MYD88L265P mutation in WM and IgM-MGUS: comparison of feasibility and benefit between ddPCR and standard ASqPCR

Waldenström Macroglobulinemia is a IgM-secreting lymphoplasmacytic B cell lymphoma. The main recognized risk factor that triggers progression to WM is a history of IgM monoclonal gammopathy of undetermined significance (IgM-MGUS). MYD88L265P mutation, identified in about 90% of WM, is important for differential diagnosis of WM and IgM-MGUS. Moreover, this mutation is both a prognostic biomarker, as well as predictive of response to targeted new drugs (i.e. Ibrutinib). Despite the detection of MYD88L265P is mainly performed by ASqPCR there are still some technical limitations as: 1) marker identification failure at low tumor level, especially after chemo-immunotherapy, 2) requirement of CD19+ cells selection, not always routinely performed in diagnostic laboratories. Recently, droplet digital PCR (ddPCR) has been proved to be suitable for WM screening and minimal residual disease monitoring. The aim of this study was to compare ASqPCR with ddPCR for MYD88L265P mutation detection in WM and IgM-MGUS, in order to identify the most suitable technique and the most useful specimen type to be used in laboratory routine. Overall, biological specimens from 228 patients (160 WM and 68 IgM-MGUS) were collected within three different cohorts: Torino (N=63), Pavia (N=67) and Varese (N=98). Method comparison was performed on 319 samples (239 WM, 80 IgM-MGUS): 117 CD19+ selected cells (90 BM, 27 PB), 53 MNC (46 BM, 7 PB), 149 WBC (73 BM, 76 PB). Additionally, 64 cfDNA from plasma (PL) were analysed for MYD88L265P with both techniques. Overall, 237/319 (74%) of samples were concordant between the two methods (182 ddPCR+/ASqPCR+ and 55 -/-), while 82/319 (26%) were discordant, mostly (81/82) mutated by ddPCR and WT by ASqPCR (23% WM and 32% IgM-MGUS). Considering BM and PB separately, we observed 162/209 (78%) concordant BM, 141 ddPCR+/ASqPCR+ [median 4,1E-02; range: 4.0E-04, 7.1E+01] and 21 ddPCR-/ASqPCR-; while 47 (22%) were discordant (46 ddPCR+/ASqPCR-, only one (CD19+) ddPCR-/ASqPCR+) [median 1.2E-03; range: 3.6E-04, 2.5E-02]. Within PB, 75/110 (68%) were concordant, 41 ddPCR+/ASqPCR+ [median 1.1E-02; range: 3.8E-04, 5.5E-01] and 34 ddPCR-/ASqPCR-; while 35 (32%) were discordant (all ddPCR+/ASqPCR-) [median 8.6E-04; range: 3.7E-04, 4.8E-02]. Focusing on CD19+ selected samples, 82/117 (70%) were concordant, 69 ddPCR+/ASqPCR+ [median 2.2E-01; range: 4.1E-04, 7.1E+01] and 13 ddPCR-/ASqPCR-, while 35 (30%) were discordant, 34 ddPCR+/ASqPCR- [median 1.5E-03; range: 3.6E-04, 4.8E-02] and only one ddPCR-/ASqPCR+. Of note, MYD88L265P level was not significantly different between MNC and WBC, however it was lower compared to CD19+ selected cells [median 1,2E-02 vs 2,2E-01]. Of note, ddPCR detected MYD88L265P in 80% (64/80) of IgM-MGUS and 83% (199/239) of WM while ASqPCR in 48% (39/80) and 60% (144/239), respectively. Interestingly, compared to WM, IgM-MGUS showed a lower median mutational level: 4.9E-03 (range: 4.1E-04, 2.6E-01) vs 5.2E-02 (range: 3.8E-04–7.1E+01) (p<0.0001). Finally, the analysis of 64 cfDNA from PL confirmed the higher sensitivity of ddPCR compared to AsqPCR (41/64 ddPCR+ vs 23/64 ASqPCR). In conclusion, this study highlights ddPCR as a feasible approach for MYD88L265P detection, more sensitive than ASqPCR across different specimen types (including cfDNA) and diseases, thus suggesting that the implementation of ddPCR assay in routine diagnostic laboratories might avoid the need of CD19+ selection. Moreover, significantly different MYD88L265P mutational levels identified between WM and IgM-MGUS highlighted the need for further studies on larger patients’ series to identify correlations between mutational levels and risk of progression to WM.