moreover, ddPCR detected MYD88 L265P in 86% (25/29) of IgM‐MGUS baseline CD19+ BM, while ASqPCR only in 52% (15/29). Interestingly, compared to WM, IgM‐MGUS showed a lower median mutational level: 1.65E‐03 (range:1.0E‐05–2.6E‐01) vs 1.68E‐01 (range:2.2E‐04–9.0E‐1) ( p  < 0.0001). Summary/Conclusion: This study highlights ddPCR as a feasible and highly sensitive approach for MYD88 L265P mutational detection, particularly in samples harboring low concentrations of tumor cells. These results suggest that the implementation of ddPCR assay in routine diagnostic laboratories might avoid the need of CD19+ selection for MYD88 L265P screening. Moreover, significantly different MYD88 L265P mutational levels identified between WM and IgM‐MGUS cases highlighted the need for further studies on larger patients’ series to identify correlations between mutational levels and risk of progression to WM.