Serum CXCL13 Level And MYD88^L265P Mutation In The Diagnosis And Disease Monitoring Of Waldenstrom Macroglobulinemia

Background and ObjectivesWaldenstrom macroglobulinemia(WM)is an indolent B cell lymphoma characterized by serum monoclonal IgM immunoglobulin.MYD88L265P and CXCR4s338X are two recurrent somatic hypermutations observed in WM patients.In clinical practice,it is difficult to differentiate WM from other indolent B cell lymphomas with monoclonal IgM immunoglobulin.Recently,Vos et al has reported an elevated serum CXCL13 level in WM patients,which might be helpful for the diagnosis of WM.We aimed to explore the value of CXCL13 in the diagnosis and assessment of WM patients,as well as the role of CXCL13 in the pathogenesis of WM.Circulating tumor DNA(ctDNA)has been proved to be a noninvasive and sensitive method in the mutation analysis and disease monitoring of many kinds of solid tumors and lymphomas.We extracted cell free DNA(cfDNA)from peripheral blood of WM patients,and detected MYD88L265P and CXCR4S338X mutations by Real-time AS-PCR.Compared to paired tumor-derived DNA,we analyzed the sensitivity and specificity of detecting MYD88L265P mutation in cfDNA,to lay the foundation for further application of cfDNA in the mutation screening and minimal residual disease monitoring in Waldenstrom macroglobulinemia.MethodsWe collected samples of WM(n=132),DLBCL with monoclonal IgM(n=13),other indolent B cell lymphoma with monoclonal IgM(n=14)and IgM-MGUS(n=10)patients from Peking Union Medical College Hospital from January 2015 to May 2018.Serum CXCL13 level were detected and analyzed combining with patients’ clinical profiles.Sixteen paraffin-embedded bone marrow tissues of WM and seven control paraffin-embedded bone marrow tissues of DLBCL(n=2),other indolent B cell lymphoma(n=2)and MGUS(n=3)patients with monoclonal IgM immunoglobulin were sectioned and stained with anti-human CXCL13 antibody,anti-human CD117 antibody and anti-human mast cell tryptase antibody.5 fresh bone marrow samples from WM patients were used for chemotaxis assay in vitro.cfDNA of 31 peripheral samples from WM patients,1 from a WM patient with transformation to DLBCL,1 from a DLBCL patient were extracted.MYD88L265P and CXCR4S338X mutations were detected by Real-time AS-PCR and compared to paired tumor-derived DNA.ResultsMedian serum level of CXCL13 was 2483.0(36.8-5644.0)pg/ml in untreated WM patients,which was significantly higher than that in sWM(35.6,27.7-549.6)pg/ml(p=0.018),IgM-MGUS patients(33.5,20.8-873.3)pg/ml(p<0.0001)and healthy donors(26.6,14.0-98.3)pg/ml(p<0.0001).Median serum level of CXCL13 were 1057.0(105.0-5204.0)pg/ml(p<0.0001)and 380.9(23.8-5518.0)pg/ml(p<0.0001)respectively in DLBCL and other indolent B cell lymphoma with monoclonal IgM,which were also significantly higher than that in healthy donors.Comparing untreated WM with other indolent B cell lymphomas with monoclonal IgM,CXCL13 was significantly higher in untreated WM patients(p=0.0099).And combining serum CXCL13 and serum M protein in the diagnosis of WM,AUC index of performance was 0.935.Patients with serum CXCL13>3250 pg/ml and serum M protein>38 g/L were diagnosed as WM with sensitivity of 98%and specificity of 85%.In WM patients,serum CXCL13 strongly correlated with hemoglobin levels(rho=0.49,p<0.0001),serum M protein(rho=0.47,p<0.0001)and IgM levels(rho=0.43,p<0.0001).After treatment,serum CXCL13 showed significant decrease;and changes in CXCL13 correlated with changes in serum M protein level.We observed that serum CXCL13 decreased earlier than M protein at the beginning of treatment and was not affected by IgM flare.By immunohistochemistry staining,we found out that CXCL13 level and number of CD117/Tryptase+ mast cells in the bone marrow of WM patients were higher than those observed in other indolent B cell lymphoma and MGUS patients.In vitro,CXCL13 induced migration of CD 19+ tumor cells and CD 117+ mast cells in WM patients.Total median cfDNA amount extracted from 31 WM patients was 95.7(19.6-4330.8)ng.cfDNA from the WM patients with DLBCL transfromation was 2054.0 ng.cfDNA from the DLBCL patient was 114.8 ng.Analyzing MYD88L265P detection by Real-time AS-PCR in 28 patients who had paired tumor-derived DNA samples,the coincidence rate of cfDNA with paired tumor-derived samples was 92.9%.The sensitivity and specificity of detecting MYD88L265P mutation in cfDNA were 95%and 83%respectively.ConclusionsSerum CXCL13 is significantly elevated in WM patients,and correlates with tumor load.Detection of serum CXCL13 is helpful for the diagnosis of WM.After treatment,serum CXCL13 decreases in accord with serum M protein,and is more sensitive and not affected by IgM flare.CXCL13 is useful in the assessment of WM.CXCL13 level and the number of mast cells found in bone marrow of WM patients are higher than in the bone marrow of other indolent B cell lymphoma and MGUS patients with monoclonal IgM.In vitro,CXCL13 induces WM cells migration.CXCL13 chemostaxis and mast cell infiltration may be related to the pathogenesis of WM.It is feasible to detect MYD88L265P and CXCR4S338X mutations in cfDNA by Real-time AS-PCR.The sensitivity and specificity of detecting MYD88L265P mutation in cfDNA are 95%and 83%respectively.cfDNA offers a convenient and less invasive way for the mutational screening and minimal residual disease monitoring in Waldenstrom macroglobulinemia.