IgM MGUS Research Articles

Ethnic Diversity in Presentation of Waldenström Macroglobulinaemia and IgM Mgcs in the United Kingdom- a Real-World Data Analysis

Introduction: Waldenström Macroglobulinaemia (WM) is a low-grade B-cell lymphoma characterised by lymphoplasmacytic marrow infiltration with a circulating IgM protein with an incidence of approximately 0.55 cases per 100,000 in the United Kingdom (UK). WM may be asymptomatic, symptomatic and/or associated with IgM-related entities (monoclonal gammopathies) of clinical significance (MGCS). A central aim of the WMUK Charity and Clinical Forum is to systematically upload demographic, clinical data and treatment outcomes from a significant proportion of the UK's WM population. Such data provide important insights on the epidemiology and clinical features of this distinct uncommon disorder. Clinical correlates with ethnicity are important but sparsely characterised. This analysis focusses on baseline demographics and outcomes across different ethnic groups in the UK. Methods: We retrospectively reviewed data from the Rory Morrison WMUK Registry, collating descriptive data of WM, non-IgM lymphoplasmacytic lymphoma and IgM MGCS from 24 centres across the UK, diagnosed between June 1978 and June 2023. Research ethics approval was obtained. Baseline characteristics were compared using χ 2 or Fisher's exact tests (categorical variables) or Wilcoxon Mann-Whitney/Kruskal-Wallis tests (continuous variables). Differences were considered significant at p-values <0.05. Statistical analyses were performed using STATA v17.0 (StataCorp, Texas, USA). Results: 1073 patients with documented self-reported ethnicity and a diagnosis of WM (n=985), non-IgM LPL (n=32) or IgM MGCS (n=88) were included. Those with asymptomatic IgM MGUS were excluded. Of 982 with a diagnosis of WM, 12% (114/985) had concurrent MGCS. Across the entire cohort, IgM MGCS comprised peripheral neuropathy including anti-MAG neuropathy (n=114), cold agglutinin disease/syndrome (n=43), AL amyloidosis (n=20), mixed autoimmune haemolytic anaemia (n=13), cryoglobulinaemia (n=8), Schnitzler syndrome (n=8) and C1 esterase deficiency (n=2). Non-IgM LPL had similar baseline characteristics to WM although a trend towards a lower presenting M-protein (7g/l vs 18g/l, p=0.07). Ethnicity was categorised in accordance with the UK National Census (ethnicity-facts-figures.service.gov.uk). The majority was white (90%; 968/1073: English/Welsh/Scottish/Irish) including 35 patients identified as ‘other’ white. The remaining 105 (10%) were from the following ethnic groups (collectively ‘ethnic minorities’, EM): 54 Asian (22 Indian, 4 Pakistani, 1 Bangladeshi, 7 Chinese, 20 other), 19 Black (5 African, 11 Caribbean, 3 other), 6 Mixed/multiple, 26 Other ethnic group. EM presented at a younger age compared to the white cohort (60 vs 65 years, p<0.001) with a lower presenting M-protein at WM diagnosis (11g/l vs 18g/l, p=0.028). A similar proportion of patients had IgM MGCS in white vs EM cohorts (11% vs 8%, p=0.69). In those with IgM MGCS alone, presenting M-protein was similar across both ethnic categories (5g/l vs 3g/l, p=0.36). MYD88 L265P mutation was assessed in 387 WM patients (36%), of which MYD88 L265P was identified in 88% (322/364) and CXCR4 mutation tested in 91 patients and mutated in 29% (26/91). MYD88-mutated WM was observed less frequently in the EM versus white cohorts (79% vs 90%, p=0.05). A similar proportion presented with symptomatic WM across ethnic groups (56% vs 53%, p=0.56). In those with WM diagnosed since 2010 (n=956), median follow up was 8 years (95% CI 7-8), median overall survival was not reached. International Prognostic Scoring System for WM (IPSSWM) was predictive of overall survival. Overall survival estimates did not differ when comparing ethnic groups. Conclusions: Analysis of the national registry for WM shows that ethnic minorities comprise 10% of WM/IgM MGCS, present with WM at a younger age, a lower M-protein and with a higher proportion of MYD88-wild type cases which may suggest different disease biology. There was no difference in overall survival in our cohort. Limitations include the retrospective analysis of real-world data and incomplete documentation of baseline data. Further analysis of molecular signatures across ethnicities, treatment and outcome is warranted and ongoing.

Single-Cell RNA Sequencing Reveals Hypo-Responsiveness of T and NK Cells to Interferon Stimulation As an Immune Hallmark in Asymptomatic Waldenstrom's Macroglobulinemia

Introduction Waldenstrom macroglobulinemia (WM) is a rare, indolent non-Hodgkin lymphoplasmacytic lymphoma of the bone marrow (BM), characterized by monoclonal IgM protein production. WM is preceded by IgM monoclonal gammopathy of undetermined significance (IgM MGUS) and smoldering WM (SWM), which are clinically detectable but asymptomatic precursor phases (collectively referred to as asymptomatic WM; AWM). Recent work has revealed significant changes in the immune microenvironment of patients with overt WM, however, less is known about immune dysregulation in patients with early-stage disease. Methods We performed 5' single-cell RNA sequencing (scRNAseq) on BM immune cells from patients with AWM (n=28), healthy donors (HD, n=23), and patients with smoldering multiple myeloma (SMM) (n=27). We also performed an in vitro stimulation experiment using universal Type I interferon (IFN) and subsequent scRNAseq on bone marrow mononuclear cells (BMMCs) and peripheral blood mononuclear cells (PBMCs) from AWM (n=3) and HD (n=3). Results Overall, we annotated 210,019 immune cells, excluding normal B cells. Despite their early stage, AWM patients showed significantly altered BM immune cell composition, including increased proportions of CD56dim NK cells, CD8+ effector memory cells, and CD16 + Monocytes and decreased proportions of pro-inflammatory CD14 + monocytes and activated and interferon (IFN)-stimulated T and NK cells (q<0.2). Low-risk AWM presented similar changes in immune cell composition as intermediate- and high-risk AWM, implying that compositional alterations may be established early in the disease course. Notably, however, the proportion of Tregs was significantly increased in patients with SWM compared to IgM MGUS (p=0.04), suggesting that Tregs may play a role in disease progression. Next, we compared BM immune cell composition between patients with AWM and SMM. In both tumor types, patients showed significantly higher proportions of CD56dim NK cells and CD16 + monocytes compared to healthy donors. However, strikingly, a significant decrease in activated and IFN-stimulated T and NK cells was only observed in AWM, suggesting it may be a hallmark of disease. Comparison of BMMCs and PBMCs from patients with AWM following in vitro culture revealed that the lack of IFN-stimulated cells was systemic-in both BMMCs and PBMCs-although the effect was significantly more pronounced in BMMCs than PBMCs. Interestingly, while in vitro stimulation with Type I IFN resulted in the generation of IFN-stimulated T and NK cells in both HD and AWM, the levels of IFN stimulation did not entirely normalize, reproducing the gradient observed in the control samples without in vitro stimulation: AWM BM < AWM PB < HD BM. To test whether the observed defect was due to a lack of IFN in AWM, we next compared the gene expression profile of monocytes from AWM and HD, which revealed significantly higher activity of the IFN signature in monocytes from AWM compared to HD (p < 10 -5). This suggests that IFN levels in patients are at least not reduced. Collectively, these results indicate that T and NK cells from AWM are hypo-responsive to IFN stimulation, despite the presence of normal or possibly increased IFN in the BM microenvironment. Conclusion Our results indicate that despite the absence of symptoms, patients with AWM already have immune dysregulation in the BM, including the absence of IFN-stimulated T and NK cells, which may provide a rationale for therapeutic intervention. Previous studies have demonstrated clinical responses to therapeutic IFN administration, suggesting that novel formulations with improved therapeutic windows may have a role in treating patients with WM in the future.

Renal Disorders Associated with Waldenström Macroglobulinaemia, IgM MGUS and IgM-Producing B-Cell Lymphoproliferative Disorders

Renal disorders are uncommonly associated with IgM MGUS and Waldenström macroglobulinaemia (WM). Data are limited to large case series that suggest that related renal involvement occurs in 5% of patients with WM. Although uncommon, there is a much greater variety of renal pathologies associated with WM and IgM MGUS than that seen in patients with multiple myeloma, where cast nephropathy predominates. In WM, uncommonly direct infiltration of the renal system by lymphoma or cast nephropathy with a high light-chain level can occur. AL amyloidosis can present with nephrotic syndrome as a feature with IgM MGUS or WM. Cryoglobulinaemia and light-chain deposition disease are other important potential causes of renal impairment with IgM MGUS and WM. There are other rarer monoclonal gammopathy of renal significance (MGRS) conditions characterised by typically isolated kidney disease that are causally related to a B-cell or plasma-cell clonal disorder usually in a precancerous MGUS state, although in some renal pathologies, the association is less clear. Central to the majority of these diagnoses is the need for an accurate renal histological diagnosis, and management requires close joint working of renal and haematology teams.

Report of consensus Panel 4 from the 11th International Workshop on Waldenstrom's macroglobulinemia on diagnostic and response criteria

Consensus Panel 4 (CP4) of the 11th International Workshop on Waldenstrom's Macroglobulinemia (IWWM-11) was tasked with reviewing the current criteria for diagnosis and response assessment. Since the initial consensus reports of the 2nd International Workshop, there have been updates in the understanding of the mutational landscape of IgM related diseases, including the discovery and prevalence of MYD88 and CXCR4 mutations; an improved recognition of disease related morbidities attributed to monoclonal IgM and tumor infiltration; and a better understanding of response assessment based on multiple, prospective trials that have evaluated diverse agents in Waldenstrom's macroglobulinemia. The key recommendations from IWWM-11 CP4 included: (1) reaffirmation of IWWM-2 consensus panel recommendations that arbitrary values for laboratory parameters such as minimal IgM level or bone marrow infiltration should not be used to distinguish Waldenstrom's macroglobulinemia from IgM MGUS; (2) delineation of IgM MGUS into 2 subclasses including a subtype characterized by clonal plasma cells and MYD88 wild-type, and the other by presence of monotypic or monoclonal B cells which may carry the MYD88 mutation; and (3) recognition of “simplified” response assessments that use serum IgM only for determining partial and very good partial responses (simplified IWWM-6/new IWWM-11 response criteria). Guidance on response determination for suspected IgM flare and IgM rebound related to treatment, as well as extramedullary disease assessment was also updated and included in this report.

Skin Punch Biopsy Findings in Patients with IgM MGUS and Waldenström Macroglobulinemia Presenting with Peripheral Neuropathy

Skin Punch Biopsy Findings in Patients with IgM MGUS and Waldenström Macroglobulinemia Presenting with Peripheral Neuropathy

S171: MOLECULAR CLUSTERS OF IGM MONOCLONAL GAMMOPATHIES PRESENT DISTINCT BIOLOGIC, IMMUNE AND METABOLIC FEATURES

Background: IgM MGUS and Waldenstrom Macroglobulinemia (WM) represent a wide range of conditions whose management varies from observation to immunochemotherapy. The current classification relies solely on clinical features and does not explain the heterogeneity that exists within each of these conditions. Aims: To shed light on the biology that may account for the clinical differences, we performed the first comprehensive multi-omics analysis of IgM monoclonal gammopathies. Methods: We used bone marrow (BM) CD19+CD138+ sorted cells and matched BM plasma from 32 pts (7 IgM MGUS, 25 WM) and 5 healthy controls to perform whole exome sequencing, RNA-seq, proteomic and metabolomic analysis. 7 matched WM and 4 IgM MGUS pts were also evaluated using mass cytometry (CyTOF). Results: Applying principal component analysis to gene expression profiling, most of WM pts clustered together, while a small subset of them grouped separately with MGUS pts, suggesting a biologic dichotomy within WM. The controls formed a group distinct from most WM and MGUS pts. Fig1A We then applied a non-negative matrix factorization consensus clustering to the gene expression data and identified three robust clusters. Cluster 1 (C1) included only pts with WM, cluster 2 (C2) included pts with both WM and MGUS, and cluster 3 (C3) included all normal controls as well as a small number of WM and MGUS pts. Fig1B-C When mutations commonly identified in WM were analyzed, there was no difference among the three groups (excluding controls) in mutation burden of MYD88 L265P and CXCR4. Interestingly, aberrant expression of TNFAIP3 was a distinct feature of C1 as deletion of 6q (which encodes for TNFAIP3) and TNFAIP3 mutations were each significantly enriched in C1 (47%) compared to C2 (0%) and C3 (20%; p=0.04). Individual clusters associated with specific transcriptional signatures and clinical features. While C1 displayed enrichment of RNA processing, downregulation of inflammatory pathways and aggressive clinical behavior, C2 showed increased inflammatory signaling and senescence with indolent clinical behavior. C3 had intermediate features with combined proliferative and antigen response signatures. Fig1D In accordance with transcriptomics, the proteomic hallmark of C1 was upregulation of proteins involved in proliferation (eg AKT, MAPK) and downregulation of inflammatory proteins (eg IL4, IL10) while the opposite was observed in C2. Once more, C3 confirmed intermediate features with combined upregulation of proliferation and inflammatory proteins. The metabolism was rewired towards mitochondrial anabolism in C1 and C3, while towards glycolysis in C2. Accordingly, C1 and C3 showed undetectable concentrations of 3-hydroxybutyric acid as opposed to C2 which had increased levels of lactic acid, as end products of fatty acid oxidation and glycolysis respectively. Next, we explored whether C1 and C2 displayed a distinct immune profile. tSNE analysis showed that CD4+ T cells were more abundant in C2 compared to C1 while the opposite was observed for CD8+ T cells. Among CD4+ T cells, activated follicular helper (TFH; p=0.02) and regulatory (Treg; p=0.008) cells were predominantly expressed in C2. In contrast, C1 showed a higher expression of senescent T effector memory (TEM) cells. Fig1E In support of this, SPADE clustering analysis identified three clusters including TFH, Treg and TEM cells. Image:Summary/Conclusion: We have identified three molecular clusters in IgM monoclonal gammopathies with distinct clinical, proteomic, metabolomic and immune features, suggesting a potential biologic classification that may have therapeutic implications.

Abstract 3487: Molecular clusters and functional drivers of IgM monoclonal gammopathies

Abstract IgM MGUS and Waldenstrom Macroglobulinemia (WM) represent a disease spectrum with highly dissimilar therapeutic management ranging from observation to chemoimmunotherapy. The current classification relies solely on clinical features and does not explain the heterogeneity that exists within each of these conditions. To shed light on the biology that may account for the clinical differences, we used bone marrow (BM) CD19+/CD138+ sorted cells and matched BM plasma from 32 patients (pts) (7 IgM MGUS, 25 WM) and 5 healthy controls to perform the first comprehensive multiomics approach including whole exome sequencing, RNA seq, proteomics and metabolomics. Applying principal component analysis to gene expression profiling, most of WM pts clustered together, while a small subset of them grouped separately with MGUS pts, suggesting a biologic dichotomy within WM. The healthy controls formed a distinct group from most WM and MGUS. We then applied a non-negative matrix factorization consensus clustering to the gene expression data and identified three robust clusters. Cluster 1 (C1) included only WM pts, cluster 2 (C2) included both WM and MGUS pts, and cluster 3 (C3) included all controls as well as a small number of WM and MGUS pts. When mutations commonly identified in WM were analyzed, there was no difference among the three groups (excluding controls) in mutation burden of MYD88 L265P and CXCR4. Interestingly, aberrant expression of TNFAIP3 seemed a distinct feature of C1 as deletion of 6q (which encodes for TNFAIP3) and TNFAIP3 mutations were each significantly enriched in C1 (47%) compared to C2 (0%) and C3 (20%; p=0.04). Individual clusters associated with specific transcriptional signature and clinical features. While C1 displayed enrichment of cell growth, downregulation of inflammatory pathways (eg IL6 and IL8 signaling) and aggressive behavior, C2 showed increased inflammatory signaling and cell survival with indolent behavior. C3 had an intermediate feature with combined proliferative and inflammatory signatures. In accordance with the transcriptomic data, the hallmark of C1 was upregulation of proliferation-associated proteins (eg AKT, MAPK) and downregulation of inflammatory proteins (eg IL4, IL10) while the opposite was observed in C2. Once more, C3 confirmed intermediate features with combined upregulation of proliferation and inflammatory proteins. The metabolism was rewired towards fatty acid catabolism in C1, glycolysis in C2 and anabolism in C3. Accordingly, C1 showed undetectable concentration of 3-hydroxybutyric acid as opposed to C2 which had increased levels of malic and lactic acids, as end products of fatty acid oxidation and glycolysis respectively. Those metabolites had intermediate levels in C3. In conclusion, we identified three molecular clusters with distinct clinical, proteomic and metabolomic features, suggesting a potential biologic classification that may have therapeutic implications. Citation Format: Patrizia Mondello, Jonas Paludo, Joseph Novak, Kerstin Wenzl, Shahrzad Jalali, Jordan Krull, Esteban Braggio, Surendra Dasari, Michelle Manske, Jithma Abeykoon, Asher Chanan-Khan, Robert Kyle, Morie Gertz, Zhi Zhang Yang, Anne Novak, Stephen Ansell. Molecular clusters and functional drivers of IgM monoclonal gammopathies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3487.

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.

OAB-040: Clonal hematopoiesis is associated with increased risk of progression of asymptomatic Waldenström Macroglobulinemia

<h3>Background</h3> Clonal 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. <h3>Methods</h3> We retrospectively reviewed clinical data of 602 patients with IgM MGUS, smoldering WM 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 (DFCI). 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. In order 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 (CH-DTA). <h3>Results</h3> The 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 time of first NGS assay. The prevalence of CH-DTA was 14% in symptomatic WM patients and did not differ significantly in MGUS and SWM (13% and 14%, respectively). Among precursor patients, there was an increased risk of progression to symptomatic WM in 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 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 OS with a relatively short median follow-up from diagnosis and NGS assay of 6.7 (95% CI: 6.1-7.6) and 2.5 (95% CI: 2.2-2.8) years, respectively. 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). <h3>Conclusion</h3> We demonstrate that CH is common in WM patients and is associated with 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.

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.

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.

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.

Differential Diagnosis of Suspected MYD88 Wild-Type Waldenstrom's Macroglobulinemia Includes Other Malignant and Non-Malignant IgM Secreting Entities, Including Frequent Cases of IgM Myeloma

MYD88 mutations are present in 95% of patients with Waldenstrom's Macroglobulinemia (WM), and permit diagnostic discrimination from other overlapping IgM-secreting B-cell malignancies. However, such diagnostic discrimination can often be difficult among suspected WM patients with wild-type MYD88 (MYD88WT), and can result in either diagnostic misclassification or mistreatment. We therefore examined a relatively large cohort of patients with suspected MYD88WT WM, and performed a systematic review of their clinical, pathologic and laboratory studies. Sixty-four patients with suspected MYD88 wild-type WM were identified. All had a monoclonal IgM protein, and bone marrow (BM) disease involvement. AS-PCR testing for MYD88 L265P mutation, and exclusion of non-L265P mutations by Sanger sequencing was performed to confirm MYD88WT status utilizing CD19-selected and unselected BM mononuclear cells (Xu et al, Blood 2013). WHO and WM consensus guidelines were used to establish clinicopathological diagnosis.Forty-six (71.8%) patients fulfilled WHO and WM consensus criteria for WM. Their median BM involvement was 37.5% (range 5-95%), serum IgM was 2,980 (range 160-9,000 mg/dL), and hemoglobin was 11 (range 4-15.5 g/dL). Sixteen (35%) and 13 (28%) had adenopathy and splenomegaly, respectively. Principal reasons for diagnosis included anemia (50%), and/or other cytopenias (13%), peripheral neuropathy (13%), adenopathy (8%), symptomatic hyperviscosity (5%), and renal failure (5%). At a median follow-up of 5.4 years, 31 (67%) patients required therapy. Notably, 7 (15.2%) MYD88WT patients were diagnosed with diffuse large B-cell lymphoma (DLBCL) during the follow-up period. Eleven (24%) patients died, 4 due to DLBCL transformation. Among the other MYD88WT patients, 7 (10.9%) had IgM multiple myeloma (IgM MM). Their median BM disease involvement was 60% (range 10%-80%), with a predominant plasma cell infiltrate. In 3(43%) patients, both clonal B-cells and plasma cells were detected, while clonal plasma cells alone were observed in the other 4 patients. FISH testing of BM mononuclear cells showed t(11;14) in 5 patients; t(14;16) in one patient; and normal cytogenetics in one patient. The median serum IgM level was significantly higher at 8,375 (range 2,530-12,000 mg/dL) versus MYD88WT WM patients (p=0.016). One (14%) patient had both adenopathy and splenomegaly. Principal reasons for diagnosis included anemia (100%), and symptomatic hyperviscosity (43%). With a median follow-up of 2.4 years, all patients required treatment and one died of myeloma. Findings consistent with marginal zone lymphoma (MZL) were observed in 6 (9%) patients. Their median BM involvement was 10% (range 5-25%), and three (50%) had detectable clonal B-cells by BM flow cytometry. Their median serum IgM level was 1,642 (95-2,800 mg/dL), and was not significantly different versus MYD88WT WM patients. Adenopathy and splenomegaly were present in 4 (67%) and 2 (33%) patients. With a median follow-up of 3.8 years, 4 (67%) required therapy, and all remain alive. For 3 (4.6%) patients, the findings favored a diagnosis of IgM MGUS with plasma cell infiltration. The median BM involvement for these patients was 5% (range 5-8%) with flow cytometry demonstrating clonal plasma cells. The median serum IgM level for these patients was 1,846 (range 1,262-2,390 mg/dL), and did not differ versus MYD88WT WM patients. With a median follow-up of 2.5 years, none progressed or received treatment, and are alive. In one patient, the findings favored the diagnosis of CLL while another patient had intravascular DLBCL. The serum IgM level was 1,822 and 355 mg/dL respectively. The BM disease burden was 5% in both patients. The CLL patient remains alive without treatment, while the patient with DLBCL required therapy and remains alive. No DLBCL transformation was observed among non-WM cases. Our findings show that up to 30% of suspected MYD88WT WM cases have an alternative clinicopathological diagnosis that includes both malignant and non-malignant entities. IgM MM was frequently present among suspected MYD88WT WM cases, and presented with very high serum IgM levels, symptomatic hyperviscosity and chr. 14 translocations. Patients with MYD88WT WM disease had a high incidence of DLBCL transformation that contributed to a third of deaths. The findings highlight the importance of diagnostic discrimination among suspected MYD88WT WM cases. DisclosuresTreon:Pharmacyclics: Consultancy, Research Funding. Castillo:Abbvie: Research Funding; Millennium: Research Funding; Janssen: Consultancy, Research Funding; Pharmacyclics: Consultancy, Research Funding.

Identification of a Candidate Gene Set Signature for the Risk of Progression in IgM MGUS to Smoldering/Symptomatic Waldenström Macroglobulinemia (WM) by a Comparative Transcriptome Analysis of B Cells and Plasma Cells.

Simple SummaryIgM monoclonal gammopathy of undetermined significance (IgM MGUS) is an early precursor stage of the rare lymphoma Waldenström Macroglobulinemia (WM). Although comparative gene expression studies on WM, IgM MGUS, and normal B-cells (CTRLs) identified several differentially expressed genes, reliable predictors of progression from IgM MGUS to WM have not yet been identified. We performed a microarray study on CD19+ and CD138+ cells of WM vs. IgM MGUS vs. CTRLs to determine gene signatures for both cell populations. We demonstrated that hematopoietic antigens, cell-adhesion molecules, Wnt-signaling, BCR-signaling, calcium signaling, coagulation cascade, and pathways responsible for cell cycle and proliferation were significantly enriched for genes expressed in B-cells of WM vs. IgM MGUS vs. CTRLs. Interestingly, we showed nine genes which displayed the same expression levels in WM and IgM MGUS compared to CTRLs, suggesting their possible role in the risk of transformation of IgM MGUS to WM.Waldenström Macroglobulinemia (WM) is a B-cell lymphoma characterized by the precursor condition IgM monoclonal gammopathies of undetermined significance (IgM MGUS). We performed a gene expression profiling study to compare the transcriptome signatures of bone marrow (BM) B-cells and plasma cells of 36 WM patients, 13 IgM MGUS cases, and 7 healthy subjects used as controls (CTRLs) by Affymetrix microarray. We determined 2038 differentially expressed genes (DEGs) in CD19+ cells and 29 DEGs genes in CD138+ cells, respectively. The DEGs identified in B-cells were associated with KEGG pathways, mainly involved in hematopoietic cell lineage antigens, cell adhesion/focal adhesion/transmembrane proteins, adherens junctions, Wnt-signaling pathway, BCR-signaling pathway, calcium signaling pathway, complement/coagulation cascade, platelet activation, cytokine-cytokine receptor interactions, and signaling pathways responsible for cell cycle, apoptosis, proliferation and survival. In conclusion, we showed the deregulation of groups of genes belonging to KEGG pathways in the comparison among WM vs. IgM MGUS vs. CTRLs in B-cells. Interestingly, a small set of genes in B-cells displayed a common transcriptome expression profile between WM and IgM MGUS compared to CTRLs, suggesting its possible role in the risk of transformation of IgM MGUS to WM.

262 Mixed Cryoglobulinemic MPGN in a Patient With Primary Sjögren's Syndrome (pSS) Complicated by IgM MGUS

262 Mixed Cryoglobulinemic MPGN in a Patient With Primary Sjögren's Syndrome (pSS) Complicated by IgM MGUS

Subepidermal Autoimmune Bullous Disease Associated With Monoclonal Gammopathy of Unknown Significance and Unexpected Positive Direct Nikolsky Phenomenon: A Case Report

Abstract Introduction: Numerous dermatoses associated with monoclonal gammopathy have been reported in the literature. Subepidermal autoimmune bullous diseases (SABD) are one of them which were not common. Here we report a case of SABD associated with MGUS who had a severe mucosal involvement and unexpected positive direct Nikolsky phenomenon. Case presentation: A 68-year-old male patient was admitted to our clinic with erosions on the oral mucosa, tense blisters, erosions, and ulcers on the trunk and extremities. Subepidermal vesicle formation was detected in the skin biopsy. Clinical examination revealed positivity for the Nikolsky phenomenon. The disease was unresponsive to conventional treatments and dysphagia and hoarseness occurred. The patient was screened for malignancy due to his unresponsiveness to the treatments and his severe oral mucosal involvement. Ig-G MGUS was detected in the patient. Discussion: The Nikolsky sign is an indicator of acantholysis and is known as a specific finding for pemphigus. However, when we look at gammopathy-associated autoimmune bullous dermatoses, skin fragility has been reported in cases. However, the meaning of fragility is not explained. The diagnosis of all these patients was Ig-M MGUS. Our patient was presented because of non-IgM MGUS, direct Nikolsky positivity, and severe mucosal involvement. Conclusion: Nikolsky positivity may be a clue for gammopathy-related subepidermal autoimmune bullous diseases.

Neuropathy with IgM Gammopathy: Incidence, Characteristics and Management, a Rory Morrison W.M.U.K Registry Analysis

Neuropathy with IgM Gammopathy: Incidence, Characteristics and Management, a Rory Morrison W.M.U.K Registry Analysis

Waldenström’s Macroglobulinemia (WM) Is Preceded By Clonal Lymphopoiesis Including MYD88 L265P in Progenitor B Cells

Waldenström’s Macroglobulinemia (WM) Is Preceded By Clonal Lymphopoiesis Including MYD88 L265P in Progenitor B Cells

Long Non-Coding RNA Expression in Waldenstrom Macroglobulinemia and IgM Monoclonal Gammopathy of Undetermined Significance

Long Non-Coding RNA Expression in Waldenstrom Macroglobulinemia and IgM Monoclonal Gammopathy of Undetermined Significance

Waldenström's Macroglobulinemia (WM) Is Preceded By Clonal Lymphopoiesis Including MYD88 L265P in Progenitor B Cells

Waldenström's Macroglobulinemia (WM) Is Preceded By Clonal Lymphopoiesis Including MYD88 L265P in Progenitor B Cells