IgM Monoclonal Gammopathy Of Undetermined Significance Research Articles

B-075 Evaluation of patients with monoclonal gammopathy of undetermined significance using Optilite Freelite assays

Abstract Background Monoclonal gammopathy of undetermined significance (MGUS) is a pre-malignant disorder with a progression risk to multiple myeloma of 1% per year. Patients with MGUS may produce monoclonal intact immunoglobulins (Ig) and/or free light chains (FLC). The International Myeloma Working Group (IMWG) recommendations for the use of FLC measurements in MGUS are to establish the diagnosis of light chain only MGUS (LC-MGUS) and further to risk-stratify newly diagnosed MGUS. Once identified, MGUS is longitudinally monitored at intervals dependent on the risk of transformation. The IMWG recommends serum protein electrophoresis (SPEP) for monitoring of known MGUS with intact Ig, however others have published benefits of additionally monitoring with FLC. In this study, we evaluated the diagnostic sensitivity and specificity of the Optilite® Freelite® Kappa Free Kit and the Optilite Freelite Lambda Free Kit (The Binding Site, part of Thermo Fisher Scientific) in patients with MGUS, and the monitoring performance of Freelite results measured in serial samples from patients with clinically stable and progressive MGUS. Methods Serum samples from 234 patients with clinically diagnosed MGUS and 140 disease controls, taken at single time points, and serial samples (≥3) from 49 patients with MGUS were tested with the Freelite assays. Intact M-protein presence and isotype was confirmed in MGUS samples with immunofixation electrophoresis (IFE). In patients clinically diagnosed with LC-MGUS, the presence of detectable M protein on IFE was not required. The disease control cohort (non-MGUS) had disorders commonly associated with elevated polyclonal immunoglobulins (e.g autoimmune, liver, and renal diseases), confirmed by total IgG and/or IgA and/or IgM results and the absence of M-protein by IFE. Patients represented a diverse population from both European and US sources. Results The overall diagnostic sensitivity of the Optilite Freelite assays was 59.4%, calculated as the frequency of abnormal free light chain ratio (rFLC) in intact immunoglobulin MGUS, and abnormal rFLC with concurrent elevated involved FLC level in LC-MGUS. The diagnostic sensitivity was: 58.0% in non-IgM MGUS, 41.7% in IgM MGUS, 66.7% in biclonal MGUS and 100% in LC-MGUS. In intact (IgG/IgA/IgM) kappa MGUS, the diagnostic sensitivity was 67.5%, versus 41.5% in intact (IgG/IgA/IgM) lambda MGUS. The diagnostic specificity was 86.4%. In the 49 patients with serial samples, concentrations of involved FLC were stable in 93.3% of patients with clinically stable MGUS, whereas in patients with clinically progressive MGUS an increasing trend was seen in 50% of cases. Conclusions These results demonstrate that the Optilite Freelite Kappa and Lambda assays produce clinically relevant results that are useful in the evaluation of MGUS.

Monoclonal Gammopathy of Undetermined Significance and the Risk of Thrombotic Events: Results from Istopmm, a Population-Based Screening Study in Iceland

Introduction: Multiple myeloma (MM), Waldenström's macroglobulinemia (WM), and related disorders have been associated with excess risk of thrombosis. Furthermore, medications used to treat these disorders, in particular immunomodulatory agents, have been associated with excess thrombotic risk. Monoclonal gammopathy of undetermined significance (MGUS), the asymptomatic precursor of MM, WM, and related disorders, has also been associated with arterial and venous thrombosis. However, prior studies have been based on cohorts where MGUS was diagnosed as part of the work-up for other unrelated medical disorders. We have shown that these cohort have biased selection of individuals with chronic disease, including autoimmune disease, heart failure, endocrine disorders and more. This selection bias may have led to false associations or overestimation of the relationship of MGUS and thrombotic events. Aim: To evaluate the relationship of MGUS and thrombosis in a screened cohort of individuals with MGUS. Methods: Data were obtained from the Iceland screens, treats, or prevents multiple myeloma study (iStopMM), a population-based screening study for MGUS and a randomized trial of follow-up strategies with the aim of evaluating the benefits and harms of MGUS screening. As part of the study 75,422 Icelandic residents 40 years and older (54% of the underlying population) have provided their informed consent for the study and were screened by serum protein electrophoresis (SPEP) and free light chain (FLC) assay. The cohort was crosslinked to population-based Icelandic government registries that includes all diagnoses made in the universal healthcare system, recorded as international classification of diseases (ICD) 10 codes. The study exposure was MGUS at screening and the outcome was venous and arterial thrombosis, defined as recorded ICD-10 codes. Events recorded within 90 days of each other were considered the same event. We further analyzed subgroups of M protein isotype and light chain (LC) MGUS. We followed participants prospectively after the date of screening to arterial and venous thrombosis as the endpoint and censoring at death, progression to MM, WM, and related disorders, or end of follow-up. Hazard ratios (HR) for time to first thrombotic event were estimated using Cox regression models, while hazard ratios for recurrent thrombotic events were estimated using mixed-effects Cox regression models with a random intercept. Both prospective analyses were adjusted for age as a non-linear variable and sex. In a secondary analysis, M-protein concentration was included as a non-linear variable into similar models that were restricted to those with MGUS with an M-protein. Results: Of the 75,422 screened, 3,353 had MGUS with an M-protein, here termed heavy chain (HC) MGUS, and 315 had LC MGUS. Over a median follow-up of 3.67 to 3.90 years, depending on the outcome of interest, 263 and 3,557 arterial events (20.4 and 13.5 per 1,000 person years) and 260 and 3,092 venous events (20.2 and 11.7 per 1,000 person years) were observed in those with and without MGUS respectively. MGUS was found to be associated with venous thrombotic events (HR: 1.31; 95% CI: 1.08-1.57), in particular deep vein thrombosis (HR:1.46; 95% CI: 1.12-1.90). Of the different MGUS subtypes, this association was only observed in non-IgM MGUS. The results were similar in the recurrent events analysis. IgM MGUS was found to be associated with a higher of peripheral arterial embolic events (HR: 2.86; 95% 1.04-7.85) but this association was not observed in the recurrent event analysis. M-protein concentration was not correlated with the risk of thrombotic events. Discussion: In this large population-based study including more than 75,000 individuals screened for MGUS, we found that MGUS, and particularly non-IgM MGUS, was associated with venous thrombotic events. Interestingly, the size of the M-protein spike was not associated with a higher risk for thromboembolic events. Further studies are needed to identify the underlying mechanisms with the goal of identifying subgroups of the MGUS population that may require interventions to prevent thrombotic events, potentially improving the survival and quality of life of those with MGUS.

Risk Factors of Smoldering Multiple Myeloma: Results from the Screened Istopmm Study

Introduction Smoldering multiple myeloma (SMM) is an asymptomatic precursor condition to multiple myeloma (MM) that carries a higher risk of progression to MM than monoclonal gammopathy of undetermined significance (MGUS). This study aimed to identify potential risk factors of SMM by analyzing the association of baseline characteristics, body mass index, smoking, alcohol consumption, prior diagnoses of autoimmune diseases and prior chronic infections and SMM in the screened Iceland Screens, Treats, or Prevents Multiple Myeloma (iStopMM) study cohort. Methods The iStopMM study is a screening study for MM precursors where 75,422 individuals over 40 years old in Iceland were screened with serum protein electrophoresis (SPEP) and free light chain (FLC) analysis. Those with a positive screening result were randomized to one of the three study arms, with clinical evaluation at the study center, including bone marrow sampling, performed in two of them. MGUS was defined by the presence of a serum M-protein concentration less than 30 g/L and less than 10% clonal plasma cells in the bone marrow, and SMM was defined as 10-60% bone marrow plasma cells and/or M-protein concentration over 30 g/L, both in the absence of MM defining events. IgM MGUS was excluded in this analysis. Height and weight were measured at the first visit to the study center, alcohol and smoking habits were evaluated with questionnaires. Information on previous diagnoses (ICD codes) was obtained from central health registries with almost 100% completeness. Logistic regression models were used to calculate odds ratio (OR) with 95% confidence interval (CI) to compare risk factors in SMM vs. MGUS and SMM vs. controls. All analyses were adjusted for age and sex. Results At the first study center evaluation, 210 individuals were diagnosed with SMM and 1,456 with MGUS. Data from 70,627 controls with negative SPEP and normal FLC analysis were included. Individuals with SMM were older and more frequently male than controls, however there was no statistically significant difference between the sex and age distribution of individuals with SMM and MGUS (Figure). Immunoparesis (OR 3.98; 95% CI: 2.74-5.76), abnormal FLC ratio (OR 3.61; 95% CI: 2.59-5.02), IgA isotype (OR 2.64; 95% CI: 1.83-3.83) and increasing M-protein levels (OR per g/L 1.33; 95% CI: 1.27-1.39) were all associated with increased odds of SMM compared to MGUS. BMI measured at the first study visit was not significantly different between individuals with MGUS and SMM (overweight vs. normal weight OR 1.08; 95% CI: 0.74-1.56). Neither current or previous smoking nor alcohol consumption more than once per week (vs. less) was associated with increased risk of SMM compared to MGUS (OR 0.88; 95% CI: 0.60-1.31, and OR 0.97; 95% CI: 0.62-1.53, respectively) or controls (OR 0.92; 95% CI: 0.64-1.33, and OR 0.85; 95% CI: 0.57-1.27, respectively). Prior diagnosis of autoimmune disease was not associated with SMM as compared to MGUS (OR 1.19; 95% CI: 0.81-1.74) or controls (OR 1.21; 95% CI: 0.85-1.73). A previous diagnosis of a chronic infection was not statistically significantly associated with increased risk of being diagnosed with SMM as compared to MGUS (OR 1.53; 95% CI: 1.00-2.34) but an association was seen when compared to controls (OR 1.54; 95% CI: 1.05-2.28). In individuals with SMM, the most common chronic infections were varicella zoster virus and herpes simplex virus, no diagnoses of hepatitis virus or chronic bacterial infections were observed. Conclusion In this large population-based screening study we did not find an association of smoking, alcohol consumption, BMI, or a prior diagnosis of autoimmune disease and SMM compared to MGUS. We found an association between a previous diagnosis of a chronic infection and SMM which could be explained by reverse causality, i.e., that SMM leads to immune impairment which increases the risk of viral infections. We found no significant age or sex difference between individuals with MGUS and SMM, but results from blood tests, including immunoparesis, abnormal FLC ratio, IgA-isotype, and higher levels of M-protein were all associated with SMM compared to MGUS. Our results indicate that neither lifestyle factors nor chronic inflammatory conditions increase the risk of SMM, and that the differences between individuals with MGUS and SMM can be detected in blood assessments associated with the plasma cell clone, but not in demographic or behavior related factors.

Genome-Wide DNA Methylation Analysis Reveals Epigenetic Influence on Intracellular and Cytokine Signaling Pathways in Waldenström Macroglobulinemia Compared to IgM-MGUS

Introduction: Waldenström macroglobulinemia (WM) is a rare B-cell lymphoma that may be preceded by an asymptomatic IgM-monoclonal gammopathy of undetermined significance (MGUS). The mechanisms underlying the progression from IgM-MGUS to WM remain to be fully elucidated; however, the stepwise accumulation of genomic abnormalities has been suggested as a potential driver. Currently, the role of epigenetic regulation of DNA remains underexplored in this disease spectrum. Aberrant DNA methylation is a hallmark of oncogenesis that plays an important role in hematologic malignancies. The aim of this study was to assess differential DNA methylation in WM compared to IgM-MGUS and identify hyper- and hypomethylated pathways that may alter key cellular functions. Methods: This study included 15 patients with WM and 6 with IgM-MGUS. Bone marrow samples were prospectively collected and CD19+ and/or CD138+ B cells were positively selected. Subsequently, genome-wide DNA methylation analysis was performed. CpG methylation ratios were segmented into 200-bp regions and differentially methylated regions (DMRs) comparing WM to IgM-MGUS were identified. DMRs with a Q-value <0.05 and absolute methylation difference of ≥20% were considered significant and utilized for analysis. DMRs were annotated according to genomic regions of interest (promotors, 3'UTR, 5'UTR, introns, exons, CpG islands, CpG shores). Next, genes with significant promotor hyper- or hypomethylation were assessed using the Ingenuity Pathway Analysis. Pathways with a corrected p-value<0.05 were considered statistically significant and a Z-value was calculated based on the degree of differential methylation. Results: Genome-wide DNA methylation analysis revealed a significant number of DMRs (n= 15691, hyper: 8248; hypo: 7443) in WM compared to IgM-MGUS. Assessing genomic regions, promotors (hyper: 1379; hypo: 2403), introns (hyper: 1548; hypo: 2292), CpG shores (hyper: 587; hypo: 849) and 3'UTR (hyper: 66; hypo: 193) regions were observed to have a significantly greater number of hypomethylated DMRs (all comparisons, p<0.001) in WM compared to IgM-MGUS (Figure A). While exons (hyper: 1686; hypo: 1378), CpG islands (hyper: 2128; hypo: 82), and 5'UTR (hyper: 854; hypo: 246) regions had a significantly greater number of hypermethylated DMRs (all comparisons, p<0.001) (Figure A). Next, promotor methylation-based pathway analysis revealed differential methylation of multiple cellular pathways important in WM pathogenesis (Figure B). These included pathways involved in intracellular signaling and the regulation of cellular proliferation, such as the STAT3 and CXCR4 pathways which were hypomethylated, and the PTEN signaling pathway which was hypermethylated. Of interest, we have previously demonstrated the transcription factor, STAT3, to have an increased expression in WM and to be implicated in IgM secretion (Elsawa, 2011). Next, multiple cytokine signaling pathways were observed to be hypomethylated in WM compared to IgM-MGUS including members of the interleukin (IL) family (IL-1, IL-8, IL-15, IL-23), VEGF, and TGF-β. Of relevance, previous studies have demonstrated the elevation of IL-8, IL-15, and increased expression of angiogenic cytokines, including VEGF, in patients with WM (Lewicki, 2010; Anagnostopoulos, 2007). Furthermore, our group has also shown miRNA-based epigenetic regulation of IL-8 and IL-15 in WM compared to IgM-MGUS (Chohan, 2023). Lastly, several pathways critical in the regulation of the cell structure and the extracellular matrix (ECM) were found to be hypomethylated including the FGF, actin cytoskeleton, PAK, and FAK signaling pathways. Of note, the FGF/FGFR axis has been demonstrated to be dysregulated in WM, and its targeting has been shown to halt WM cell growth (Sacco, 2021). Conclusions: This study demonstrates differential DNA methylation of key genomic regions in WM compared to IgM-MGUS. Promotor methylation-based analysis reveals hypomethylation of several important cellular pathways involved in cytokine signaling, cell structure/ECM, and intracellular signaling. These results underscore the influence of epigenetics in the IgM-gammopathy disease spectrum and suggest the potential role of aberrant DNA methylation underlying the malignant progression from IgM-MGUS to WM.

Case Report of Anti-DPPX Encephalitis: Diagnostic Challenges in Hematologic-Oncologic Comorbidities (P5-5.024)

<h3>Objective:</h3> Describe a case of anti-dipeptidyl-peptidase-like protein-6 (DPPX) encephalitis with attention to hematologic-oncologic comorbidities. <h3>Background:</h3> Anti-DPPX encephalitis is a rare autoimmune encephalitis with prodromal features of weight loss and gastrointestinal symptoms, followed by cognitive impairment and central nervous system hyperexcitability. Due to atypical and variable presentation, diagnosis is frequently delayed. Autoimmune encephalitis is associated with malignancies, and three cases of anti-DPPX encephalitis have been linked to hematologic malignancies. <h3>Design/Methods:</h3> Consent was obtained from patient’s wife. PubMed and Ovid databases were searched using terms (“DPPX” and “lymphoma”) as well as “DPPX”. <h3>Results:</h3> A 54-year-old male presented with two months of metallic taste and unintentional weight loss followed by confusion, memory impairment, hallucinations, and sleep disturbances. Neurologic exam was significant for encephalopathy, ataxia, and stimulus-induced myoclonus. Initial workup revealed mildly elevated protein in cerebrospinal fluid but no pleocytosis, MRI brain with nonspecific white matter hyperintensities, and electroencephalogram without epileptiform activity. Initial basic chemistries were within normal limits, and no vitamin deficiencies were identified. Intravenous methylprednisolone (1000 mg) was initiated for suspected autoimmune encephalitis. After anti-DPPX antibody returned positive, he received additional first- and second-line treatment with intravenous immunoglobulins, plasmapheresis and rituximab with modest improvements in encephalopathy and myoclonus. The patient had a history of monoclonal gammopathy of undetermined significance (MGUS) and tobacco use. He underwent serum protein electrophoresis, confirming IgM MGUS with low to intermediate risk of malignant transformation. However, peripheral flow cytometry revealed an indeterminate kappa monotypic restricted B cell population (CD19 positive, CD5 and CD10 negative) without corresponding findings in the CSF or lymphadenopathy. In discussion with hematology-oncology, the B cell population was favored to be reactive to the anti-DPPX encephalitis, rather than a primary malignant hematologic condition. Other malignancy screening was performed and negative. <h3>Conclusions:</h3> This case highlights the challenges of determining association of anti-DPPX encephalitis with hematologic malignancy and importance of interdisciplinary collaboration. <b>Disclosure:</b> Dr. Fisher has nothing to disclose. Dr. Misiolek has nothing to disclose. Dr. Buckley has nothing to disclose.

IgM monoclonal gammopathy of undetermined significance: clinicopathologic features with and without IgM-related disorders.

A subset of patients with immunoglobulin M (IgM) monoclonal gammopathy of undetermined significance (MGUS) develop IgM-related disorders (IgM-RD) including peripheral neuropathy, cryoglobulinemia and/or cold agglutinin disease (CAD). We examined the clinical and bone marrow pathologic findings in 191 IgM MGUS patients (2016 World Health Oragnization criteria). Clonal plasma cells were identified in 41 of 171 (24%) cases by immunohistochemistry (IHC) and clonal B cells in 43 of 157 (27%). IgM-RD was identified in 82 (43%) cases, including peripheral neuropathy (n=67, 35%), cryoglobulinemia (n=21, 11%), and CAD (n=10, 5%). Cases of CAD showed distinctive features including lack of MYD88 mutations (P=0.048), supporting the concept of primary CAD as a distinct clinicopathologic disorder. Following exclusion of CAD, comparison of the remaining cases with (n=72) or without (n=109) IgM-RD showed IgM-RD to be more frequent in men than women (P=0.02) and to be more highly associated with MYD88 L265P (P=0.011). Cases with and without IgM-RD otherwise showed similar features including serum IgM concentrations, presence of lymphoid aggregates, clonal B cells by flow cytometry or clonal plasma cells by IHC. No differences were observed in overall survival between cases with and without IgM-RD. No cases in this series met criteria for plasma cell type IgM MGUS as defined in the 2022 International Consensus Classification of lymphoid neoplasms. These results show IgM-RD to be common in patients with IgM MGUS. While CAD shows distinctive features, the remaining cases of IgM-RD largely show pathologic findings similar to IgM MGUS without IgM-RD.

Abstract 3811: Micro-RNA induced inactivation of inflammatory cytokine signaling pathways in Waldenstrom macroglobulinemia compared to IgM-MGUS

Abstract Introduction: Cytokines are protein factors that regulate diverse cellular processes. The malignant progression of immunoglobulin M monoclonal gammopathy of undetermined significance (IgM-MGUS) to Waldenstrom macroglobulinemia (WM) is in part cytokine mediated, and we have previously shown IL-6 mediates IgM secretion in WM via the JAK/STAT pathway. Cytokine signaling can be epigenetically modulated by micro-RNAs (miRs). We aimed to identify the role of miRs in the regulation of inflammatory cytokine pathways between these diseases. Methods: Bone marrow samples of patients with IgM-MGUS (n=7) and WM (n=25) were prospectively collected and sorted for CD19+ and/or CD138+ malignant cells. Total RNA extraction and sequencing were performed, miR and mRNA profiling was conducted, and differential expression was calculated between WM and IgM-MGUS using a log2 fold change (FC)&amp;gt;0.5 and ←0.5, false discovery rate&amp;lt;0.05. Experimental differentially expressed miR-mRNA pairing was performed, and pairs were filtered for correlated expression (i.e., upregulated miR/downregulated mRNA, and vice-versa). Filtered pairs were analyzed using the Ingenuity Pathway Analysis to identify pathways between WM and IgM-MGUS with absolute activation Z-score ≥1. Results: We identified 34 differentially expressed miRs experimentally targeting 1132 mRNAs. Pathway analysis showed overall miR-based inactivation of inflammatory cytokine signaling pathways in WM compared to IgM-MGUS, especially of the interleukin (IL) family: IL-2, IL-6, IL-8, IL-9, IL-15, and IL-17. Underlying multiple pathways was upregulation of miRs-146a, 150, 194, all targets of Signal Transducer and Activator of Transcription 1 (STAT1), observed to be downregulated. Of relevance, miR-146a has been previously shown to be an inhibitor of inflammatory cytokines, including IL-6. Next, we observed upregulation of miR-142 which targets phosphoinositide-3-kinase regulatory subunit 6 (PIK3R6), observed to be downregulated. Both the STAT and PIK3 families are important cytokine signal transducers, and we have previously shown the PI3k-AKT-IkB-p65 pathway induces expression of IL-6 via a GLI2-mediated mechanism. We additionally observed granulocyte-macrophage colony-stimulating factor (GM-CSF) signaling to be inactivated in WM. Here, miR-19b and miR-30e were upregulated, both target CSF 2 receptor subunit beta (CSF2RB), found to be downregulated. CSF2RB is the common receptor subunit of GM-CSF, IL-3, and IL-5. Conclusion: Our study identifies multiple cytokines pathways involved in inflammation and the tumor microenvironment to be potentially inactivated by differentially expressed miRs in WM compared to IgM-MGUS. Downregulation of these pathways and targeting of the STAT and PIK3 family indicates a possible miR-based negative feedback loop to down-modulate inflammatory cytokine signaling in WM. Citation Format: Karan Chohan, Jonas Paludo, Surendra Dasari, Jithma P. Abeykoon, Prashant Kapoor, Esteban Braggio, Michelle K. Manske, Aneel Paulus, Craig B. Reeder, Sikander Ailawadhi, Asher A. Chanan-Khan, Robert A. Kyle, Morie A. Gertz, Anne J. Novak, Stephen M. Ansell. Micro-RNA induced inactivation of inflammatory cytokine signaling pathways in Waldenstrom macroglobulinemia compared to IgM-MGUS. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3811.

Progression Risk and Long-Term Survival Trends of 915 Patients with Asymptomatic IgM Monoclonal Gammopathy

BACKGROUND: Waldenström macroglobulinemia (WM) is a lymphoplasmacytic lymphoma with a serum monoclonal IgM. Early asymptomatic stages include IgM monoclonal gammopathy of undetermined significance (MGUS) and smoldering WM (SWM). While most IgM MGUS patients (pts) will not progress during follow-up, a greater proportion of SWM pts will do. Most prognostic models identify high-risk SWM; however, they fail to characterize IgM MGUS and low-risk SWM. Moreover, data is scarce regarding how IgM MGUS and SWM survival trends have evolved during the last decades. We wondered whether inclusion of new biomarkers could improve progression risk models using the largest cohort of IgM MGUS and SWM reported. Here, we describe a comprehensive proposal to model progression risk and give more insight into the survival trends of IgM MGUS and SWM using retrospective data from 13 Spanish hospitals. METHODS: Diagnostic criteria were defined by the Second International Workshop. MYD88 L265P was analyzed by allele-specific or digital droplet polymerase chain reaction. Progression free survival (PFS) was calculated from the date of diagnosis to last follow-up or disease progression (symptomatic WM, other non-Hodgkin lymphoma or AL amyloidosis). Overall survival (OS) was calculated up to death from any cause. To find the best model, we first used a backward stepwise regression with meaningful variables as continuous. Also, a logistic and lasso regression (least shrinkage and selection operator) selected important variables that predicted progression at 10 years. We then categorized continuous data according to Liu and Youden methods. Relative survival (RS) was calculated using the Ederer II method leveraging the Human Mortality Database. RESULTS: 915 pts (462 IgM MGUS and 453 SWM) were included. Median age at diagnosis was 70 years. 102 (11%) pts were diagnosed before the year 2000. MYD88 L265P (available in 465 pts) was detected in 107 (54%) IgM MGUS and 217 (81%) SWM pts. After a median follow-up of 6 years, disease progression was documented in 250 pts (16% in IgM MGUS and 39% in SWM). The incidence rate of progression per 100 person-years was 2.9 (95% confidence interval [CI] 2.4 - 3.7) and 7.0 (95% CI 6.1 - 8.2) in IgM MGUS and SWM, respectively. Continuous (serum IgM, IgG, IgA, albumin, β2-microglobulin, bone marrow infiltration [BM]) and categorical (MYD88 L265P) candidates to clinical significance were fitted into a backward stepwise regression. Low IgG (hazard ratio [HR] 0.92; p=0.03), high IgM (HR 1.03; p<0.01), high BM infiltration (HR 1.02; p=0.01), and low albumin (HR 0.37; p<0.01) were selected. As the median follow-up of our series was 6 years, we also performed lasso to select predictors of progression at 10 years. Here, all previous variables were selected plus low IgA. We then chose the 4 best predictors between the two methods (BM infiltration, IgM, decreased IgG/IgA, and albumin). To fit into an easy-to-use model, we categorized continuous data as meaningful for clinicians. Albumin (<3.5 g/L) and IgG/IgA (<6.8/0.6 g/L) were categorized below the normal values; BM infiltration (≥ 20%) and IgM (≥ 10 g/L) cutpoints were established as previously described. 553 pts were introduced in the final model. Each variable was assigned a point so that low (0-1), intermediate (2) and high-risk (3-4) groups were able to discriminate pts. 84% of IgM MGUS pts were categorized as low-risk, while 57% of SWM were intermediate or high-risk. Regarding OS, there were no differences between IgM MGUS and SWM; however, each stage clearly differed from that of series of symptomatic WM pts from the same hospitals (p<0.01) (Figure 1A). Regarding survival trends, IgM MGUS had a RS slightly higher than that of the matched general population during the first 10 years of diagnosis, with a subsequent decline especially at 20 years. SWM had a RS lower than the general population, with a difference observed from the first 10-15 years of diagnosis (Figure 1B). CONCLUSION: We developed a new single ready-to-use risk model accounting for both IgM MGUS and SWM. Most IgM MGUS pts were categorized in the low-risk group, while SWM accounted for the vast majority of intermediate and high-risk groups. There were no differences in OS between IgM MGUS and SWM. However, RS of IgM MGUS was higher in the first 10 years from diagnosis, probably due to both early access to public health and follow-up, while SWM showed a constant decreasing RS. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Genomic Pathways Differentiating IgM-MGUS from Waldenstrom Macroglobulinemia: The Integrated microRNA and Gene Expression Landscape

Introduction Waldenstrom Macroglobulinemia (WM) is a rare non-Hodgkin lymphoma that is preceded by an IgM monoclonal gammopathy of undetermined significance (IgM-MGUS), a premalignant, clinically asymptomatic disorder. The factors underlying this malignant transformation remain poorly understood, but the stepwise accumulation of genomic abnormalities has been identified as a potential mechanism. Non-coding aspects of the genome are increasingly recognized as drivers of disease pathogenesis but remain under-explored in this disease spectrum. Previously, we have shown that several oncogenes are in close genomic proximity to overexpressed long non-coding RNAs in WM compared to IgM-MGUS (Paludo et al. 2019). In this study, we aimed to identify the microRNAs (miR) and genomic pathways which may facilitate the IgM-MGUS to WM malignant progression. Methodology Sorted CD19+ and CD138+ B cells from prospectively collected bone marrow samples of patients with IgM-MGUS (n=7) and WM (n=25) underwent total RNA extraction and sequencing. Profiling of miRNAs was conducted, and selection of targets of interest was performed through identifying differentially expressed miRNA between IgM-MGUS and WM with a log2 fold change (FC)>0.5 or <-0.5 and false discovery rate (FDR) <0.05. Next, mRNA sequencing was conducted on the same patient samples, and differential expression was performed the same way as the miRNA data to identify mRNA candidates. Differentially expressed miRNA/mRNA pairs between WM and IgM-MGUS with correlated biological expression, i.e. either upregulated (up) miRNA experimentally predicted to regulate downregulated (down) mRNA or vice versa, were selected. The miRNA/mRNA pairs identified here were evaluated using Ingenuity Pathway Analysis (IPA) to discover dysregulated genomic pathways between IgM-MGUS and WM. Only pathways with an absolute activation Z-score of ≥2 were selected, and dysregulated miRNA and associated genes involved in altered pathways were identified. Results There were 34 miRNAs (16 up/18 down) found to be significantly differentially expressed between IgM-MGUS and WM (Figure). Among these, miR-4532, miR-192, miR-3195, miR-6761, miR-1295, and miR-3934 were found to have the highest FC between these two conditions. After correlation of miRNA/mRNA pairs, the most differentially regulated pathways with recognized oncological relations were identified, among which, the ID-1, IL-8 and STAT3 pathways were found to be downregulated in WM compared to IgM-MGUS, while the PTEN pathway was found to be upregulated. More broadly, the G protein-coupled receptor (GPCR), and the tumor microenvironment (TM) pathways were both found to be downregulated in WM compared to IgM-MGUS. Some of the main miRNAs driving multiple pathways with key associated genes included: miR-20 (GPCR, TM, IL8, ID1) through regulation of cyclin D1 (CCND1), matrix metalloproteinase-2 (MMP2), and leukemia inhibitory factor (LIF), miR-1260 (ID1, GPCR, STAT3, TM) through regulation of B-cell lymphoma 2 (BCL-2), chemokine receptor 6 (CCR6), and ETS proto-oncogene 1 (ETS1), and miR-1295 (IL8, TM, GPCR) through regulation of vascular cell adhesion molecule 1 (VCAM1), matrix metallopeptidase 19 (MMP19), and hydroxycarboxylic acid receptor 2 (HCAR2). Conclusion Our study is one of the first to identify the altered non-coded transcriptomics in WM and MGUS. We found several differentially expressed miRNAs with a known oncological role, such as miR-4532, miR-20 and miR-1260, highlighting the role of miRNAs as potentially important epigenetic regulators in these two diseases. The genomic pathways found to be potentially regulated by miRNA may underlie the transition between the premalignant to malignant disease of IgM-MGUS to WM. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Single-Cell Transcriptome Analysis Reveals Stem Cell-like Subsets in the Progression of Waldenström's Macroglobulinemia

Waldenström's macroglobulinemia (WM) is an uncommon lymphoproliferative disorder characterized by monoclonal immunoglobulin M protein production and bone marrow infiltration by lymphoplasmacytoid cells. It remains an incurable disease. IgM monoclonal gammopathy of undetermined significance (MGUS) is the premalignant condition and is associated with the risk of developing WM. MYD88L265P is a recurrent mutation found in more than 90% of WM patients, and this genetic event is detectable in at least half of IgM MGUS patients. However, the precise cellular landscape and the mechanisms of progression from IgM MGUS to WM remain unclear. Single-cell RNA sequencing (scRNA-seq) provides us a powerful approach to explore tumor heterogeneity and identify rare malignant cells. Anti-CD38 monoclonal antibodies have been introduced into the therapeutic arsenal for plasma cell diseases. Therefore, we analyzed CD38+ immune microenvironment together with B cells and plasma cells to illustrate the first cellular landscape of IgM MGUS and WM in single cell resolution. We isolated bone marrow CD19+ and CD19-CD38+ cells from WM patients(n=3), IgM MGUS patients(n=3) and healthy donors(n=3) and employed the 10x Genomics platform to perform single-cell transcriptomic sequencing. After quality control, our dataset contained a total of 73024 cells. We used UMAP to visualize the cell superclusters and identified clusters including B cells, plasma cells, CD3+CD20+ (CD19+) cells, T cells and NK cells based on the expression of canonical lineage markers and cluster-specific markers. We found that healthy donors had the highest percentage of B cells (72.7% in healthy donors, 19.0% in IgM MGUS patients, 60.5% in WM patients; p<0.001) and that IgM MGUS and WM patients had more NK and T cells than healthy controls (0.06% and 17.7%, respectively, in healthy donors; 24.1% and 20.5% in IgM MGUS patients; 8.5% and 24.0% in WM patients; p<0.001). We also noticed that CD3+CD20+(CD19+) cells were present in both IgM MGUS and WM patients. First, we investigated the cell clusters correlated with WM pathology including B cells and plasma cells. We compared the B-cell gene expression profiles after removing the early B cells. Compared with those of IgM MGUS patients, mature B cells of WM patients showed upregulation of PIM1, DUSP22, HES1, NEAT1, RGS1, RGS16, and GADD45B. Light chain restriction and higher CNV levels were found in B cells of WM patients. SCENIC analysis showed that FOS, FOSB, EGR1 and JUNB were downregulated in B cells from WM and IgM MGUS patients. REL, a proto-oncogene promoting the survival and proliferation of B lymphocytes, was upregulated in B cells of WM patients, and IRF8 and ELF1 were upregulated in B cells of IgM MGUS patients. Nevertheless, plasma cells of WM patients, IgM MGUS patients and healthy donors were colocalized in one cluster, which might indicate their similar gene expression patterns. Next, we further determined the characteristics of B-cell subpopulation with aberrant T-cell antigen expression. Trajectory analysis revealed that CD3+CD20+(CD19+) cells and pro-B cells were located at the same end of the branched structure and were then directed toward mature B cell and plasma cell fates, respectively (Figure. 1A). This inferred developmental trajectory suggested that CD3+CD20+(CD19+) cells may be tumor stem cell-like subset. Using flow cytometry, we validated that CD3 was expressed in a fraction of CD19+ cells (mean: 3.69%, range: 0.28%-15.10%) of bone marrow samples from 8 WM patients. In addition, targeted gene sequencing was performed on CD3+CD19+ and CD3-CD19+ cells from 6 WM patients. MYD88 were proved to be the most frequently mutated gene in this cell subset. And we inferred that MYD88 mutation might be the early events in tumorigenesis by VAF analysis (Figure. 1B). Additional subclonal hits, such as CXCR4 and MAP2K1 mutations, could be acquired during tumor progression. In conclusion, our study provides comprehensive insights into mechanisms of progression from IgM MGUS to WM. We identified the rare CD19+ cell subpopulation with T-cell antigen expression in WM patients. It will be interesting to explore novel therapeutic strategies targeting rare potential cancer-initiating cells in future. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Monoclonal Gammopathy of Undetermined Significance and Risk of Chronic Kidney Disease: Results of the Population-Based Iceland Screens, Treats, or Prevents Multiple Myeloma (iStopMM) Study

Background: Monoclonal gammopathy of undetermined significance (MGUS) and chronic kidney disease (CKD) are common conditions. When monoclonal gammopathy causes CKD it is referred to as monoclonal gammopathy of renal significance (MGRS) and a kidney biopsy is required to confirm the diagnosis. Because of this, some have speculated that MGRS may be severely underdiagnosed and have recommended considering a kidney biopsy in all individuals with MGUS who have microscopic hematuria, proteinuria, or declining estimated glomerular filtration rate (eGFR) of <60 mL/min/1.73m2. If MGRS were more common than previously thought, the prevalence of CKD in persons with MGUS would be expected to be higher than persons without MGUS. We aimed to compare the prevalence of CKD in individuals with and without MGUS in a large, prospective, screened, population-based cohort. Furthermore, we aimed to assess possible association between M protein concentration and kidney function. Methods: A total of 75,422 participants in the Iceland Screens, Treats or Prevents Multiple Myeloma (iStopMM) study were screened for MGUS with serum protein electrophoresis (SPEP), serum free light chains (FLC), and immunofixation (IFE). All previous clinical laboratory samples were obtained, including serum creatinine (SCr), urine albumin/creatinine ratio (ACR) and urinalysis. CKD was defined as SCr-based eGFR of <60 mL/min/1.73m2, or proteinuria or hematuria, present for >90 days. Participants diagnosed with lymphoproliferative disorders or without data on kidney function were excluded. Power analysis demonstrated that this sample size yielded 90% power to detect a 1.5% absolute difference in the prevalence of CKD in persons with and without MGUS. Prevalence of CKD was evaluated using multivariable logistic regression adjusted for age, sex and MGUS status. The association between eGFR and M protein concentration was assessed with linear regression for continuous eGFR and ordinal logistic regression for CKD categories. All models included age and M protein concentration as four knot restricted cubic splines. Sensitivity analyses were performed where CKD was defined solely by persistently lowered eGFR and as a combination of persistently lowered eGFR, proteinuria, and hematuria. Results: A total of 69,120 participants had SCr measurements and 34,294 had urine studies available. MGUS was present in 4,547 (6.6%) participants, of which 2,242 (49.3%) were non-IgM; 703 (15.5%) were IgM; 1,295 (28.5%) were light chain (LC-MGUS); and 307 (6.7%) were biclonal MGUS. A total of 6,680 (9.7%) participants had CKD defined either as eGFR <60 mL/min/1.73 m2 (N=5,915) or persistent proteinuria or hematuria (N=693), or were receiving treatment for end-stage kidney disease (N=74). Participants with MGUS were significantly older (median [IQR] age 69 [60-76] vs 60 [52-69] years) and more predominantly male (55% vs 41%) compared with participants without MGUS. After adjusting for age and sex, no significant association was found between heavy chain MGUS and CKD (odds ratio [OR] 1.00, 95% confidence interval [CI] 0.95-1.17). This also remained true for subgroups of non-IgM MGUS, IgM MGUS, and biclonal MGUS (Table). Participants with LC-MGUS were less likely to have CKD than participants without MGUS (OR 0.39, 95% CI 0.31-0.49, p <0.001). Sensitivity analyses of CKD definition yielded similar results. M protein concentration had a small negative correlation with eGFR (r= -0.08, p<0.001) but when stratified by age and sex category no significant correlation remained. No significant association was found between M protein concentration and worse eGFR category on ordinal logistic regression model adjusted for nonlinear age and sex (OR 1.01, 95% CI, 0.28-3.66). No significant correlation was found between M protein concentration and severity of proteinuria assessed with urine ACR. Conclusion: In this nationwide prospective population-based screening study of nearly 70,000 individuals, we found no association between MGUS and CKD, regardless of whether CKD was defined based on eGFR, urinary findings, or both. Furthermore, we found no association between M protein concentration and eGFR or severity of albuminuria. We conclude that in the absence of multiple myeloma, the results of SPEP, IFE, and serum FLC measurements are of little value in the evaluation of CKD. Furthermore, our results are not consistent with substantial underdiagnosis of MGRS. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

VEGF and IL-6 As Markers of Severity and Response to Treatment in Waldenstrom Macroglobulinemia and IgM-Monoclonal Gammopathy of Undetermined Significance: A Monocentric Experience

Background: Angiogenesis plays an important role in the development and the maintenance of hematolymphoid malignancies. Serum levels of angiogenic cytokines, such as vascular endothelial growth factor (VEGF) and interleukin 6 (IL-6), are known to be increased in patients with Waldenstrom Macroglobulinemia (WM) and IgM monoclonal gammopathy of undetermined significance (MGUS). Even if the role of IL-6 and VEGF as markers of disease status and severity has been recognized, only a limited number of studies have addressed the role of cytokines in WM and there is no proof about any correlation between their serum levels in WM and IgM-MGUS patients. Nevertheless, their dosage at various stages of the disease aims to understand if they have a comparable pattern, and therefore they could be used in clinical practice for disease monitoring. Aims: This study aims to find a correlation of serum levels of VEGF and IL-6 with the burden of disease in our patients with IgM-MGUS and WM and to evaluate a possible relationship between the two angiogenic cytokines, in an attempt to identify potential markers of severity. Methods: We performed a retrospective monocentric study of 18 patients (ten with WM and eight with IgM-MGUS) with a median age of 73 years (range: 52-90), whose 66% were male. Patients with WM were at different stages of disease (four were naive, four in remission, two relapsed/refractory). A total of 18 controls with a similar age and gender distribution were also tested. Controls were selected among patients with neuropathies in the absence of any hematological condition. We used the 1:1 ratio for the selection of controls: for every male or female WM patient, one control of the same gender and of similar age was included. In order to find a relationship of VEGF and IL-6 with a laboratory parameter, IgM levels dosage for each patient have been measured at the same time-point of the measurement of the cytokines. In all patients and controls, we evaluated serum levels of VEGF and IL-6 using Immunoassay panels (i.e., anti-IL-6 and anti-VEGF ELISA kit). Results: All patients showed elevated values of angiogenic cytokines when compared with controls (P value < 0.05). Both VEGF and IL-6 serum levels (median values: 623,5 pg/mL and 4,5 pg/mL, respectively; ranges: 113,4-1981 pg/mL and 0,3-21,9 pg/mL, respectively) showed a positive correlation with IgM serum levels (median value: 2055 mg/dL, range: 300-6123 pg/mL) (Fig. 1a and 1b). Furthermore, the relationship between IL-6 and VEGF was statistically significant and could be explained by a linear model (P value = 0,007) (Fig. 1c). No statistically significant differences were observed between the different phases of WM. Conclusions: Evidence from our sample shows that serum levels of angiogenic cytokines as VEGF and IL-6 have a comparable pattern with IgM levels. Therefore, it seems reasonable to suggest that they could be used in clinical practice as markers of severity of disease and response to treatment. Moreover, since the cytokines profiles are comparable, IL-6 can be used interchangeably with VEGF, allowing the monitoring of the disease with easily availability and unexpensive techniques. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Prevalence of MGUS Is High in the Istopmm Study but the Prevalence of IgA MGUS Does Not Increase with Age in the Way Other Immunoglobulin Subtypes Do

Introduction: Monoclonal gammopathy of undetermined significance (MGUS) is an asymptomatic plasma cell disorder that precedes the onset of multiple myeloma (MM) and other lymphoproliferative disorders. Detailed data on the prevalence and natural course of MGUS are key to understanding the risk of MGUS progression, as well as providing relevant clinical advice to individuals with MGUS on their personal risk and optimal follow-up strategy. Data on the prevalence of MGUS in the general population collected at the end of the 20th century have served as the key reference for physicians and researchers seeking to understand the epidemiology of MGUS in the general population. Nearly a quarter century later we are now able to use data from the Iceland Screens, Treats, or Prevents Multiple Myeloma (iStopMM) study to refine our understanding of MGUS further and provide a more detailed picture of the prevalence and characteristics of MGUS in a screened population. Aims: Describe the population prevalence of MGUS by age and sex, the prevalence of individual immunoglobulin subtypes, and the distribution of risk groups. Methods: The iStopMM study has screened 75,422 individuals, representing 51% of all Icelanders aged 40 and over at the time of study initiation. To detect and quantify monoclonal proteins, serum protein electrophoresis (SPEP) was performed on all samples followed by immunofixation electrophoresis (IFE) if a monoclonal spike was found or if the free light chain (FLC) ratio was abnormal. Age was based on the date of sample collection. We used these data to calculate the population prevalence of MGUS, age- and sex- standardized to the invited study population of all 148,704 individuals aged 40 and over living in Iceland at the start of the study. We calculated separate prevalence rates for the whole study group and for individuals over 50 to allow for direct comparison to prior studies. Then we plotted the prevalence in 10-year age groups by sex with 95% confidence intervals (CI). In a second graph we used 5-year age groups and plotted the prevalence of MGUS as well as of each of three subtypes of immunoglobulins, IgG, IgM, and IgA and multiclonal MGUS. Age groups over 85 were collapsed into one. We added cubic smoothing splines with bootstrapped 95% confidence intervals (CI) to both plots. Finally, we used the Mayo Clinic risk stratification model to assign all individuals diagnosed with MGUS to risk groups. Results: In 75,422 screened samples we diagnosed MGUS in 3,358, with IgG in 1,923 (57%), IgM in 721 (21%), IgA in 400 (12%), and more than one clonal spike in 314 (9%). The crude prevalence of MGUS in the study group was 4.5% (95% CI 4.3%-4.6%). The age and sex standardized prevalence was 3.9% (95% CI 3.8%-4.0%) for the whole group, and 5.0% (95% CI 4.9%-5.2%) when limited to age over 50. The crude prevalence rates with 95% CIs are shown for 10-year age groups divided by sex in Figure 1. Figure 2 shows the crude prevalence rates by 5-year age groups for MGUS and subtypes of immunoglobulin monoclonal spikes. Based on the Mayo Clinic risk stratification model, 1,445 (43.0%) were low risk, 1,356 (40.4%) were low-intermediate risk, 547 (16.3%) were high-intermediate risk, and 10 (0.30%) were at high risk for progression. Discussion: The results presented here show an age- and sex- standardized prevalence of 5% among individuals over 50 years of age in the largest population-based screening study of MGUS among individuals of Western European descent to date. This is a higher population standardized prevalence of MGUS than previously reported for this age group, but this study confirms the pattern of higher prevalence with increasing age and male gender seen in other studies. Our data show, for the first time, that IgA MGUS behaves in sharp contrast to other immunoglobulin subtypes, with prevalence rising slowly with advancing age, if at all after age 70. As previous studies show that immunosenescence involves an increased amount of overall IgA secretion, a phenomenon also observed in the iStopMM study (data not shown), it is important to consider several potential explanations, including that IgA M-proteins are more often transient, arise less frequently later in life, or are associated with a shortened lifespan. Follow-up data from the iStopMM study will provide further insights into this new discovery. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Type 1 Cryoglobulinaemia Associated with Waldenström Macroglobulinemia, IgM MGUS or Non-Hodgkin Lymphoma

Introduction: Type 1 cryoglobulinaemia is defined by monoclonal immunoglobulins which precipitate at temperatures <37°C and redissolve on warming. IgM monoclonal protein may be associated with type I cryoglobulinaemia with underlying Waldenström macroglobulinemia (WM), non-Hodgkin lymphoma (NHL) or monoclonal gammopathy of undetermined significance (MGUS). Due to its rarity, the incidence and clinical outcomes are poorly understood, with fewer than 40 patients characterised in the largest series. Clonal characteristics have not previously been described. We analysed the incidence, clinical and clonal characteristics of the largest reported series of IgM-associated type I cryoglobulinaemia. Methods: Data for consecutive adult patients (> 18 years) with IgM-associated type I cryoglobulinaemia identified between 2013-2022 from 2 centres (University College London Hospital [UCLH], UK and AMC, Netherlands) were retrospectively analysed and followed up to June 2022. In order to calculate the incidence of type I cryoglobulinaemia, the total number of requests performed at UCLH was also determined. Results: 423 samples were screened for cryoglobulinaemia between 2013-2022 from all patients with an IgM-associated disorder at the specialist centre, UCLH. 140 (33%) were positive for all-type cryoglobulinaemia; 94 (22%) patients had type I cryoglobulinaemia. Four additional patients were recruited from Amsterdam UMC, without incidence data. In total, 98 patients (58 male, 40 female) were identified with type I cryoglobulinaemia; the majority with underlying WM (defined as >10% bone marrow infiltration) (77; 79%), and remaining IgM MGUS (16; 16%) and NHL (4 marginal zone lymphoma, 1 CLL; 5%). The median age at cryoglobulin diagnosis was 66 years (range 39-90). There was a kappa light-chain predominance in the majority (85%). MYD88L265Pwas present in 44/49 (90%) of WM and 3/8 (38%) MGUS cases. CXCR4 was mutated in 5/13 (38%) WM cases. Coexistent IgM-associated disorders were present in 25: cold agglutinin disease/syndrome (CAD/CAS, 15%), Bing-Neel syndrome (7%), anti-MAG antibodies (5%), Schnitzler syndrome (1%). Those with coexistent CAD/CAS had characteristics of a CAD clone, significantly associated with MGUS (vs WM/NHL, p<0.0001) and MYD88-wild type (p<0.0001). Patients with co-existent anti-MAG antibodies were significantly more likely to have MGUS (p=0.0245). Median M-protein at diagnosis of cryoglobulinaemia was 12g/l (range 0-63) and lowest in the MGUS cohort vs WM vs NHL (4g/l vs 16g/l vs 10g/l, p=0.0003). The majority were diagnosed with cryoglobulinaemia >3 months after diagnosis of the underlying IgM disorder (60/98; 61%), at a median of 46 months (range 4-442) and longer in those with WM compared with MGUS or NHL (51 vs 17 vs 13 months, p=0.023). Forty nine percent were symptomatic at cryoglobulinaemia diagnosis, with a trend towards a greater proportion in those with MGUS/NHL vs WM (75%/60% vs 43%, p=0.0569). The most common symptoms were cutaneous/vasomotor symptoms (28%), neuropathy (21%), hyperviscosity (10%), arthralgia (7%) and renal involvement (2%). No cases had cardiac or pulmonary involvement. At a median follow up of 2.4 years (range 0-9.4), 2-year overall survival (OS) was 89% (95% CI 80-94), 5-year OS was 73% (95% CI 56-84). Age was the only predictor of OS (Hazard ratio [HR] 1.07, 95% CI 1.01-1.12, p=0.006). Fourteen patients required chemoimmunotherapy for cryoglobulinaemia at a median of 1 month (0-7) from diagnosis for cutaneous manifestations (5/14; 36%), cryoglobulinaemic glomerulopathy (2/14; 14%) or hyperviscosity (9/14; 64%). Independent predictors of cryoglobulinaemia-indicated treatment-free survival/death included cutaneous involvement (HR 1.48, 95% CI 1.21-7.7.92, p=0.018) and hyperviscosity (HR 5.21 95% CI 4.21-27.8, p<0.0001) at cryoglobulinaemia diagnosis on multivariate analysis. Conclusions: This is the largest reported series describing the characteristics of IgM-associated type I cryoglobulinaemia. It is common amongst patients with monoclonal IgM disorders and approximately half of patients may be symptomatic. Distinct clonal populations are present in which type I cryoglobulinaemia may develop including an MYD88L265P WM clone and MYD88-wild type CAD/anti-MAG clone. Treatment may be required selectively in those with hyperviscosity, cutaneous or renal involvement, in a short time from diagnosis. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Patients with IgM Monoclonal Gammopathy of Undetermined Significance (MGUS) or IgA with an M Band < 1.0 g/DL Have the Same Low Risk of Progression to Lymphoplasmacytic Neoplasm (LPN) As Those with IgG MGUS and an M-Band < 1.5 g/DL

Background Patients with MGUS can progress to LPN. Non-IgG subtype and the size of the M-band are known to correlate with the risk of progression. Consensus guidelines recommend that patients be monitored on an annual basis, but that IgG MGUS with an M-band < 1.5 g/dL, and a normal serum free light chain ratio is at low risk and can be managed with less intensive monitoring. There are limited data on the risk of progression to LPN in non-IgG MGUS with small M-bands. Methods Kaiser Permanente Northern California (KPNC) is a large integrated health care system serving more than 4 million members representative of Northern California residents. We identified a cohort of 3,303 KPNC health plan members who had at least one serum protein electrophoresis (SPEP) performed between January 1997 and December 2006 and extracted follow up data through December 2016. We excluded those with an M-Band > 3 g/dL, absence of a confirmatory immunofixation, or LPN before through 3 months after initial SPEP. The primary end point was progression to LPN, defined as multiple myeloma (MM), non-Hodgkin's lymphoma, or amyloidosis, and identified via an integrated tumor registry and, in the case of amyloidosis, by clinic diagnosis. Findings The median age of this cohort was 73 (IQR 63-80), 54.5% were male, and the ethnicity of this group was White 66.9%, Black 17.2%, Asian 6.7%, Hispanic 7.7%, and others 1.6%. Sixty six percent had an IgG gammopathy and 73% of gammopathies were < 1.0 g/dL. Overall, there were 2.1 (2.0-2.3) progressions per 100 patient years. The number of progressions per 100 patient years for an M-band < 1.0 g/dL, 1.0-1.5 g/dL, and 1.5-3.0 g/dL were 1.4 (1.2-1.6), 3.5(2.9-4.1), and 6.6 (5.5-7.7) respectively. As can be seen in the Figure, the cumulative risk of progression to LPN was clinically similar for patients with an IgG ≥ 1.5 g/dL and IgA ≥ 1.0 g/dL and considerably increased compared to those with an IgG gammopathy < 1.5 gm/dL, IgA gammopathy < 1.0 g/dL, or an IgM of any size. Discussion It has been estimated that there are several million individuals in the United States with MGUS. Following a patient with MGUS indefinitely is a financial burden and creates a substantial psychologic burden for the patient. Others have found that the effect of an MGUS diagnosis on quality of life is greater than a new diagnosis of symptomatic MM. Therefore, identifying additional categories of MGUS that could be assigned to a low-risk category could potentially diminish the burden and stress of ongoing monitoring. Limitations of our study are that, owing to the period from which our cohort was accrued, and the nature by which they were identified, free light chain levels were not obtained, bone marrow biopsies were done infrequently, and modern advanced imaging was not performed. In the modern era, many of our cohort would likely be identified with more advanced neoplasia such as smoldering or frank MM accounting for the higher rates of progression compared with other published reports. We found that during much of the period covered by our study, the clinical diagnosis of Waldenstrom's Macroglobulinemia was used in a non-discriminatory manner. We therefore limited our definition of progression only to those subjects developing frank malignancy that qualified for inclusion in our tumor registry rather than the less rigorously defined entity of "Waldenstrom's Macroglobulinemia.". This likely accounts for the lower risk of progression to LPN among our cohort with IgM gammopathy than has been described elsewhere. Conclusion Patients with either IgM MGUS of any size or IgA MGUS with an M-band < 1.0 g/dL have an equivalent clinical risk of progression as patients with an IgG MGUS < 1.0 g/dL and should potentially be assigned to a low-risk category to decrease the burden and stress of ongoing monitoring. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Prognostic impact of MYD88 and CXCR4 mutations assessed by droplet digital polymerase chain reaction in IgM monoclonal gammopathy of undetermined significance and smouldering Waldenström macroglobulinaemia.

Waldenström macroglobulinaemia (WM) is characterized by recurrent somatic mutations in MYD88 and CXCR4 genes. However, limitations arise when analysing these mutations in IgM monoclonal gammopathy of undetermined significance (MGUS) or smouldering WM (SWM) given the lower tumour load. Here, we used droplet digital polymerase chain reaction (ddPCR) to analyse MYD88 L265P and CXCR4 S338* mutations (C1013G and C1013A) in unsorted bone marrow (BM) or cell-free DNA (cfDNA) samples from 101 IgM MGUS and 69 SWM patients. ddPCR was more sensitive to assess MYD88 L265P compared to allele-specific PCR, especially in IgM MGUS (64% vs 39%). MYD88 mutation burden correlated with other laboratory biomarkers, particularly BM infiltration (r=0.8; p < 0.001). CXCR4 C1013G was analysed in MYD88-mutated samples with available genomic DNA and was detected in 19/54 (35%) and 18/42 (43%) IgM MGUS and SWM cases respectively, also showing correlation with BM involvement (r=0.9; p < 0.001). ddPCR also detected 8 (38%) and 10 (63%) MYD88-mutated cfDNA samples in IgM MGUS and SWM respectively. Moreover, high BM mutation burden (≥8% MYD88 and ≥2% CXCR4) was associated with an increased risk of progression to symptomatic WM. We show the clinical applicability of ddPCR to assess MYD88 and CXCR4 in IgM MGUS and SWM and provide a molecular-based risk classification.

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.

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.

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.

IgM Paraprotein-Associated Type 1 Cryoglobulinaemia: Clinical Characteristics and Outcomes

IntroductionType 1 cryoglobulinaemia (CG) is characterised by monoclonal immunoglobulins which precipitate at temperatures below 37°C and redissolve on warming. They may be associated with lymphoproliferative disorders including Waldenström's macroglobulinemia (WM), other non-Hodgkin lymphoma (NHL), chronic lymphocytic leukaemia (CLL) or monoclonal gammopathy of undetermined significance (MGUS). Due to their rarity and heterogeneous clinical manifestations, incidence and outcomes are not well characterised and they are likely underdiagnosed. We retrospectively reviewed our cohort of patients (pts) with IgM paraprotein-associated type 1 CG.MethodsData from consecutive pts during 2013 and 2021, aged >18 years were extracted from clinical databases at two specialist centres [UCLH, United Kingdom (UK) and AMC, Netherlands].ResultsA total of 62 pts (38 male, 24 female) were identified (Table 1); 59 from UK and 4 from Netherlands: 49 (79%) had WM, 7 (11%) IgM MGUS and 6 (10%) NHL (5 other lymphoma, 1 CLL). Median age at CG diagnosis was 66 (range 39-90) years; 32 (52%) were >65 years. MYD88 was mutated in 23/25 (92%) evaluable cases of WM. All cases were negative for hepatitis C.CG was detected after the monoclonal disorder in 46 (74%), with a median time to CG diagnosis of 8 (range 0-1390) months, concurrently in 11 (18%) pts and at a median of 1 month (range 0-4) in 5 (8%) pts prior to the monoclonal disorder. These patients were diagnosed due to CG symptoms. Eight pts (of which 50% had WM) also had active cold agglutinin disease (CAD).CG symptoms were present at time of testing in 25 (40%) pts; the others were diagnosed as a part of asymptomatic screening. CG symptoms were more common in those with MGUS / NHL compared to WM, most frequently in MGUS compared to WM (33% v 71%, p=0.05). Skin manifestations including acrocyanosis, purpura, ulcer and necrosis were noted in 14 (23%); 11 (18%) had peripheral neuropathy (6 sensory, 5 mixed) and 8 (13%) hyperviscosity. Median plasma viscosity was >7 (range 4.7 - >7) mPa of 5/8 pts measured with hyperviscosity and a median paraprotein of 29 (range 5-63) g/l. One patient with WM had cryoglobulinaemic glomerulopathy demonstrated by renal biopsy.In all, 53 (85%) pts received treatment, 10 (16%) for the CG and 43 for the monoclonal disorder, including plasma exchange (11/53). Thirty had Rituximab-based therapy (Table 1), and one received Ibrutinib. All achieved complete resolution of symptoms and 3/6 (50%) treated for CG had complete biochemical response with cryoglobulins undetectable after treatment. Two (20%) required further lines of therapy >4 years later. Overall at a follow up of 21 (range 0-94) months, median survival was not reached. Nine (14%) pts died, with 1 (2%) CG-related death due to relapse disease. Estimated 5-year overall survival (OS) was 67% (95% CI 40-84%) (figure 1).ConclusionsIn our cohort of 62 pts with type I IgM paraprotein-associated CG, the majority had WM compared to other NHL and MGUS, likely reflecting the clinical bias of the centres and our policy of screening for CG at first visit. A greater proportion of cases (40%) were symptomatic than previous reports (16%; Néel et al, 2014); when present, symptoms were dominated by skin manifestations, neuropathy and hyperviscosity. Patients tested for CG with IgM MGUS were more likely to be symptomatic compared to WM. CAD co-existed in a proportion. Those with CG symptoms treated had good clinical responses; treatment subgroups were too small to draw conclusions as to relative efficacy, but Rituximab-based therapy appeared effective in most cases. CG-related mortality was low with an estimated 5-year OS 67%. [Display omitted] DisclosuresVos: Sanofi: Membership on an entity's Board of Directors or advisory committees; Celgene: Other: Travel reimbursement. D'Sa: Janssen Cilag: Honoraria, Research Funding; BeiGene: Honoraria, Research Funding; Sanofi: Honoraria.