IgM Monoclonal Gammopathy Research Articles

MYD88 Mutations: Transforming the Landscape of IgM Monoclonal Gammopathies.

The MYD88 gene has a physiological role in the innate immune system. Somatic mutations in MYD88, including the most common L265P, have been associated with the development of certain types of lymphoma. MYD88L265P is present in more than 90% of patients with Waldenström’s macroglobulinemia (WM) and IgM monoclonal gammopathy of undetermined significance (IgM-MGUS). The absence of MYD88 mutations in WM patients has been associated with a higher risk of transformation into aggressive lymphoma, resistance to certain therapies (BTK inhibitors), and shorter overall survival. The MyD88 signaling pathway has also been used as a target for specific therapies. In this review, we summarize the clinical applications of MYD88 testing in the diagnosis, prognosis, follow-up, and treatment of patients. Although MYD88L265P is not specific to WM, few tumors present a single causative mutation in a recurrent position. The role of the oncogene in the pathogenesis of WM is still unclear, especially considering that the mutation can be found in normal B cells of patients, as recently reported. This may have important implications for early lymphoma detection in healthy elderly individuals and for the treatment response assessment based on a MYD88L265P analysis.

Revisiting the spectrum of IgM-related neuropathies in a large cohort of IgM monoclonal gammopathy.

A significant number of patients with a peripheral neuropathy have IgM monoclonal gammopathy (IgM-MG). In this work, we encompassed the spectrum and outcome of IgM-related neuropathies (IgM-NP) in a large monocentric cohort of patients with IgM-MG. We retrospectively reviewed the neurological and hematological findings and the course of neuropathy in all patients with IgM-MG over a five-year period in our center (Henri Mondor hospital, Assistance Publique Hôpitaux de Paris (APHP), France). Among 550 patients with IgM-MG, 83 patients (15%) had IgM-NP (55 males, mean age 67 y.o.). The median serum level of IgM-MG was 3.4g/L, mostly kappa light chain component. The hematological diagnosis was Monoclonal Gammopathy of Undetermined Significance (MGUS) in 62 patients. Anti-MAG antibodies were detected in 38 patients with heterogeneous clinical and neurophysiological features. Four patients had neurolymphomatosis presenting as a non-length dependent predominantly motor neuropathy, which occurred long after the finding of IgM-MG and was responsive to hematological treatment. Five patients had an AL amyloid neuropathy revealed by a small fiber neuropathy. Finally, 30 patients were classified as "Neuropathy of Uncertain Relationship with the IgM" (NURIM) with characteristics close to those of an anti-MAG-NP at the time of diagnosis, except for the neurophysiological features with a predominant axonal pattern. This study emphasizes the wide spectrum of IgM-NP associated with a variety of hematological diagnoses. In particular, the course and prognosis vary considerably. In this setting, further studies are needed to unravel the group of patients classified as NURIM.

MO256: IGM in Fibrillary Glomerulopathy Linked to Monoclonal Gammopathy

Abstract BACKGROUND AND AIMS Fibrillary glomerulopathy (FGN) is a rare glomerular deposit disease accounting for less than 1% of annual native kidney biopsies. Its pathogenesis is still not fully understood. Immunofluorescence (IF) techniques in the vast majority of cases reveal the presence of smudgy polytypic IgG deposits associated with C3 and, anecdotally, of scarce IgM or IgA. The majority of cases are idiopathic, but up to one-third of patients may present with a secondary cause including monoclonal gammopathy, which in previously reported series ranged from 4% to 16% of the cases. The association of FGN and monoclonal gammopathy remains controversial, nonetheless monotypic cases in the absence of multiple myeloma, wäldestrom macroglobulinemia or high-grade lymphoma are currently included in the spectrum of monoclonal gammopathy of renal significance (MGRS). The aim of the study was to describe the role of IgM in FGN as a possible marker of an underlying MGRS. METHOD A single-center, retrospective and observational study of FGN cases diagnosed during the period of 2012–2019 was performed. Kidney biopsies were retrospectively reviewed. All cases had a confirmatory diagnosis made with electron microscopy (EM). Due to a lack of material and the novelty of the marker, DNAJB9 was not performed in all samples. IF on frozen tissue was done in all cases; digestion with proteinase K was done in cases of light chain restriction to unmask possible deposits. Demographics, clinical presentations, associated conditions and outcomes were reviewed. Data were expressed as mean standard deviation (SD). RESULTS Our cohort consisted of 17 patients, all but 2 caucasians, 9 males and 8 women, with a mean age of 63.1 ± 13.2. The mean follow-up was of 34.6 months. At the time of the diagnosis, most patients presented with hypertension 70% (12). All patients had proteinuria (mean 24-h protein excretion of 4.84 g ± 5.2) and renal impairment with a medium creatinine of 203.8 ± 202. Full nephrotic syndrome was present in 29% (5) of the cases and microhematuria in 52.9% (9); complement was normal in all cases. As for related medical conditions: 23% (4) had history of autoimmune diseases, 17% (3) had diagnosis of a solid tumor, 76.5% (13) were smokers (2 with diagnosed chronic obstructive pulmonary disease) and 29% (5) had diabetes mellitus. Monoclonal gammopathy was present in 35.3% (6/17) of the cases, all of which had an identified monoclonal component detected by electrophoresis with immunofixation (SPEPE/SIF). Three of them (50%) progressed (2 to multiple myeloma, 1 to amyloidosis AL) (Fig. 1). Light microscopic study showed three distinct patterns of glomerular injury, being mesangial pattern the most common 82% (14/17). Staining with congo red was negative in all cases, 17 cases. By immunofluorescence only 82% (13) showed glomerular positivity for IgG. Unexpectedly 23% (4) showed exclusively IgM deposits; three of them had monoclonal gammopathy. Looking in depth, 5/6 of the patients with monoclonal gammopathy showed IgM deposits in contrast to the non-associated FGN cases (3/10) (Fig. 2). No cases of GNF with IgM deposits had underlying cryoglobulinemia; there was one case of diabetes mellitus. A total of 41% (7) end up needing renal replacement therapy (RRT). No differences in the rate of RRT were observed between patients with monoclonal gammopathy and without. CONCLUSION IgM deposits were observed in patients with FGN cases associated with monoclonal gammopathy. IgM deposition is a finding rarely described in the literature up to the date. In this regard, we hypothesize that IgM monoclonal gammopathy due to a conformational alteration in the protein could generate deposits in the form of fibrils triggering a possible GNF. Thus, IgM deposition in the context of GNF could be a marker of a possible underlying MGRS. Future studies proving IgM conformational alterations in these patients could corroborate our hypothesis.

Nucleic Acid Biomarkers in Waldenström Macroglobulinemia and IgM-MGUS: Current Insights and Clinical Relevance

Waldenström Macroglobulinemia (WM) is an indolent lymphoplasmacytic lymphoma, characterized by the production of excess immunoglobulin M monoclonal protein. WM belongs to the spectrum of IgM gammopathies, ranging from asymptomatic IgM monoclonal gammopathy of undetermined significance (IgM-MGUS), through IgM-related disorders and asymptomatic WM to symptomatic WM. In recent years, its complex genomic and transcriptomic landscape has been extensively explored, hereby elucidating the biological mechanisms underlying disease onset, progression and therapy response. An increasing number of mutations, cytogenetic abnormalities, and molecular signatures have been described that have diagnostic, phenotype defining or prognostic implications. Moreover, cell-free nucleic acid biomarkers are increasingly being investigated, benefiting the patient in a minimally invasive way. This review aims to provide an extensive overview of molecular biomarkers in WM and IgM-MGUS, considering current shortcomings, as well as potential future applications in a precision medicine approach.

Refractory immune thrombocytopenic purpura associated with IgM monoclonal gammopathy of undetermined significance: Successful treatment with tirabrutinib plus conventional therapies.

When immune thrombocytopenia (ITP) is secondary to malignant diseases, chemotherapy is expected to improve the platelet count (PC) as well. Herein, we report a case of a 72‐year‐old man with ITP refractory to standard therapies. IgM monoclonal gammopathy of undetermined significance (MGUS) was determined as an underlying disease. After bendamustine and rituximab (BR) therapy was found inadequately effective, tirabrutinib, a novel Bruton's tyrosine kinase inhibitor, was initiated, and the PC normalised subsequently. Surveillance of underlying diseases with which effective therapies are available may help manage refractory ITP, and IgM‐MGUS is potentially a targetable underlying disease with this newly available drug.

TEMPI syndrome associated with IgM monoclonal gammopathy

TEMPI syndrome associated with IgM monoclonal gammopathy

Serum neurofilament light chain, contactin-1 and complement activation in anti-MAG IgM paraprotein-related peripheral neuropathy

IntroductionIn anti-myelin-associated glycoprotein IgM paraprotein-related peripheral neuropathy (anti-MAG PN), there is a lack of reliable biomarkers to select patients eligible for therapy and for evaluating treatment effects, both in routine practice and in clinical trials. Neurofilament light chain (NfL) and contactin-1 (CNTN1) can serve as markers of axonal and paranodal damage. Complement activation is involved in the pathogenesis in anti-MAG PN. We, therefore, hypothesized that serum NfL, CNTN1, C3b/c and C4b/c may function as biomarkers of disease activity in anti-MAG PN.MethodsIn this prospective cohort study, we included 24 treatment-naïve patients with anti-MAG PN (mean age 69 years, 57% male) that had IgM paraproteinemia, a high IgM MAG-antibody, and clinical diagnosis of anti-MAG PN by a neurologist specialized in peripheral nerve disorders. We measured serum NfL, CNTN1, C3b/c and C4b/c, reference values were based on healthy controls. As controls, 10 treatment-naïve patients with IgM Monoclonal gammopathy of undetermined significance (MGUS) or Waldenström’s Macroglobulinemia (mean age 69 years, 60% male) without signs of neuropathy were included (non-PN).ResultsNfL, CNTN1 levels in serum were mostly normal in anti-MAG PN patients and comparable to non-PN patients. C3b/c and C4b/c levels were normal in anti-MAG PN patients.ConclusionOur results do not support serum NfL, CNTN1, and C3b/c and C4b/c as potential biomarkers in anti-MAG PN, although we cannot exclude that subgroups or subtle abnormalities could be found in a much larger cohort with longitudinal follow-up.

Clonal hematopoiesis is associated with increased risk of progression of asymptomatic Waldenström macroglobulinemia

AbstractClonal hematopoiesis (CH) is associated with adverse outcomes in patients with non-Hodgkin lymphoma (NHL) and multiple myeloma undergoing autologous stem cell transplantation. Still, its implications for patients with indolent NHL have not been well studied. We report the prevalence of CH in patients with Waldenström macroglobulinemia (WM) and its association with clinical outcomes. To unambiguously differentiate CH mutations from those in the WM clone, CH was defined by the presence of somatic mutations in DNMT3A, TET2, or ASXL1 (DTA) and was detected in 14% of 587 patients with IgM monoclonal gammopathy of undetermined significance (MGUS), smoldering WM (SWM) or WM. The presence and size of DTA clones were associated with older age. Patients with CH had an increased risk of progression from MGUS or SWM to WM, but not worse overall survival in this cohort. These findings further illuminate the clinical effects of CH in patients with indolent NHL such as WM.

Prognostic Impact of MYD88 L265P Mutation By Droplet Digital PCR in IgM MGUS and Smoldering Waldenström Macroglobulinemia

BACKGROUND: MYD88 L265P mutation is highly prevalent in IgM monoclonal gammopathy of undetermined significance (MGUS), smoldering Waldenström macroglobulinemia (SWM) and symptomatic WM. Allele-specific PCR (AS-PCR) has been used routinely to assess MYD88 mutation; however, with the advent of more precise high-throughput technologies such as droplet digital PCR (ddPCR), absolute quantification can be achieved. There is no data regarding ddPCR applicability in asymptomatic IgM monoclonal gammopathies or as a prognostic biomarker. Here, we aimed to compare MYD88 quantification by ddPCR with clinical and laboratory features and to analyze the prognostic impact in a series of patients (pts) with IgM MGUS and SWM.METHODS: We analyzed bone marrow (BM) and peripheral blood (PB) samples stored from pts diagnosed with IgM MGUS and SWM at our institution from 1980 to 2020. DNA extraction methods followed manufacturer instructions (Qiagen) to obtain genomic DNA from unsorted BM samples and cell-free DNA (cfDNA) from PB. MYD88 L265P mutation was quantified by ddPCR using a Bio-Rad commercial assay (HEX-labeled wild-type allele; FAM-labeled mutant allele). We used OCI-Ly3 DLBCL ABC cell line, homozygous for MYD88 L265P, as a positive control. ddPCR was performed following Bio-Rad technical specifications using the QX200 droplet reader. Data was analyzed using QuantaSoft v.1.0 software (Bio-Rad). Absolute quantification of the mutation was expressed as percentage of fractional abundance. For survival analysis, we used a competing risk analysis to evaluate the prognostic impact of MYD88 mutation on progression to symptomatic WM.RESULTS: A total of 217 unsorted samples were analyzed (187 BM and 30 PB). Genomic DNA from unsorted BM samples was extracted from pts diagnosed with IgM MGUS (46%), SWM (44%), and symptomatic WM (10%). cfDNA was obtained from a subgroup of pts with IgM MGUS (52%) and SWM (48%). Median age at diagnosis was 68 (range 61 to 76). AS-PCR could detect the mutation in 22 (31%) pts with IgM MGUS and 49 (75%) with SWM. ddPCR improved precision detection up to 48 (55%) pts with IgM MGUS and 68 (83%) with SWM. All pts with symptomatic WM harbored the MYD88 mutation, as identified by both techniques. Median absolute quantification from BM was 2.3% and 7% for pts with IgM MGUS and SWM, respectively (p<0.001).Pearson correlation coefficients comparing BM MYD88 mutation quantification by ddPCR with serum M-protein size, IgM concentration, BM lymphoplasmacytic infiltration rate and BM CD19+ cells were 0.3, 0.4, 0.6, and 0.9 (p<0.0004), respectively. Similar coefficients were observed in symptomatic WM regarding BM infiltration rate (0.6; p=0.001) and BM CD19+ cells (0.9; p<0.0001). Spearman correlation coefficients comparing cfDNA MYD88 mutation quantification with BM lymphoplasmacytic infiltration rate and BM CD19+ cells were 0.4 and 0.5 (p<0.008), respectively. Agreement regarding MYD88 mutation detection by ddPCR in BM DNA and cfDNA samples was 82% (Cohen kappa index 0.6).With a median overall survival of 13 years in pts with IgM MGUS and SWM, 13% of them progressed to symptomatic WM while 22% died without progression. Cox univariate analysis using continuous values for MYD88 quantification (p=0.004), serum IgM (p<0.001), BM lymphoplasmacytic infiltration (p<0.001), and serum albumin (p=0.04) were significant. X-tile software was used to find the optimal cutoff point of MYD88 quantification as a biomarker. 4.5% was established for pts with IgM MGUS while 25% for SWM. Using the Fine and Gray regression model in a competing risk analysis taking death without progression as a competing event, higher MYD88 mutation burden negatively impacted the risk of progression of IgM MGUS (SHR 4.6; p=0.003) and SWM (SHR 6; p<0.001) (Figure 1).CONCLUSION: Quantification of the MYD88 L265P mutation by ddPCR has higher precision and sensitivity compared to AS-PCR; thus ddPCR could be used as a potential new and useful biomarker. MYD88 tumor burden correlated with well-known laboratory parameters used for diagnosis and risk stratification, whether using genomic DNA from unsorted BM samples or cfDNA. Risk of progression was higher in patients harboring an increased mutant allele burden. This is the first report showing the prognostic impact of MYD88 quantification in a series of patients with asymptomatic IgM gammopathy and long-term follow up. [Display omitted] DisclosuresCibeira: Akcea: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees. Bladé Creixenti: Janssen, Celgene, Takeda, Amgen and Oncopeptides: Honoraria. Rosinol: Janssen, Celgene, Amgen and Takeda: Honoraria. Fernandez de Larrea: BMS: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Takeda: Honoraria, Research Funding; GSK: Honoraria; Sanofi: Consultancy; Janssen: Consultancy, Honoraria, Research Funding.

Evaluating Front Line Treatment Regimens for Waldenstrom Macroglobulinaemia: A Systematic Review and Meta-Analysis

BackgroundWaldenstrom macroglobulinemia (WM) is a rare haematological malignancy characterised by infiltration of the bone marrow by clonal lymphoplasmocytic cells and serum monoclonal IgM gammopathy. Rituximab based chemo-immunotherapy has long been the backbone of WM treatment. Bendamustine rituximab (BR), cyclophosphamide, dexamethasone rituximab (CRD) and bortezomib dexamethasone rituximab (BDR) are currently considered frontline options. The Bruton tyrosine kinase inhibitor (BTKi) ibrutinib in combination with rituximab (IR) has recently shown efficacy in treatment naïve WM and has been approved for use in this setting. With the expanding number of treatment options and absence of randomised trials (RCTs) comparing IR with chemo immunotherapy, there is uncertainty as to the optimal frontline treatment for WM.MethodsThe aim of this systematic review was to compare the efficacy and safety of rituximab-based chemoimmunotherapy combinations and IR, in treatment naïve WM. Using a pre-specified protocol in accordance with Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA), MEDLINE, Embase, BIOSIS and Cochrane Library databases were searched for articles published from inception of each database to present. We included trials studying newly diagnosed WM patients treated with rituximab-based chemo-immunotherapy or BTKi based regimens. Primary outcome measures were progression free and overall survival (PFS and OS) while secondary outcomes included response rates and adverse events. A meta-analysis of proportions (expressed as a percentage), with their 95% CI were calculated.ResultsOf the 753 unique records identified, 8 single arm studies, one phase II non-randomised non inferiority study and 3 Phase III RCTs met the inclusion criteria. The sample size ranged from 23 to 261. PFS at 1- and 2-years were higher in BR(91%, 87%) and IR (93%, 82%), compared to BDR (87.5%, 66.8%) and CRD (82%, 64%) . Combined complete response (CR) and very good partial response (VGPR) rates were also superior with BR (35%) and IR ( 26%) compared to BDR (9%), Bortezomib- Rituximab (8% ) and CRD ( 7%) (Figure 1). The bortezomib bendamustine rituximab ( BBR) regimen was evaluated in only one study but showed an impressive CR/VGPR rate of 47% and a PFS at 3 years of 75%. Bleeding and atrial fibrillation were more common with IR, while neuropathy was more prevalent with bortezomib based regimens.ConclusionWe present the first systematic review and meta-analysis evaluating frontline treatment options for WM in the BTKi era. Our findings suggest that bendamustine-based regimens may be superior in terms of depth of response and PFS. Longer follow up of the IR treated patients on the INNOVATE trial is however required to perform a definitive comparison of the regimens. The differential efficacy of these treatments in genomic subtypes of WM (based on MYD88 and CXCR4 mutational status) is an important question to be answered in future trials. While chemo-immunotherapy remains a potent, fixed duration option for newly diagnosed WM, BTKi based regimens are a valid alternative for patients who prefer an oral treatment or who are unfit for cytotoxic therapy. With the expanding number of treatment options for WM, RCTs comparing BTKi/immunotherapy with chemo-immunotherapy regimens are called for. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Clonal Hematopoiesis Is Associated with Increased Risk of Progression of Asymptomatic Waldenström Macroglobulinemia

▪IntroductionClonal hematopoiesis (CH) is associated with adverse outcomes in patients with non-Hodgkin lymphoma (NHL) and multiple myeloma undergoing autologous stem cell transplantation. Still, its implications for patients with indolent NHL have not been well studied. Here, we report the prevalence of CH in patients with Waldenström Macroglobulinemia (WM) and its association with clinical outcomes.MethodsWe retrospectively reviewed clinical data of 602 patients with IgM monoclonal gammopathy of undetermined significance (MGUS), smoldering WM (SWM), and WM who had clinical next-generation sequencing (NGS) performed on bone marrow aspirates or peripheral blood obtained between October 2014 and February 2020 at the Dana-Farber Cancer Institute. An Illumina Truseq amplicon-based NGS assay of 95 genes recurrently mutated in myeloid and lymphoid neoplasms was utilized. Each specimen yielded ~2 million reads and ~1500X average coverage, with 90% of amplicons having >200X coverage. Pathogenic driver variants were identified based on mutation type, position, and frequency in published reports and public databases. To unambiguously differentiate CH mutations from those in the WM clone, CH was defined by the presence of somatic mutations in DNMT3A, TET2, or ASXL1 (DTA).ResultsThe cohort included 147 patients with MGUS or asymptomatic WM and 453 patients with symptomatic WM, with a median age of 66 years (range = 40-89) and 68 years (range = 33-93), respectively, at the time of first NGS assay. The prevalence of CH-DTA was 14% in symptomatic WM patients and did not differ significantly in MGUS (13%) or SWM (14%). Among precursor patients, there was an increased risk of progression to symptomatic WM for those with CH-DTA [7/20 patients with vs. 11/116 without CH-DTA progressed over a median of 54 months (P = 0.002)]. In symptomatic WM patients, CH-DTA was positively associated with older age (P < 0.001) at the time of NGS, with a median age of 72 vs. 67 years for patients with versus those without CH-DTA. CH-DTA was not associated with inferior overall survival (OS) with a relatively short median follow-up from diagnosis and NGS assay. OS was 6.7 years (95% CI = 6.1-7.6) for patients with CH-DTA and 2.5 years for patients without DH-DTA (95% CI = 2.2-2.8). The most common cause of death was disease progression, with no significant difference between those with or without CH-DTA. Patients with CH-DTA had an increased risk of cardiovascular disease (30% vs. 18%, P = 0.036).ConclusionsWe demonstrate that CH is common in WM patients and is associated with an increased risk of progression from precursor states but not with inferior survival. Further work is needed to determine how the presence of CH might promote progression to WM and whether it can be incorporated into future risk stratification models. Importantly, our data do not support changes in clinical management or alterations in therapy for patients with WM and coexistent CH and reinforce the need to interpret NGS results within their specific clinical context. DisclosuresSteensma: Novartis: Current Employment. Castillo: Abbvie: Consultancy, Research Funding; BeiGene: Consultancy, Research Funding; Pharmacyclics: Consultancy, Research Funding; Janssen: Consultancy; Roche: Consultancy; TG Therapeutics: Research Funding. Treon: X4: Research Funding; Dana Farber Cancer Institute: Current Employment; Janssen: Consultancy, Research Funding; Pharmacyclics: Consultancy, Research Funding; BeiGene: Consultancy, Research Funding; Self: Patents & Royalties: Holder of multiple patents related to testing and treatment of MYD88 and CXCR4 mutated B-cell malignancies; BMS: Consultancy, Research Funding; AbbVie: Consultancy, Research Funding. Ghobrial: AbbVie, Adaptive, Aptitude Health, BMS, Cellectar, Curio Science, Genetch, Janssen, Janssen Central American and Caribbean, Karyopharm, Medscape, Oncopeptides, Sanofi, Takeda, The Binding Site, GNS, GSK: Consultancy. Sperling: Adaptive: Consultancy.

The Genomic Landscape of Waldenström Macroglobulinemia Reveals Sustained Germinal Center Activity and Late-Developing Copy Number Aberrations

The Genomic Landscape of Waldenström Macroglobulinemia Reveals Sustained Germinal Center Activity and Late-Developing Copy Number Aberrations

Common Gene Expression Signature of B-Cells of Waldenström Macroglobulinemia (WM) and IgM Monoclonal Gammopathies of Undetermined Significance (IgM MGUS) Compared to Healthy Subjects

We performed a comparative gene expression profiling (GEP) study on B-cells and plasma cells of Waldenström Macroglobulinemia (WM), IgM monoclonal gammopathies of undetermined significance (IgMMGUS), and normal individuals (CTRLs) to identify GEP changes as reliable predictors of progression of IgMMGUS to WM.We analyzed bone marrow B-cells and plasma cells from 36 WM patients, 13 IgMMGUS subjects, and 7 CTRLs by Affymetrix microarray, respectively (Table 1). GEP experiments were performed on the CD19+ and CD138+ cells using GeneChip-HGU133 Plus 2.0. Data were preprocessed and normalized by Robust Multi-Array Average and ComBat. Selection of the different expressed genes was performed separately for CD19+ and CD138+ cells, using Significance Analysis of Microarrays (SAM) on the 3 groups and a false discovery rate threshold of 5%, followed, for significance comparisons, by a pair-wise SAM test corrected for multiple testing.We focused on the comparison of the CD19+ cells of WM vs. IgMMGUS vs. CTRLs which highlighted 2038 unique genes whereas the same comparison of the CD138+ cells determined 29 unique genes (Trojani et.al. Cancers 2021). Among the 2038 DEGs, 115 genes were grouped in KEGG pathways involved in Wnt-signaling, BCR-signaling, calcium signaling, hematopoietic cell antigens, cell adhesion, adherens junctions, coagulation cascade, platelet activation, cytokine receptor, and signaling pathways responsible for cell cycle, apoptosis, and survival.Interestingly, most of the 115 DEGs in B-cells were different expressed in WM vs. IgMMGUS and CTRLs. Only 9/115 DEGs were significantly different expressed in WM vs. CTRLs and in IgMMGUS vs. CTRLs, but no significant expression changes were noted between WM and IgMMGUS (Table 2).To further inspect the similarities and the differences among WM and IgMMGUS, we computed the Euclidean pair-wise distance between subjects and, using this distance as weight, constructed a minimum spanning tree (MST) (Figure 1).Considerably, four probesets identified ADRB2 (transmembrane Beta adrenergic receptor) which was up regulated in WM and IgMMGUS compared to CTRLs. The over expression of ADRB2 was also demonstrated in Mantle Cell Lymphoma cell lines and in Diffuse large B-cell lymphoma (DLBCL) lymphocytes compared to normal B-cells (doi10.1016/j.cellsig.2017.08.002), and in most malignancies (doi10.1007/s11033-021-06250-y) .As far as we know, ADAM23 (ADAM Metallopeptidase Domain23) has not been found in WM, whereas we suggest its possible role in WM patients with Sjogren's syndrome (SS). ADAM23 plays a role in the peripheral neuropathy by controlling the activity of potassium channels in SS (doi10.1007/s10067-016-3499-z). Some authors found that sensory/motor neuropathies were associated with MGUS patients (doi10.1017/s0317167100011483). We strongly believe that the down regulation of ADAM23 in WM and IgMMGUS has a good chance to be associated with clinical neuropathy in WM and IgMMGUS.RASGRP3 (RAS Guanyl Releasing Protein3) and PIK3AP1 (Phosphoinositide 3 Kinase Adaptor Protein1) play crucial roles in BCR signaling pathway: PIK3AP1 activates the PI3K-Akt signaling while RASGRP3 stimulates MAPK signaling pathway.The deregulation of LEF1 (Lymphoid Enhancer Binding Factor1) and genes of Wnt-pathway were previously demonstrated in B-cell disorders and multiple myeloma (doi10.1007/s00277-017-3207-3, doi10.1016/j.pathol.2019.09.009). According to these studies, we showed the under expression of LEF1 in WM and IgMMGUS compared to CTRLs.We identified the down regulation of EZH2 (Enhancer Of Zeste2 Polycomb Repressive Complex2Subunit) in WM and IgMMGUS compared to CTRLs. EZH2 is involved in Follicular lymphoma and DLBCL (doi10.1080/2162402X.2017.1321184).CDHR3 (Cadherin Related Family Member3), CHEK1 (Checkpoint Kinase1), and HIST1H1B (Histone-H1.5) were over expressed in CTRLs compared to IgMMGUS and WM.In conclusion, the common gene-set in WM and IgMMGUS could suggest two-hit hypothesis. First, the gene-set could play a role in the risk of progression of IgMMGUS to WM. Until now, all the IgMMGUS subjects have not been transformed in WM or other NHL, but they have been monitored every 6 months, and their possible transformation to lymphoma could highlight new insights. The second hypothesis suggests their involvement in the biological processes of leukemogenesis in WM and IgMMGUS which will be further investigated. [Display omitted] DisclosuresTedeschi: AbbVie: Honoraria, Speakers Bureau; AstraZeneca: Honoraria, Speakers Bureau; Beigene: Honoraria, Speakers Bureau; Janssen: Honoraria, Speakers Bureau.

P-034: MAGNAZ trial - A prospective phase II study in patients with monoclonal gammopathy of unknown significance (MGUS) and anti-Myelin Associated Glycoprotein (MAG) Neuropathy and Zanubrutinib Treatment

<h3>Introduciton</h3> Polyneuropathy (PNP) associated with IgM monoclonal gammopathy (MGUS), also called IgM-related PNP, is mediated by the anti-neural effect of the M-protein component and is classified as one of the MGUS-related diseases. In around 70% of patients with IgM-related PNP anti-myelin associated glycoprotein (MAG) antibodies are detected. There is no established treatment for IgM-related PNP except anti-CD20 monoclonal antibody treatment with 30% or less clinical responses. There is increasing interest to use Bruton's tyrosine kinase (BTK) inhibitors, approved for the treatment of Waldenstrom Macroglobulinemia, for IgM-related PNP treatment but a formal study is currently lacking. We therefore designed a phase 2 clinical trial to investigate the effect of zanubrutinib, a next generation BTK inhibitor, combined with anti-CD20 monoclonal antibody treatment, in IgM-related PNP with anti MAG antibodies. The primary study endpoint is change from baseline in the Rasch-built Overall Disability Scale (RODS) for inflammatory neuropathies (iRODS) at the end of Cycle 12. The main secondary endpoint is to assess the safety of zanubrutinib treatment in IgM-related PNP as measured by CTCAE, version 5.0. <h3>Methods</h3> Patients will be treated for a minimum of 6 cycles; patients experiencing hematological response continue until 12 cycles of treatment. All patients will be followed for the duration of 12 cycles for the primary endpoint analysis. Patients who have an anti MAG titer > 10.000 BTU, adequate hematological, renal and hepatic function tests, no hemorrhagic disorder and no New York Heart Association (NYHA) grade 3 or 4 cardiac disease can be included after signing informed consent. Explorative analysis will consist of Next Generation Sequencing of MYD88 and CXCR4 mutations after CD19 selection of the bone marrow aspirate at start. During study participation extensive neurological testing and serum IgM and anti MAG testing will be performed. In total 40 patients will be included and the MAGNAZ study expects to start in Q4 2021.

Antibody testing in neuropathy associated with anti-Myelin-Associated Glycoprotein antibodies: where we are after 40 years

The diagnosis of Myelin-Associated Glycoprotein (MAG) neuropathy is based on the presence of elevated titers of IgM anti-MAG antibodies, which are typically associated with IgM monoclonal gammopathy, and a slowly progressive, distal demyelinating phenotype. The condition, however, can be under or over diagnosed in patients with mildly elevated antibody titers, absent monoclonal gammopathy, or an atypical presentation. The purpose of this paper is to examine recent advances in our understanding of the currently available anti-MAG antibody assays, their reliability, and their use in deciding treatment or monitoring the response to therapy. Higher titers of anti-MAG antibodies are more likely to be associated with the typical MAG phenotype or response to therapy. Mildly elevated antibody levels can occur in patients with chronic inflammatory demyelinating polyneuropathy. Testing for cross-reactivity with HNK1 can add to the specificity of the antibody assays. Patients with MAG neuropathy can present with an atypical phenotype and in the absence of a detectable monoclonal gammopathy. Assays for anti-MAG antibodies by Enzyme-Linked Immunosorbent Assay can be improved by testing for antibody binding at multiple serum dilutions, the inclusion of antigen-negative microwells as internal controls for each sample, testing for cross-reactivity with HNK1, and formal validation. The diagnosis needs to be considered in patients with demyelinating neuropathy, even in the absence of a monoclonal gammopathy or typical phenotype. The change in antibody levels needs to be considered in evaluating the response to therapy with B-cell depleting agents.

SY7.5. Diagnosis and treatment of paraproteinemic neuropathies

There are several neuropathies associated with paraproteinemia: AL-amyloidosis, anti-myelin associated glycoprotein (MAG) neuropathy, and POEMS syndrome. They can be sometimes diagnosed as chronic inflammatory demyelinating polyneuropathy (CIDP), but their therapeutic strategies are clearly different from those for CIDP. Appropriate diagnosis and early treatment are essential to improve prognosis of paraproteinemic neuropathies. AL-amyloidosis is a systemic and fatal disorder associated with plasma cell dyscrasia. Its neuropathy is characterized by length-dependent axonal polyneuropathy and autonomic neuropathy. Because diffuse mild conduction slowing and/or prolonged distal latency in the median nerve, which is caused by deposition of amyloid in the carpal tunnel, are sometimes found, AL-amyloidosis can be initially diagnosed as CIDP. Therapeutic approach to myeloma with autologous stem-cell transplantation or proteasome inhibitors can be effective. AL- amyloidosis usually deteriorates subacutely and delay in starting treatment can progress to fatal outcome. Similar to AL-amyloidosis, POEMS syndrome is a systemic and fatal disorder based on monoclonal plasma cell proliferation. POEMS neuropathy can be summarized as diffusely distributed demyelinating polyneuropathy and it can be frequently diagnosed as CIDP. It is not usually associated with dysautonomia. Disease progression leads to multiple organ failure and long-lasting neurological sequelae. Early therapeutic intervention is a key to improve its prognosis. As with AL-amyloidosis, treatments for myeloma can be used for POEMS syndrome. The efficacy of autologous stem-cell transplantation, immunomodulatory drugs, and proteasome inhibitors have been shown so far. Anti-MAG neuropathy is accompanied by IgM monoclonal gammopathy. Distal dominant demyelinating neuropathy is a common feature of its neuropathy. Anti-MAG neuropathy is often refractory to conventional treatment such as steroid and immunoglobulin. Rituximab might be effective in some cases.

Natural History and Outcomes of Asymptomatic IgM Monoclonal Gammopathy

Waldenström's macroglobulinemia (WM) is a distinct clinical entity defined by the presence of a serum monoclonal IgM and bone marrow (BM) infiltration of ≥10% lymphoplasmacytic cells. Asymptomatic (smoldering) WM (aWM) is defined by the presence of serum IgM ≥3 g/dL and/or ≥10% BM lymphoplasmacytic infiltration but no evidence of end-organ damage (symptomatic anemia/cytopenias, constitutional symptoms, lymphadenopathy, hepatosplenomegaly or IgM-related symptoms such as hyperviscosity, severe neuropathy etc) that can be attributed to the underlying clonal disorder (Kyle et al; Blood 2012). IgM monoclonal gammopathy of undetermined gammopathy (MGUS) is defined by the presence of a monoclonal IgM of <3 g/dL, <10% of clonal lymphoplasmacytic cells in the BM and the absence of symptoms related to the underlying clonal disorder or monoclonal IgM. Herein, we report the outcomes of IgM MGUS and aWM in a well-defined cohort of 103 consecutive subjects with asymptomatic IgM monoclonal gammopathy.All patients were diagnosed and followed in the Department of Clinical Therapeutics (Athens, Greece) since 1995 and had BM biopsies available. Patients who presented with IgM-related complications (neuropathy, Schnitzler syndrome, IgM related amyloidosis etc) were not included. At the time of diagnosis of the IgM gammopathy, median age of all subjects was 71 years (range 25-86), 76% had IgMκ and 24% IgMλ; only one had a biclonal gammopathy (IgM and IgG). Median IgM was 1024 mg/dL (range 104 - 5320) and the monoclonal spike in the SPEP was 0.84 g/dL (ranging <0.1-4.35) and only 3% had M-monoclonal protein ≥3 g/dl. Median hemoglobin was 12.8 gr/dl, median platelet counts was 271 x 109/L (range 104-499), median peripheral blood lymphocytes 1850/uL (range 220 - 5620) and median β2-microglobulin (β2M) was 2 mg/L (range 1.1-16.8). Among 71 patients with available free light chains at the time of initial diagnosis, 44 (63%) had abnormal FLC ratio. The median clonal cell BM infiltration was 20% (range 0-90%) and there was an association of serum IgM levels and extent of clonal cell infiltration (r=0.416, p<0.001). Immunoparesis (low levels of non-involved immunoglobulins) was present in 32% of the subjects; 22% hag low IgG, 28% low IgA and 18% had both IgG and IgA suppressed.According to the definition by Kyle et al, 62 (61%) subjects had aWM and 41 (39%) IgM MGUS. Patients with aWM had higher M-protein (median 1.24 vs 0.55 g/dL) and more often monoclonal protein ≥1 g/dl (58% vs 13%, p<0.001) so that 42% of patients with <1 g/dL of monoclonal IgM had ≥10% clonal lymphoplasmacytic cells. There were no differences in other characteristics such as gender, age, hemoglobin levels or β2M. Median follow up of the whole cohort is 5 years (range 1-21); 15 (14%) patients have progressed to develop symptomatic disease and 8 (7%) have died without developing symptomatic disease. For patients with aWM the respective rate of PD to symptomatic disease and of unrelated death was 20% and 3% at 2 years and 39% and 8% at 5 years respectively. For those with IgM MGUS the PD rate and unrelated death rates were 0% and 3% at 2 years and 3% and 7% at 5 years. Thus, aWM was associated with a 7-fold increase of risk of progression to symptomatic disease compared to IgM MGUS (95% CI 3-27, p=0.001). Symptoms defining progression included peripheral neuropathy in 4 patients, B-symptoms in 5, anemia (<10 g/dl) in 8 and organomegaly (splenomegaly and lymphadenopathy) in two. The overall survival for all patients is 91% at 5 years, similar for IgM MGUS and aWM. In patients with aWM, hemoglobin <12 g/dl (p=0.004) and BM clonal cells ≥50% (p=0.04) were associated with higher risk of progression but not IgM levels or abnormal FLCs ratio or the presence of immunoparesis. In multivariate analysis, hemoglobin <12 g/dl (p=0.016) and BM clonal cells ≥50% (p=0.051) were independently associated with shorter time to PD.In conclusion, patients with aWM should be followed without treatment unless symptoms develop. Bone marrow infiltration ≥50% and a hemoglobin <12 g/dL are the major risk factors for progression to symptomatic disease. For those with IgM MGUS the risk of progression is low but they should be evaluated periodically for the development of symptoms related to the underlying dyscrasia. Because 42% of patients with monoclonal IgM <1 g/dL had ≥10% lymphoplasmacytic cells, bone marrow biopsy is indicated in all patients. [Display omitted] DisclosuresKastritis:Amgen: Research Funding; Takeda: Honoraria; Genesis: Honoraria; Janssen: Honoraria, Research Funding. Terpos:Genesis/Celgene: Honoraria, Other: DMC member, Research Funding; BMS: Honoraria; GSK: Honoraria; Abbvie: Honoraria; Takeda: Honoraria, Other: SC member; Janssen: Honoraria, Research Funding; Amgen: Honoraria, Other: SC member, Research Funding. Dimopoulos:Novartis: Consultancy, Honoraria; Genesis Pharma: Research Funding; 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.

Molecular Characterization of Waldenström's Macroglobulinemia Patients Using Peripheral Blood Cell-Free Tumor DNA

BackgroundMutational 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.MethodsA 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.ResultsThe 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).ConclusionsOur 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. DisclosuresTerpos: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.

Detection of MYD88 L265P Mutation By Next Generation Deep Sequencing in Peripheral Blood Mononuclear Cells of Waldenstrom Macroguloblinemia and IgM Monoclonal Gammopathy of Undetermined Significance

Introduction: Whole genome sequencing has recently revealed MYD88 L265P somatic mutations in Waldenstrom macroguloblinemia (WM) tumor cells. Allele-specific polymerase chain reaction (AS-PCR) using bone marrow (BM) aspirate and peripheral blood (PB) is the most frequently used technique to detect the specific mutation. In search for a non-invasive, simple, and more sensitive detection of MYD88 L265P mutation, we performed next generation deep sequencing (NGS) technique using unselected peripheral blood mononuclear cells (PBMCs) collected from WM and IgM monoclonal gammopathy of undetermined significance (IgM MGUS) patients. The main purpose of this study is to assess the clinical implication of MYD88 L265P mutation burden detected by NGS technique using unselected PBMCs.Patients and methods: The study was approved from the Institutional Review Board. After informed consent was obtained in accordance with the Declaration of Helsinki, PBMCs were collected from 38 patients with WM and 20 patients with IgM MGUS. The median time from diagnosis to PBMC collection was 2.2 years (range, 0-25 years). Twenty-nine (76.3%) of WM patients were previously treated. The overall response (CR+PR+MR) rate at the time of analysis was 75.8% (22/29). Genomic DNA was extracted from unselected PBMCs and 300 ng of DNA was used for NGS analysis. Variation rates of MYD88 at the nucleotide position corresponding to L265P were measured with an Illumina MiSeq DNA sequencer by molecular barcoding technology essentially as previously reported (Nature Protocols 12, 664:2017). PCR products were sequenced on a MiSeq DNA sequencer with a Reagent kit v2. for 300 cycles. Serial dilution assessment was carried out to investigate the sensitivity of the method by analyzing the DNA of BM sample from a MYD88 mutation positive patient diluted in DNA from a healthy control.Results: Dilution assessment demonstrated that NGS technique was detectable to a sensitivity of 0.02%, ×5 higher than previously reported sensitivity by AS-PCR. MYD88 L265P mutation was detected from 6/9 (66.7%), 8/29 (27.6%), and 11/20 (55%) in patients with untreated WM, previously treated WM, and IgM MGUS, respectively. Among the patients who were positive forMYD88 L265P mutations, the median percentage of mutant allele relative to WT was 3.0% (0.14-32.3%), 0.24% (0.02-33.8%), and 0.55% (0.06-2.85%), in untreated WM, previously treated WM, and IgM MGUS, respectively. Patients with Untreated WM and those with lymphadenopathy showed a trend toward higher mutant allele burden in PBMCs, but the difference was not statistically significant. Among 26 previously treated WM patients, those with at least minimal response showed significantly lower detection rate of MYD88 L265P mutation (13.6 vs. 75.0%, P=0.02) and mutant allele burden (median, 0.00% vs. 0.23%, P=0.01), compared to patients with less than minimal response.Conclusion: This study is the first to report NGS analysis of MYD88 L265P mutation using unselected PBMCs of WM and IgM MGUS patients. The sensitivity for MYD88 L265P mutation in unselected PBMCs from IgM MGUS patients was comparable with that detected by AS-PCR using CD19-selected PB cells (Leukemia, 2014;28:1698-704). Although its specificity is in need for further investigations, NGS technique successfully detected the mutation carried by minimal tumor cells included in unselected PBMCs with high sensitivity as AS-PCR using CD19-selected PB cells. The mutation detection rate of previously treated WM patients was lower than that of untreated patients in our study and previously reported cohort; however, this in turn may potentially indicate that NGS detection of MYD88 L265P mutation in PBMC be clinically utilized in assessing treatment response by monitoring tumor burden during patients' clinical course. DisclosuresNo relevant conflicts of interest to declare.

MYD88L265P Detection in IgM Monoclonal Gammopathies: Methodological Considerations for Routine Implementation.

In IgM monoclonal gammopathies MYD88L265P is a prognostic and predictive biomarker of therapy response. MYD88L265P detection is mainly performed by allele-specific quantitative PCR (ASqPCR), however recently, droplet digital PCR (ddPCR) has been proved to be suitable for MYD88L265P screening and minimal residual disease monitoring (MRD). This study compared ASqPCR and ddPCR to define the most sensitive method for MYD88L265P detection in bone marrow (BM), peripheral blood (PB) sorted or unsorted CD19+ cells, and in plasma cell-free DNA (cfDNA). Overall, the analysis showed a good concordance rate (74%) between the two methods, especially in BM samples, while discordances (26%) were mostly in favor of ddPCR (ddPCR+ vs. ASqPCR-) and were particularly evident in samples with low mutational burden, such as PB and cfDNA. This study highlights ddPCR as a feasible approach for MYD88L265P detection across different specimen types (including cfDNA). Interestingly, its high sensitivity makes CD19+ selection dispensable. On the other hand, our results showed that MYD88L265P detection on PB samples, especially with ASqPCR, is suboptimal for screening and MRD analysis. Finally, significantly different MYD88L265P mutational levels observed between Waldenström Macroglobulinemia and IgM monoclonal gammopathy of undetermined significance patients suggest the need for further studies in order to identify possible correlations between mutational levels and risk of progression to Waldenström.