Cell‐free <scp>DNA</scp> analysis for the detection of <scp>MYD88</scp> and <scp>CXCR4</scp> mutations in <scp>IgM</scp> monoclonal gammopathies; an update with clinicopathological correlations
Authors: Ioannis Ntanasis‐Stathopoulos, Tina Bagratuni, Maria Gavriatopoulou, Dimitrios Patseas, Christine Liacos, Nikolaos Kanellias, Despina Fotiou, Eleni Tsiligkeridou, Amalia Andreatou, Nefeli Mavrianou‐Koutsoukou, Magdalini Migkou, Evangelos Eleutherakis‐Papaiakovou, Maria Roussou, Evangelos Terpos, Efstathios Kastritis, Meletios A. Dimopoulos
Journal: American Journal of Hematology (2020)

The current management of Waldenström's macroglobulinemia (WM) and IgM monoclonal gammopathies necessitates the determination of the mutational status of the MYD88 and the CXCR4 genes.1 This procedure may include the selection of CD19+ cells derived from bone marrow (BM) aspirates, which is an invasive technique, potentially associated with patient discomfort. Taking these into consideration, we have previously shown that peripheral blood cell-free tumor DNA (cfDNA), also known as liquid biopsy, may constitute a feasible alternative method of evaluating the genomic landscape.2 In this extension study, we aimed to characterize the mutational status of MYD88 and CXCR4 among patients with WM and IgM MGUS by using peripheral blood plasma-derived cfDNA and matched tumor DNA (tDNA) from BM-CD19+ selected cells. We sought to determine whether cfDNA can be used as an adjunct and/or alternative diagnostic tool in identifying the mutational profile of IgM monoclonal gammopathies. Briefly, peripheral blood (10-12 mL) was collected in EDTA tubes and DNA was extracted using the MagMax cell free DNA isolation kit. At the same time point, BM aspirates were collected and processed for CD19 enrichment. The tDNA and cfDNA samples were analyzed for the MYD88 L265P and for CXCR4 mutations. Allele-specific PCR and direct sequencing were performed in order to detect the MYD88 and CXCR4 mutations. The study was approved by the institutional review board and all patients provided written informed consent for sample collection and analysis. The detailed methodology has been previously described.2 Overall, 207 patients were included in the study; in 33 patients only cfDNA collection was performed. The MYD88 mutations were detected in 91% (147/162) and 89% (168/188) of the patients according to the tDNA and cfDNA analysis, respectively. The prevalence is comparable to the frequency reported in other studies based on CD19-selected cells from the bone marrow or the peripheral blood.1 Note, tDNA could not be isolated from the BM aspirates in 12 patients with low infiltration by malignant lymphoplasmacytic cells, whereas cfDNA isolation was not feasible in 19 patients. A group of 150 patients had paired tDNA and cfDNA informative samples. The MYD88 L265P mutation was detected in 135/150 patients (90%) in both tDNA and cfDNA; in five (3%) the mutation was seen in tDNA but not in cfDNA and 10 (7%) patients harbored the MYD88 WT genotype both in tDNA and cfDNA. Thus, the overall concordance between tDNA and cfDNA for MYD88 genotype was 97% (P < .001, Spearman's rho). Among patients with IgM MGUS, WM in remission and sWM/ newly diagnosed (ND)WM/relapsed (RR) WM, the concordance rates were 96% (27/28), 97% (36/37) and 96% (76/79), respectively. In all discordant cases the MYD88 L265P mutation was detectable in the BM but not in the cfDNA. Therefore, the need for bone marrow sampling could be restricted only to cases with indeterminate cfDNA results. Interestingly, the amount of cfDNA was not correlated with IgM levels (P = .717, Spearman's rho, 2-tailed), bone marrow invasion (P = .439) or CD19+ yield (P = .65). Inversely, the CD19+ yield was associated with both IgM levels (P = .01) and bone marrow invasion (P < .001). The detection of the MYD88 L265P in tDNA was significantly associated with the CD19+ yield (P = .007), marginally associated with the IgM levels (P = .048) and showed a trend for association with the percentage of BM infiltration (P = .057). The detection of the MYD88 L265P in the cfDNA was not associated with the IgM levels (P = .071), the percentage of BM infiltration (P = .634) or the amount of the isolated cfDNA (P = .601) and the yield of CD19+ cells (=.334). Regarding the evaluation of CXCR4 mutations, 131 patients had paired tDNA and cfDNA samples. In 15 patients (12%) the same CXCR4 mutations were detected in both paired samples, whereas 11 patients (8%) showed discordant mutations between the tDNA and the cfDNA. In 13 (10%) and 8 (6%) patients mutations in the CXCR4 were detected only in the tDNA and the cfDNA, respectively. In a large proportion of the participants (64%) no CXCR4 mutations were revealed by either tDNA or cfDNA; thus, 36% of patients had a CXCR4 mutation detected by either tDNA or cfDNA. Overall, the pathogenic mutation S338X was present in five patients (three in both tDNA and cfDNA, two in tDNA only) and the L50X in one (tDNA only) (Table 1). The concordance rate regarding the presence or absence of CXCR4 mutations between tDNA and cfDNA was 84% (P < .001), which was consistent among patients with IgM MGUS (88%), WM in remission (82%), and sWM/NDWM/RRWM (85%). If we exclude the cases with discordant CXCR4 mutations, the overall concordance rate was 76%. The detection of CXCR4 mutations in tDNA was not associated with the CD19+ yield (P = .530), IgM levels (P = .458) or BM infiltration (P = .909). The detection of CXCR4 mutations in the cfDNA was not associated with the amount of isolated cfDNA (P = .820), IgM levels (P = .113), BM infiltration (P = .091) or the CD19+ yield (P = .184). In accordance to our findings, it has been shown that cfDNA shows a superior relative enrichment of MYD88 L265P DNA compared with peripheral blood CD19+ selected cells, peripheral blood mononuclear cells and paired unselected bone marrow mononuclear cells.3 Furthermore, the feasibility of detecting MYD88 mutations in the cfDNA has been demonstrated using another technique, the droplet digital PCR (ddPCR).4 Among 60 patients with paired bone marrow and cfDNA samples, an encouraging concordance regarding MYD88 L265P detection was reported with a median mutational value of 1.92 × 10−2 and 1.4 × 10−2 for bone marrow DNA and ctDNA, respectively. Detecting MYD88 L265P in the cfDNA by ddPCR showed also a good concordance (r2 = 0.64, P < .001) with the IgH-based minimal residual disease (MRD) assessment by ddPCR among 10 patients with available serial samples. The IgH-based MRD evaluation is a standardized technique and, although it has not been validated in the context of WM, it can serve as a reliable comparator to assess the role of novel liquid biopsies for MRD monitoring in future studies. Interestingly, ddPCR can be effectively applied in other liquid biopsies as well, such as in determining the MYD88 mutational status in the cerebrospinal fluid of patients with suspected Bing-Neel syndrome.5 In conclusion, peripheral blood cfDNA is a reliable, minimally invasive approach for the detection of MYD88 and CXCR4 mutations in patients with IgM monoclonal gammopathies irrespective of the disease burden. However, bone marrow biopsy remains the gold standard for establishing the diagnosis of WM and other IgM monoclonal gammopathies, and evaluating for concurrent primary bone marrow diseases. In line with the integration of liquid biopsies in the management of patients with solid malignancies,6 we encourage the integration of cfDNA-directed endpoints in prospective studies in order to assess the evolving genomic dynamics during the disease course and establish the cfDNA analysis in the clinical practice. None. None relevant to declare.