Mutational characterization of Waldenström's macroglobulinemia (WM) currently relies on DNA from CD19-selected cells derived from bone marrow (BM) aspirates, which is an invasive technique, associated with significant patient discomfort. Peripheral blood cell-free tumour DNA (cf or ctDNA) has been recently shown to be a powerful non-invasive molecular biomarker in monitoring tumor status in several cancers. In this study, we aimed to characterize the mutational status of WM/IgM MGUS patients by using peripheral blood plasma-derived cfDNA and using matched tumor DNA (tDNA) from BM-CD19+ selected cells as a control, in order to determine whether peripheral blood , by means of cfDNA, can be used as an additional diagnostic tool in identifying the mutational profile of WM.


A total of 30 consecutive patients with IgM monoclonal gammopathies were included in this study. All patients gave a written informed consent for sample collection and analysis. Peripheral blood (10-12mL) was collected in EDTA tubes and DNA was extracted using the MagMax cell free DNA isolation kit (ThermoFisher Scientific). cfDNA concentration was measured using the Qubit fluorometer. The quantities of cfDNA varied between 1.5 ng/μL and 8 ng/μL. BM aspirates were collected at the same time with peripheral blood, and were processed for CD19 enrichment, using CD19 magnetic beads (Miltenyi Biotech), followed by DNA extraction (Invitrogen). The paired tDNA and cfDNA samples from the 30 patients were analyzed for the MYD88 L265P and for CXCR4 mutations. The presence of L265P mutation was initially assessed by Allele-Specific PCR (AS-PCR) and then confirmed with direct sequencing. The presence of CXCR4 mutations was assessed with direct sequencing.


The study included patients with symptomatic WM before initiation of any therapy (N=4) , with WM under therapy (N=4) or after completion of therapy (N=12), with asymptomatic WM (N=8) and with IgM MGUS (N=2). The median age of the patients was 70 years (range 22-87). The median bone marrow infiltration of the tested samples was 28% (range 5 to 90%) and median IgM levels were 1510 mg/dL (range 80 to 7310 mg/dL).

In all, but one patient, (29/30) the L265P mutation was detected in both cfDNA and tDNA (100% concordance). This mutation was initially detected by AS-PCR which was validated by direct sequencing in all patients. Assessment of CXCR4 mutation status was feasible in 29 patients; in one patient CXCR4 sequencing was unsuccessful in both cfDNA and tDNA. Mutations in CXCR4 were detected in 24% (7/29) of patients. The pathogenic mutation S338X (rs104893626) was present in 2 (7%) patients and was identified in both cfDNA and tDNA. In one patient the L50X (1/29) truncating mutation was found in tDNA but not in cfDNA. Two patients harbored the F29L (2/29) mutation and another two (2/29) the P31T mutation, both in cfDNA and in tDNA; these two mutations have not been previously reported. Synonymous mutations were also found in 10% of the tested patients in both cfDNA and tDNA. Thus, the concordance for CXCR4 mutations identified by direct sequencing of cfDNA and tDNA was 97% (in 28/29 patients).


Our study demonstrates that in patients with IgM monoclonal gammopathies peripheral blood cell-free DNA can be used as a non-invasive method to detect the MYD88 L265P mutation with excellent concordance to BM CD19+ derived tumor DNA. Furthermore, CXCR4 mutations could also be detected with high concordance to tDNA by direct sequencing. This method could be more cost effective, as it avoids the expenses and time needed for CD19 selection, either BM or peripheral blood derived. Peripheral blood cfDNA could be used as an initial test to assess the presence of MYD88 L265P and CXCR4 mutations, so that BM testing can be limited to patients with indeterminate results. Updated results will be presented in the meeting. Further investigation is ongoing in order to assess tumor dynamics using peripheral blood.


Terpos: Janssen: Honoraria, Research Funding; Genesis/Celgene: Honoraria, Other: DMC member, Research Funding; Abbvie: Honoraria; Takeda: Honoraria, Other: SC member; Amgen: Honoraria, Other: SC member, Research Funding; GSK: Honoraria; BMS: Honoraria. Kastritis: Prothena: Honoraria; Janssen: Honoraria; Genesis pharma: Honoraria; Takeda: Honoraria; Amgen: Honoraria, Research Funding. Dimopoulos: Amgen Inc, Celgene Corporation, Janssen Biotech Inc, Onyx Pharmaceuticals, an Amgen subsidiary, Takeda Oncology: Consultancy, Honoraria, Other: Advisory Committee: Amgen Inc, Celgene Corporation, Janssen Biotech Inc, Onyx Pharmaceuticals, an Amgen subsidiary, Takeda Oncology; Novartis: Consultancy, Honoraria; Genesis Pharma: Research Funding.

Author notes


Asterisk with author names denotes non-ASH members.