BACKGROUND:Monoclonal proteins are detected using electrophoretic techniques and used to help diagnose plasma cell dyscrasias. The Immunoglobulin Isotypes (GAM) for the EXENT® analyser (EXENT assay) is an automated MALDI-ToF mass spectrometry immunoassay, designed for use with Optilite® turbidimetric assays for the identification and measurement of monoclonal immunoglobulins (M-proteins). The assay reports M-protein presence, concentration, isotype, and mass/charge ratio (m/z). METHODS:The EXENT assay precision performance, linearity, lower limit of the measuring interval (LLMI) and interference were established per CLSI guidelines. M-proteins were evaluated using electrophoretic methods or the EXENT assay in clinical samples from 718 patients with various monoclonal gammopathies, 417 disease controls and 364 healthy samples at 3 different laboratories. RESULTS:The EXENT assay demonstrated a lower measuring limit of 0.308 g/L, 0.073 g/L and 0.054 g/L for IgG, IgA, and IgM in samples with normal polyclonal levels. Total imprecision ranged from 3.7 to 12.8% across all isotypes, and between-laboratory was 5.1-14.3%. Therapeutic monoclonal antibodies were flagged from endogenous IgG kappa M-proteins and showed no interference when m/z thresholds were met. Clinical cut-offs were established at 0.359 g/L (IgG), 0.325 g/L (IgA), and 0.197 g/L (IgM). The EXENT assay detected M-proteins in 92.6% of monoclonal gammopathy patients versus 85.1% by serum protein electrophoresis (SPE), with strong concentration correlations (r = 0.874) and 97% isotype concordance in positive samples. CONCLUSIONS:The EXENT assay demonstrated comparability to SPE and immunofixation electrophoresis (IFE) for M-protein detection and the evaluation of monoclonal gammopathies.

Detection of light chain (LC) monoclonal gammopathies (MGs) traditionally relies on serum free LC (FLC) kappa, lambda, and their ratio (kappa/lambda) reference ranges based on a mostly White population. We investigated FLC values in a racially diverse population by screening 10 035 individuals for heavy chain MG, identifying 9028 negative cases whose FLC were measured. Participants included 4149 from the PROMISE study (United States, n = 2383; South Africa, n = 1766) and 4879 from the Mass General Brigham Biobank, with 44% self-identifying as Black. Using standard FLC reference ranges, 1074 of 10 035 individuals (10.7%) were diagnosed with LC monoclonal gammopathy of undetermined significance (MGUS), with 99% being kappa-restricted. In the United States, 14.8% of Black and 4% of White individuals were diagnosed (P < .01). Among US participants of African (AFR) and European (EUR) genetic ancestry, 14.4% AFR and 2.9% EUR were diagnosed (P < .01). Among South Africans (100% Black), 27.8% were diagnosed using standard ranges. To avoid overdiagnosis, we propose a new kappa/lambda ratio reference range (0.686 to 2.10) for populations of AFR descent with normal renal function, with standard values for kappa and lambda being 7.97 to 77.50 mg/L and 6.20 to 49.20 mg/L, respectively. This reduces LC-MGUS overdiagnosis by 91% (10.7% vs 0.97%). Using the new reference, LC-MGUS accounts for 8.8% of MGUS cases, with 74% being kappa-restricted, consistent with LC myeloma rates. These findings highlight the importance of basing disease definitions, such as MGUS, on diverse populations. Adopting our proposed FLC reference values would reduce MGUS overdiagnosis among Black individuals, avoiding unnecessary financial, psychological, and medical consequences. This study includes data from NCT03689595.

Background We previously demonstrated that monoclonal gammopathies can be detected in a population at risk for myeloma using mass spectrometry (MS), identifying two entities: monoclonal gammopathy of indeterminate potential (MGIP) and MS detected MGUS (MS-MGUS). However, it remains unclear whether these early clones progress to clinically detected MGUS or multiple myeloma (MM). To investigate this, we utilized archived blood samples from individuals in the screening arm of the National Cancer Institute Prostate, Lung, Colorectal, and Ovarian Cancer Screening (PLCO) Trial, collected up to 22 years before their clinical diagnosis with MGUS or MM. Methods Samples were obtained from healthy adults aged 55-74 enrolled between Nov 1993 and July 2001. We focused on participants who developed either MM or MGUS based on clinical records of serum protein electrophoresis (SPEP and IFX) and serum free light chain (sFLC) tests conducted by Landgren et al. We used matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) MS coupled with turbidimetry (EXENT and Optilite, Binding Site part of Thermo Fisher Scientific) to detect and quantify monoclonal peaks in the serum. Monoclonal peaks were categorized as previously described: MGIP (0.015-0.2 g/L) or MS-MGUS (>0.2 g/L). Participants were classified into: MS-MGUS, MGIP or Negative. Results We analyzed 2,219 serial samples from 509 participants (median 5 samples, range 1-6) who later developed MM (n=134; median 13 yrs [IQR: 10-17]) or MGUS (n=375: median 17 yrs [IQR: 13-19]). Among the 128 participants who progressed to MM (excluding 6 with light chain only disease), 123 (96%) had at least one sample positive for MGIP or MS-MGUS before clinical diagnosis. Of these, 90 cases (73%) showed an MS-MGUS peak, while 33 cases (27%) an MGIP peak at the earliest sample. MGIP (n=23) or MS-MGUS (n=31) was detected in 54 out of 56 patients (96%) who had available samples before the clinical MGUS diagnosis (median 3 yrs, range 1-6 yrs). A log-rank test showed no significant difference in time of progression to MM between individuals whose first sample had a monoclonal protein detected at concentrations of MGIP (median: 13, IQR 10-17 yrs) vs. MS-MGUS (median: 14, IQR 11-17 yrs) (p=0.92). We captured progression of the MGIP to MS-MGUS in 6 and 26 participants, before MM or MGUS clinical diagnosis, respectively. We leveraged the sensitivity of MS to determine the number of monoclonal peaks present in each sample, their isotype, and mass-to-change ratio and then followed those clones across time in the serial samples. The purpose of this was to gain insight into early clonal dynamics among expanded B cell/plasma cell clones and elucidate how these monoclonal populations compete over time. Several distinct patterns were observed in 422 individuals who had at least 2 positive samples serially tested. In 324/422 participants (77%), there was a dominant clone present in all samples and had minimal dynamic alterations over time. In 127 of the 324 (39%) participants with a persistent dominant clone, MS detected only one clone in the first sample, which remained dominant with follow up until clinical detection. For the remaining cases, 197/324 (61%) participants, MS demonstrated the presence of multiple monoclonal clones (median of 2 clones, range: 2-13) in which one clone remained dominant. In the remaining 98/422 (23%) participants, clonal dominance varied significantly over time with different clones becoming dominant at various timepoints. When clones were competing for dominance, IgM isotype clones were the first o be detected at the MGIP stages and were replaced by IgG clones later becoming the dominant clone in the majority (43%) of the observed cases during follow up over time. This phenomenon of transitioning clones was observed at similar rates among participants regardless of their progression to MGUS or MM. The clonal transition was similar in all cases regardless of the clone's concentration at first available sample (MGIP or MS-MGUS) or isotype (IgG, IgA, or IgM). Conclusion Our data demonstrates that monoclonal gammopathies detected by MS precede the clinical diagnosis of MGUS or MM by up to 22 years. In most cases, a dominant clone persists over time, while in 23%, there is notable clonal transition. Further investigation is needed to determine if these IgM to IgG changes constitute isotype class switch within the same B cell clone during plasma cell differentiation.

Abstract Monoclonal gammopathy of undetermined significance (MGUS) is a premalignant condition of multiple myeloma with few known risk factors. The emergence of mass spectrometry (MS) for the detection of MGUS has provided new opportunities to evaluate its risk factors. In total, 2628 individuals at elevated risk for multiple myeloma were enrolled in a screening study and completed an exposure survey (PROMISE trial). Participant samples were screened by MS, and monoclonal proteins (M-proteins) with concentrations of ≥0.2 g/L were categorized as MS-MGUS. Multivariable logistic models evaluated associations between exposures and MS outcomes. Compared with normal weight (body mass index [BMI] of 18.5 to <25 kg/m2), obesity (BMI of ≥30 kg/m2) was associated with MS-MGUS, adjusting for age, sex, Black race, education, and income (odds ratio [OR], 1.73; 95% confidence interval [CI], 1.21-2.47; P = .003). High physical activity (≥73.5 metabolic equivalent of task (MET)-hours per week vs <10.5 MET-hours per week) had a decreased likelihood of MS-MGUS (OR, 0.45, 95% CI, 0.24-0.80; P = .009), whereas heavy smoking and short sleep had increased likelihood of MS-MGUS (>30 pack-years vs never smoker: OR, 2.19; 95% CI, 1.24-3.74; P = .005, and sleep <6 vs ≥6 hours per day: OR, 2.11; 95% CI, 1.26-3.42; P = .003). In the analysis of all MS-detected monoclonal gammopathies, which are inclusive of M-proteins with concentrations of <0.2 g/L, elevated BMI and smoking were associated with all MS-positive cases. Findings suggest MS-detected monoclonal gammopathies are associated with a broader range of modifiable risk factors than what has been previously identified. This trial was registered at www.clinicaltrials.gov as #NCT03689595.

Introduction:The EXENT® Solution, a fully automated system, is a recent advancement for identifying and quantifying monoclonal immunoglobulins in serum. It combines immunoprecipitation with MALDI-TOF mass spectrometry. Compared to gel-based methods, like SPEP and IFE, it has demonstrated the ability to detect monoclonal immunoglobulins in serum at lower levels. In this study, samples that tested negative using EXENT® were reflexed to LC-MS to determine if the more sensitive LC-MS method could identify monoclonal immunoglobulins missed by EXENT®.Objectives:To assess whether monoclonal immunoglobulins that are not detected by EXENT® can be detected by LC-MS using a low flow LC system coupled to a Q-TOF mass spectrometer.Methods:Samples obtained from patients confirmed to have multiple myeloma (MM) were diluted with pooled polyclonal human serum and analyzed using EXENT®. If a specific monoclonal immunoglobulin was not detected by EXENT®, the sample was then subjected to analysis by LC-MS. For the LC-MS analysis, the sample eluate, obtained after the MALDI-TOF MS spotting step, was collected and transferred to an autosampler tray for subsequent analysis using LC-MS.Conclusion:LC-MS has the capability to detect monoclonal immunoglobulins that are no longer detected by EXENT®. Reflexing samples to LC-MS for analysis does not involve additional sample handling, allowing for a faster time-to-result compared to current approaches, such as Next-Generation Sequencing, Next-Generation Flow, and clonotypic peptide methods. Notably, LC-MS offers equivalent sensitivity in detecting these specific monoclonal immunoglobulins.

fracture and show reduced overall survival due to aging co-morbidities.Since oncogenes and DNA damage drive senescent growth arrest, we hypothesize that stable MGUS and SMM pre-malignant PCs exhibit senescence features.Methods: We performed gene set enrichment analysis (GSEA) of a published human PC gene array dataset (GSE5900).Immunostaining and fluorescent in situ hybridization were performed to evaluate senescence in MGUS, SMM and MM patient PCs and trephine bone biopsies from MGUS and SMM patients that progressed or not to MM. Lastly, quantitative PCR was used to validate senescence gene expression changes in MGUS, SMM, and MM PCs.Results: MGUS and SMM PCs exhibited significant enrichment for senescence phenotyping gene sets that were distinct from aging.PCs from bone marrow aspirates were identified as senescent based on the Loss of LMNB1 (LoL, < 50%) and senescence associated distension of satellites (SADS, ≥3/cell).PCs from patients with stable MGUS (N=4) exhibited increased percentage of senescent PCs compared to SMM and MM, while stable vs progressing SMM PCs (stable vs progressed ≤5 years, n=6-8) were unchanged.However, PCs from stable, but not progressing SMM, had increased expression of senescence genes (CDKN2A, TP53, BCL2, and IL1B).Both stable MGUS and SMM PCs showed a significant increase in the expression of the retrotransposable element, L1HS, which is increased with senescence, compared to PCs from SMM progressors.Trephine biopsy cells were scored as senescent based on LoL and loss of nuclear HMGB1.Stable MGUS patients exhibited increased percentage of senescent PCs compared to MM and MGUS patients that progressed ≤10 years (n=20-40).Senescent PCs in trephine biopsies correlated with senescence in the bone marrow microenvironment (BMME).Of interest, MGUS and SMM PCs from patients that progressed ≤10 years both exhibited a significant loss in neighboring senescent BMME compared to stable MGUS and SMM PCs.Conclusions: Overall, we demonstrate the presence of stage-specific senescence features in PCs from stable MGUS and SMM patients; these features are decreased in PCs from SMM progressors and MM, consistent with the protective effect of senescence against tumorigenesis.Thus, evaluating PC senescence in MGUS and SMM may have prognostic value to identify patients that will progress to MM. Importantly, given the increased risk for osteoporotic fracture and reduced overall survival in stable MGUS patients, the senescence-related protection against MM may come at the expense of premature aging.Ongoing studies are evaluating the safety and therapeutic utility of ablating pre-malignant senescent cells in MGUS and SMM.

Background Prevalence estimates for monoclonal gammopathy of undetermined significance (MGUS) are based on predominantly White study populations screened by serum protein electrophoresis supplemented with immunofixation electrophoresis. A prevalence of 3% is reported for MGUS in the general population of European ancestry aged 50 years or older. MGUS prevalence is two times higher in individuals of African descent or with a family history of conditions related to multiple myeloma. We aimed to evaluate the prevalence and clinical implications of monoclonal gaminopathies in a high-risk US population screened by quantitative mass spectrometry. Methods We used quantitative matrix-assisted laser desorption ionisation-time of flight (MALDI-TOF) mass spectrometry and EXENT-iQ software to screen for and quantify monoclonal gammopathies in serum from 7622 individuals who consented to the PROMISE screening study between Feb 26, 2019, and Nov 4, 2021, and the Mass General Brigham Biobank (MGBB) between July 28, 2010, and July 1, 2021. M-protein concentrations at the monoclonal gammopathy of indeterminate potential (MGIP) level were confirmed by liquid chromatography mass spectrometry testing. 6305 (83%; 2211 from PROMISE, 4094 from MGBB) of 7622 participants in the cohorts were at high risk for developing a monoclonal gammopathy on the basis of Black race or a family history of haematological malignancies and fell within the eligible high-risk age range (30 years or older for PROMISE cohort and 18 years or older for MGBB cohort); those over 18 years were also eligible if they had two or more family members with a blood cancer (PROMISE cohort). Participants with a plasma cell malignancy diagnosed before screening were excluded. Longitudinal clinical data were available for MGBB participants with a median follow-up time from serum sample screening of 4.5 years (IQR 2.4-6.7). The PROMISE study is registered with ClinicalTrials.gov , NCT03689595. Findings The median age at time of screening was 56.0 years (IQR 46.8-64-1). 5013 (66%) of 7622 participants were female, 2570 (34%) male, and 39 (<1%) unknown. 2439 (32%) self-identified as Black, 4986 (65%) as White, 119 (2%) as other, and 78 (1%) unknown. Using serum protein electrophoresis with immunofixation electrophoresis, the MGUS prevalence was 6% (101 of 1714) in high-risk individuals aged 50 years or older. Using mass spectrometry, we observed a total prevalence of monoclonal gammopathies of 43% (1788 of 4207) in this group. We termed monoclonal gammopathies below the clinical immunofixation electrophoresis detection level (<0. 2 g/L) MGIPs, to differentiate them from those with higher concentrations, termed mass-spectrometry MGUS, which had a 13% (592 of 4207) prevalence by mass spectrometry in high-risk individuals aged 50 years or older. MGIP was predominantly of immunoglobulin M isotype, and its prevalence increased with age (19% [488 of 2564] for individuals aged <50 years, 29% [1464 of 5058] for those aged >= 50 years, and 37% [347 of 946] for those aged X70 years). Mass-spectrometry MGUS prevalence increased with age (5% [127 of 2564] for individuals aged <50 years, 13% [678 of 5058] for those aged a50 years, and 18% [173 of 946] for those aged >= 70 years) and was higher in men (314 [12%] of 2570) compared with women (485 [10%] 5013; p=0.0002), whereas MGIP prevalence did not differ significantly by gender. In those aged 50 years or older, the prevalence of mass spectrometry was significantly higher in Black participants (224 [17%] of 1356) compared with the controls (p=0.0012) but not in those with family history (368 [13%] of 2851) compared with the controls (p=0.1008). Screen-detected monoclonal gammopathies correlated with increased all-cause mortality in MGBB participants (hazard ratio 1.55, 95% CI 1-16-2.08; p=0.0035). All monoclonal gammopathies were associated with an increased likelihood of comorbidities, including myocardial infarction (odds ratio 1.60, 95% CI 1.26-2.02; p=0.00016 for MGIP-high and 1- 39, 1-07-1.80; p=0.015 for mass-spectrometry MGUS). Interpretation We detected a high prevalence of monoclonal gammopathies, including age-associated MGIP, and made more precise estimates of mass-spectrometry MGUS compared with conventional gel-based methods. The use of mass spectrometry also highlighted the potential hidden clinical significance of MGIP. Our study suggests the association of monoclonal gammopathies with a variety of clinical phenotypes and decreased overall survival. Copyright (C) 2022 Elsevier Ltd. All rights reserved.

Background: Monoclonal gammopathy of undetermined significance (MGUS) is a premalignant condition of multiple myeloma with few known risk factors. The emergence of mass spectrometry (MS) for the detection of MGUS has provided new opportunities to evaluate its risk factors.Methods: 2628 individuals at elevated risk for multiple myeloma were enrolled in a prospective screening study and completed an exposure survey. Participants were screened concurrently by MS and serum protein electrophoresis/immunofixation (SPEP/IFX). Monoclonal proteins (M-proteins) detected by MS with concentrations >0·2 g/L were categorized as MS-MGUS. Multivariable logistic regression models evaluated associations between exposures and MS and SPEP/IFX outcomes.Findings: Prevalence of MS-MGUS and SPEP/IFX-detected MGUS were 9% and 4%. Compared to having a normal weight (BMI: 18·5 to <25 kg/m2), obesity (BMI: >30 kg/m2) was associated with MS-MGUS, adjusting for age, sex, Black race, education, and income (odds ratio [OR] 1·73, 95% CI 1·21—2·47; p=0·0025). High physical activity (>73·5 MET-hours/week vs. <10·5 MET-hours/week) had a decreased likelihood of MS-MGUS (OR 0·45, 95% CI 0·24—0·80; p=0·0091), whereas heavy smoking and short sleep had an increased likelihood of MS-MGUS (>20 pack-years vs. never smoker: OR 2·26, 95% CI 1·13—4·31; p=0·017 and sleep <6 vs. >6 hours/day: OR 2·11, 95% CI 1·26—3·42; p=0·0031). Associations between exposures and SPEP/IFX-detected MGUS did not reach statistical significance.Interpretation: MS-MGUS is associated with a broader range of modifiable risk factors than previously identified. Findings may inform targeted screening and prevention strategies.Funding Information: This study was funded in part by the National Institutes of Health (F32 CA220859, K22 CA251648, R25 AI147393, R38 HL150212). This research was also supported by a Stand Up To Cancer Dream Team Research Grant (Grant Number: SU2C-AACR-DT-28-18).Declaration of Interests: DS, DB, MCP are current employees of The Binding Site. SH is a current employee, member of the Board of Directors, and holds patents related to The Binding Site. IMG has served as a consultant for AbbVie, Adaptive, Aptitude Health, BMS, Cellectar, CurioScience, Genetch, Janssen, Janssen Central American and Caribbean, Karyopharm, Medscape, Oncopeptides, Sanofi, Takeda, The Binding Site, Gene Network Sciences Healthcare, and GSK. IMG’s spouse, William Savage, is CMO and equity holder at Disc Medicine. All other authors declare no competing interest.Ethics Approval Statement: This study was conducted upon approval of protocol 18-370 by the institutional review board of the Dana-Farber Cancer Institute.

Introduction Serum free light chain (sFLC) are important for diagnosis and prognosis of plasma cell dyscrasias and are known to be affected by kidney function and inflammatory conditions. However, African Americans (AA) have been reported to have significantly higher Kappa (κ) and Lambda (λ) sFLC levels than European Americans (EA) in a biobanked population, even after adjusting for kidney function (El-Khoury et al., Lancet Haem 2022). Data on race-adjusted reference ranges for sFLC and sFLC ratio (sFLCr) remain limited. We hypothesize that an adjustment in sFLC reference ranges for individuals of African and European ancestry is needed. Methods We analyzed serum samples from 738 healthy Black South African (SA) and 3402 US individuals (AA+EA) as part of an international study screening for individuals at high risk of multiple myeloma (MM) and precursor conditions. Self-identification as Black or having family history of hematologic cancer or MM precursor were key eligible criteria. sFLC levels were measured using the Optilite® Freelite assay (The Biding Site). All participants were screened for Monoclonal Gammopathy of Undetermined Significance (MGUS; M protein concentration ≥ 0.2 g/L) by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. Serum Cystatin C (Optilite®) was used to calculate estimated Glomerular Filtration Rate (eGFR) with CKD-EPI Cystatin C Equation (2012) formula for 752 individuals (568 SA, 184 AA). Clinical data was collected using validated questionnaires at study enrollment. Group medians were compared with Wilcoxon tests. Abnormal sFLCr was defined as any value outside the normal reference range with the involved sFLC above the manufacturer's normal reference range. We used 95% central interval (CI) values to compute new sFLCr reference values for each group. Results After excluding individuals with Heavy Chain-MGUS, our study population consisted of 654 SA, 326 AA, and 2,551 EA. Median age was 52 (IQR 46-59), 56 (IQR 50-65, and 58 (IQR 50-64) for the three groups, respectively. There was a female predominance in the AA (74%) and EA (73%) groups compared to SA (52%). Chronic kidney disease (CKD) was self-reported in 0.2%, 1% and 1% of the three groups, respectively. Calculated median eGFR was 118 ml/min (IQR 106-134) for SA and 112 (IQR 98-123) for AA. HIV positivity was higher (23%) in SA than the other groups (<1%) and 99% were under treatment (Tenofovir 92%, which is known to be nephrotoxic). The median free κ was higher in SA (32 mg/l, IQR 24.8-46) than AA (18.1 mg/l, IQR 14.4-23, +80%, p<0.01) and EA (13.7 mg/l, IQR 10.8 - 17.1, +133%, p<0.01). Similarly, median free λ was higher in SA (24.6 mg/l, IQR 18.8-33.4) than AA (15 mg/l, IQR 11.7-19.3, +64%, p<0.01) and EA (11.9, mg/l IQR 9.1-15.5, +106%, p<0.01). 92% of SA, 43% of AA, and 16% of EA had κ values above the normal range. Similarly, 43% of SA, 10% of AA, and 3% of EA had λ values outside the normal range. Median sFLCr was higher in SA (1.34, IQR 1.13-1.65) than AA (1.25, IQR 1.03-1.48, p<0.01), and EA (1.18, IQR 0.99-1.39, p<0.01) (Fig 1). Using the standard manufacturer ranges, 25% of SA, 11% of AA and 3% of EA had abnormal sFLCr and involved FLC. Decreasing eGFR, increasing age, HIV positivity, and the cohort (SA, AA, or EA) were all significant predictors of changes in sFLCr in univariate analysis. In multivariate logistic regression, increasing age, HIV positivity, and decreasing eGFR were independent predictors of abnormal sFLCr (Table 1) for SA or AA compared to EA. In SA with normal renal function (eGFR >60 ml/min), excluding those with free κ and λ outside the 95% CI, we identified a new reference (95% CI) for FLCr of 0.80-2.38. Conclusions This is the first report of sFLC measurements in an African population screened for MG and compared to AA and EA. We observe a significantly higher sFLC for Black (SA and AA) than White individuals (EA), independent of renal function. This difference could be due to genetic background and socioeconomic and environmental factors (e.g., HIV epidemiology). In contrast with previous reports (Zemlin, et al, J Clin Pathol 2015), HIV was associated with higher FLCr, maybe with a possible role of nephrotoxic HIV drugs. Here, standard reference ranges do not apply to SA participants. While further validation is needed, we propose to use a different sFLC range for African ancestry populations with normal renal function. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Background Monoclonal gammopathy of undetermined significance (MGUS) is a premalignant expansion of plasma cells that always precedes the development of multiple myeloma. Recent U.S.-based screening studies utilizing matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) reported a high prevalence of monoclonal gammopathies (MGs) among Blacks. One study observed among Blacks over age 50 years, a high prevalence of MGUS (17%) as well as low-level MGs below the detection threshold of serum protein electrophoresis/immunofixation (SPEP/IFX) assays, termed monoclonal gammopathy of indeterminate potential (MGIP, 31% prevalence). In the current study, we aimed to define the prevalence of MGs by MALDI-TOF MS in a Black population in South Africa and evaluate its association with risk factors (e.g., obesity). Methods Individuals who self-identify as Black African and aged >=40 years were recruited from Soweto, Johannesburg, South Africa to participate in the study. Those with a prior diagnosis of a plasma cell dyscrasia and/or any cancer history requiring active therapy were excluded. Serum samples of participants were analyzed using the Binding Site Group's EXENT® assay based on MALDI-TOF MS for the quantification of M-proteins. M-proteins >=0.2 g/L were categorized as MGUS, and those <0.2 g/L were MGIP. This concentration threshold of 0.2 g/L was the lower detection limit of SPEP/IFX-determined by serial dilution sensitivity testing comparing MALDI-TOF MS and SPEP/IFX assays, performed and provided by the manufacturer and reported in a prior study. Participants’ heights and weights were measured at enrollment and used to calculate body mass index (BMI, kg/m2). All participants completed a survey querying sociodemographic factors, clinician-diagnosed comorbidities, and lifestyle factors. Logistic regression models estimated odds ratios (OR) and 95% confidence intervals (CI) for exposure and MGUS/MGIP associations. Results As of June 2022, 738 participants enrolled in the study. Median age was 52 years (range, 40-79), and 52% were female. 3% reported an educational status of college graduate or above. Ethnic background of participants included 38% IsiZulu, 20% Sesotho, 13% IsiXhosa, 13% Setswana, 5% Tsonga, 4% Sepedi, 4% Seswati, and 1% Tshivenda. The prevalence of MGUS and MGIP was 12% and 17% in the entire cohort and 13% and 18% in those aged >=50 years. MGUS was predominantly IgG (89%), followed by IgA (7%) and IgM (4%). MGIP was predominantly IgM (81%), followed by IgA (15%) and IgG (4%). In univariate analysis, MGs were associated with increasing 10-year age intervals (OR, 1.40; 95% CI, 1.17-1.67). Mean BMI was 27 kg/m2, and 27% of participants were obese (>=30 kg/m2). Adjusting for age, sex, and education, obesity was associated with all MGs (OR, 1.49; 95% CI, 1.01-2.20). Obesity was associated with MGIP (OR, 1.94; 95% CI, 1.20-3.12) and not with MGUS (OR, 1.04; 95% CI, 0.58-1.82). Diabetes was also associated with MGIP (OR, 2.98; 95% CI, 1.19-7.19). HIV was reported in 23% of participants, among whom 99% reported current HIV treatment. HIV was not associated with MGUS or MGIP. All other comorbidities, including cardiovascular disease, stroke, COPD/emphysema, arthritis, liver disease, and chronic kidney disease, were reported in <=1% of participants. As for lifestyle factors, heavy alcohol consumption (>=30 g/day) was associated with MGUS (OR, 2.76; 95% CI, 1.47-5.51). Heavy smoking (>10 pack-years) had a trend toward association with MGUS (OR, 1.81; 95% CI, 0.98-3.32). Short sleep (<=6 hours/day) was associated with MGIP (OR, 2.11; 95% CI, 1.22-3.59). Conclusion These results are the first to describe a high prevalence of MGUS detected by MS (13%) for a Black African cohort aged >=50 years, consistent with high prevalence estimates of MS-detected MGUS recently reported in U.S. Blacks. Obesity and diabetes were associated with MGIP, an entity that currently bears unknown etiology. We, however, did not observe associations for obesity and related comorbidities with MGUS, possibly due to limited sample size. Nonetheless, these first results along with other observed novel risk factor associations (e.g., MGUS and heavy alcohol consumption) in our expanding South African cohort provide clues to pathophysiological mechanisms of MGs and may inform interception strategies in preventing their progression.

Background: Monoclonal gammopathy of undetermined significance (MGUS) is a premalignant condition of multiple myeloma (MM) traditionally identified by serum protein electrophoresis (SPEP) and immunofixation (IFX). Recently, novel, ultra-sensitive mass spectrometry (MS)-based approaches have allowed for monoclonal (M)-protein detection at concentrations below SPEP levels. MGUS has only few known risk factors implicated in its development and progression. Thus, we analyze MS results of the PROMISE nationwide U.S. screening study to evaluate risk factors for (1) MGUS at traditional SPEP/IFX levels of detection and (2) low-level monoclonal gammopathies, which bear uncertain etiology and clinical significance. Methods: PROMISE enrolled individuals age ≥40 who are Black and/or have a first-degree relative with a blood cancer or MM precursor condition. Those with ≥2 first-degree relatives were eligible at age ≥18. Participants were screened for monoclonal gammopathy by MALDI-TOF MS and provided a survey querying metabolic comorbidities and lifestyle. M-protein concentrations ≥0.02 g/dL were considered traditionally-defined MGUS, whereas M-proteins <0.02 g/dL are hereafter referred to as monoclonal gammopathy of indeterminate potential (MGIP). Multivariable logistic regression models estimated odds ratios (OR) and 95% confidence intervals (CI) for exposure and MGUS/MGIP associations. Results: 1,893 screened participants completed the survey. MGUS and MGIP were detected in 13.4% and 22.0% of Blacks and 8.6% and 26.7% of individuals with family history. Adjusting for sex, age at screening, income, and education, obesity or BMI of ≥30 was associated with MGUS (OR, 1.55; 95% CI, 1.08-2.21), compared to BMI <30. Clinician-diagnosed hypertension (yes/no) and diabetes mellitus (yes/no) were associated with MGUS with ORs 1.44 (95% CI, 1.01-2.04) and 1.88 (95% CI, 1.004-3.51). There were no significant associations between MGUS and high cholesterol, high triglycerides, heart disease, myocardial infarction, or stroke. Short sleep of ≤6 hours/day was associated with MGUS (OR, 1.43; 95% CI, 1.01-2.03), compared to >6 hours/day. Physical activity (metabolic equivalents/week), smoking status (current, past, never), alcohol consumption (g/day) had no associations with MGUS. No risk factor associations were found for MGIP. Conclusion: In screening a high-risk population by mass spectrometry, we found associations of both traditionally established (obesity) and novel risk factors (diabetes, hypertension, short sleep) with MGUS. None of these exposures were associated with MGIP despite finding a high prevalence of MGIP in Blacks and individuals with family history, suggesting that these risk factors may not be etiologically involved in MGIP development but possibly its clonal expansion to more advanced stages. Citation Format: David J. Lee, Habib El-Khoury, Jean-Baptiste Alberge, D.J. Sakrikar, David Barnidge, Mark C. Perkins, Stephen Harding, Jacqueline Perry, Maya I. Davis, Julia Amstutz, Erica Horowitz, Timothy R. Rebbeck, Irene M. Ghobrial, Catherine R. Marinac. Obesity, metabolic comorbidities, and lifestyle factors and their association with monoclonal gammopathies in a high-risk screened population: Results of the PROMISE study [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 3651.

Background: serum protein electrophoresis (SPEP) and B-cell receptor next generation gene sequencing (NGS) are methodologies used to monitor the amount of a monoclonal protein in multiple myeloma (MM) patients and to evaluate minimal residual disease (MRD), respectively. Mass spectrometry (MS) is a methodology currently being explored to provide a serum/plasma-based quantitative assessment of M-protein, which could also serve as a surrogate of MRD. Here we present the analytical performance of SPEP and MRD compared to the MALDI-TOF MS-based assay EXENT. Methods: SPEP, NGS (Clonoseq v 2.0, Adaptive Biotechnologies), and MS (EXENT®, The Binding Site) assays were performed to assess M-protein in 65 serum samples from 22 MM patients who participated in a phase 3 clinical trial (CASTOR, NCT02136134), in which patients received Daratumumab, Bortezomib and Dexamethasone (DVd) or Bortezomib and Dexamethasone (Vd). Irrespective of treatment, serum samples collected at three time points were selected from each subject, including 1) baseline prior to treatment, 2) available sample at or most approximately prior to the first confirmed complete response (CR) according to IMWG criteria with SPEP equaled 0, and 3) last available sample prior to disease progression (PD) with SPEP equaled 0. M-protein concentration measured by EXENT was compared to that measured by SPEP. Data was analyzed by using either all obtained values (n=62) or only non-zero values (n=18) by EXENT or SPEP. NGS-based MRD results (n=26) used for the comparison were those sampled within 60 days of the samples used for the EXENT analysis. Results: M-protein concentration by EXENT significantly correlated with SPEP measurements by using all obtained values (r=0.986, p<0.0001). The correlation was also significant when non-zero values were used in the analysis (r=0.969, P<0.0001). The lower limit of quantification (LLOQ) of EXENT was 0.015 g/L, while that of SPEP was approximately 1 g/L. With the better sensitivity, EXENT was able to detect M-protein up to 167 days prior to PD when SPEP detected none (figure). MS-based assessments were concordant with NGS-based MRD results. A 76.9% (20 of 26) agreement was observed when comparing EXENT with MRD at a sensitivity of 10-5. EXENT purified samples were reflexed to analysis by the more sensitive MS-based methodology LC-MS. The agreement between LC-MS and MRD at a sensitivity of 10-6 was 92.3% (24 of 26). In addition, we observed several useful features unique to MS-based assays. These include distinguishing daratumumab from the endogenous M-protein, detecting isotype switching, and the ability of detecting light chain glycosylation. Conclusion: EXENT significantly correlated with SPEP while EXENT demonstrated a higher sensitivity in quantification of M-protein in serum than SPEP. A high degree of agreement was observed between EXENT/LC-MS and NGS-based MRD. With its non-invasive nature and early detection of relapse, EXENT can be used as a complementary assay in MM patient management to guide bone marrow based MRD assessment. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Background: The prevalence of monoclonal gammopathy of undetermined significance (MGUS) in a population at high risk of developing multiple myeloma (MM) was recently described in a US screening study. In a high-risk population, MGUS prevalence was measured as 6% by serum protein electrophoresis with immunofixation (SPEP/IFX) and 13% by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Screening by MALDI-TOF MS allowed for the detection of monoclonal proteins (M-proteins) below the threshold of detection of gel-based assays, that was referred to as Monoclonal Gammopathy of Indeterminate Potential (MGIP). MGIP was found in 25% of the screened cohort and was significantly associated with aging. Other studies indicated that MGUS and MGIP detected by MS may be transient and their detection may not be critical for long term outcomes. Therefore, we aimed to determine the fate of M-proteins detected by MS in a US population-based study. Methods: High-risk individuals enrolled in the study include those who self-identify as Black or have a family history of hematologic malignancies or a precursor condition to MM. Samples that tested positive for MGUS (M-protein >= 0.2 g/L) by MALDI-TOF MS were confirmed using clinically approved assays and results were returned to participants. All participants who had an M-protein detected at baseline by MALDI-TOF MS were invited to submit follow-up samples to investigate the fate of the M-proteins detected at the MGIP and MGUS levels. Serum was analyzed using the Binding Site Group's EXENT® assay based on MALDI-TOF MS for the identification and quantification of M-proteins. Persistence of the M-protein detected at baseline was defined as the presence of the same isotype M-protein on a serial sample and within +/-5 mass to charge ratio on mass spectra. All serial samples where the M-protein was not detected by MALDI-TOF MS were reflex tested to liquid chromatography (LC) MS. Results: We analyzed 262 multi-timepoint samples collected from 102 participants who tested positive for an M-protein, with a median follow-up of 336 days (Range 36 - 1,240; IQR 141 - 737). The median age at screening was 60.8 years, 65 (64%) participants were female, 9 (9%) self-identified as Black, and 93 (91%) were non-Black and reported a family history of hematologic malignancy. At baseline, 66 (65%) participants had MGIP (M-protein <0.2g/L) and 36 (35%) had MGUS (M-protein >=0.2/L) measured by MALDI-TOF MS. All participants with MGIP were negative at baseline by SPEP/IFX, while among those with MGUS at baseline, 25/36 (69%) participants had an M-protein detected by SPEP/IFX. At baseline, the distribution of isotypes was 21% IgG, 17% IgA, and 62% IgM for MGIP and 69% IgG, 17% IgA, and 14% IgM for MGUS. For all 102 cases, their most recent serial samples were tested by MALDI-TOF MS or LC-MS. 60/66 (91%) of the MGIP cases were re-confirmed on serial sampling, with a median follow-up time of 504 days (Range 41 - 1,240; IQR 149 - 888). As for MGUS, 36/36 (100%) of cases detected at baseline persisted on serial sampling, with a median follow-up time of 239 days (Range 36 - 1105; IQR 103 - 628) (Figure 1). Time from baseline to serial sampling was not associated with persistence. In 12 participants so far, M-proteins detected by MS were seen to persist for more than 3 years after baseline testing. In 5 of the MGIP cases, M-protein concentration measured by MALDI-TOF MS increased from MGIP level to above the MGUS threshold. These participants had a median baseline M-protein concentration of 0.14 g/L (Range 0.13 - 0.17 g/L; IQR 0.13 - 0.14), which was in the upper MGIP concentration level, and had a median follow-up time of 532 days (Range 171 - 1,127; IQR 302 - 570). Of these, 3 persisted as IgG, 1 IgA, and 1 IgM isotypes. Conclusion: We present the first longitudinal analysis of M-proteins detected by MS, including those below the threshold of detection of gel-based assays, in a US population-based screening study. Results confirm the persistence of 91% of MGIP and 100% of MGUS cases detected by MS. Some cases showed increasing M-protein concentration, crossing the threshold of detection by gel-based assays. Additional studies are underway to further validate our findings in a larger cohort with longer follow-up time. Testing serial samples with increasing M-protein concentration by gel-based assays will help better understand the significance of early detection of MGIP cases by MS. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Abstract Background Multiple myeloma (MM) evolves from monoclonal gammopathy of undetermined significance (MGUS), a clinically detectable but asymptomatic premalignant phase seen in ~3% of the general population 50 years of age or older. The prevalence of MGUS has not been described in a population at high risk of developing MM, specifically Black/African American (AA) individuals or first-degree relatives of patients with hematologic malignancies (HM). In 2019, we launched the first nationwide US screening study for individuals at high risk of MM to help better identify what population would benefit most from screening and early intervention for precursor MM stages. We aim to assess the prevalence of MGUS in a population at high risk of MM and characterize clinical variables of individuals who screen positive. Here, we report interim screening data on the first 2,960 participants. Methods Individuals aged 40 or older with an additional MM risk factor are eligible to be screened in the PROMISE Study. High-risk individuals include Black/AAs and those with a first-degree relative diagnosed with a hematologic malignancy or a precursor condition to MM. Blood from all participants was analyzed via serum protein electrophoresis, immunofixation, and Optilite® to measure the serum free light chains (sFLC), IgG, IgA and IgM. Results were returned to all participants, and those who tested positive for a monoclonal gammopathy (MGUS/SMM) were referred to a hematologist for clinical follow-up and invited to periodically complete epidemiologic exposure and psychosocial questionaries, including a 4-item cancer worry questionnaire and the RAND 36-item Short Form Survey (SF-36). To investigate the use of the higher-sensitivity matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) with the Optilite® IgG, IgA, IgM and sFLC results as a screening test for all participants, we rescreened 1,092 samples from PROMISE. The Binding Site Group proprietary software was used for the analysis of the combined MS/Optilite® results, allowing for the detection and quantification of M-protein. Heavy-Chain MGUS (HC-MGUS) was defined by the presence of one or more paired heavy and light chain monoclonal peaks detected by MS. Pairing was based on mass to charge ratio of identified peaks. To enrich the PROMISE cohort with Black/AA individuals, we identified and screened 1,868 Black/AA additional individuals from the Mass General Brigham (MGB) Biobank who met the PROMISE enrollment criteria. Screening was performed by MS/Optilite®, and results have not been returned to participants. Results We screened 2,960 participants with the combined MS/Optilite® approach. We report here the prevalence of HC-MGUS and plan on presenting the estimated rate of light chain MGUS in our cohort, at the meeting. We detected HC-MGUS in 9.6% (95% CI: 8.6-11%) of our cohort, with a prevalence of 10% (95% CI: 8.3-12%) in the PROMISE cohort and 9.4% (95% CI: 8.1-11%) in the MGB cohort (Table 1 and Figure 1). HC-MGUS prevalence increased with age in high-risk individuals from 4.9% (CI: 3.3-6.9%) for participants aged 40-49 to 13% (CI: 10-17%) in the 70-79 range (P < 1.2E-5). Among monoclonal HC-MGUS, we found 65% IgG, 18% IgM, and 18% IgA. M-spike was quantified in 97% of samples. Median M-spike concentration was, 0.058g/dL (max. 2.6g/dL) for IgG, 0.0043g/dL (max. 0.6g/dL) for IgM, and 0.067g/dL (max. 0.8g/dL) for IgA. In the Promise cohort, no significant change in cancer worry was observed across the pre- and post-screening interval among participants who screened positive (P = 0.52). Health-related quality of life, as measured by the SF-36, was not significantly different in screen-positive vs. screen-negative individuals for any of the eight subscales (all P > 0.20). Conclusions We present the largest dataset on monoclonal gammopathy prevalence and screening in individuals at high risk for MM, and more specifically the largest cohort of Black/AA, using a novel high-sensitivity testing approach. Our results confirm that older adults who are Black/AA or have a first-degree relative with an HM have a high prevalence MGUS and may benefit from precision screening approaches to allow for early detection and clinical intervention. Preliminary data on cancer worry and quality of life indicates that the psychosocial burden of screening in this population is likely minimal. Figure 1 Figure 1. Disclosures Sakrikar: The Binding Site: Current Employment. Krause: The Binding Site: Current Employment. Barnidge: The Binding Site: Current Employment. Bustoros: Takeda: Consultancy, Honoraria; Janssen, Bristol Myers Squibb: Honoraria, Speakers Bureau. Perkins: The Binding Site: Current Employment. Harding: The Binding Site: Current Employment, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Kapoor: Ichnos Sciences: Research Funding; Amgen: Research Funding; Pharmacyclics: Consultancy; Takeda: Research Funding; Sanofi: Consultancy; Regeneron Pharmaceuticals: Research Funding; AbbVie: Research Funding; BeiGene: Consultancy; Sanofi: Research Funding; Karyopharm: Research Funding; Glaxo SmithKline: Research Funding; Karyopharm: Consultancy; Cellectar: Consultancy. Mo: Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees; GSK: Consultancy, Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; Epizyme: Consultancy; Eli Lilly: Consultancy; Janssen: Honoraria; Karyopharm: Honoraria, Membership on an entity's Board of Directors or advisory committees. Murray: The Binding Site: Patents & Royalties: Potential Royalties for use of mass spectrometry in M-protein detection. Getz: Scorpion Therapeutics: Consultancy, Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees; IBM, Pharmacyclics: Research Funding. Marinac: JBF Legal: Consultancy; GRAIL Inc: Research Funding. Ghobrial: AbbVie, Adaptive, Aptitude Health, BMS, Cellectar, Curio Science, Genetch, Janssen, Janssen Central American and Caribbean, Karyopharm, Medscape, Oncopeptides, Sanofi, Takeda, The Binding Site, GNS, GSK: Consultancy.

Background: Monoclonal gammopathy of undetermined significance (MGUS) prevalence estimates are currently based on predominantly White study populations screened by serum protein electrophoresis/immunofixation (SPEP/IFX). A 3% MGUS prevalence is reported in the general population aged ≥50 years.Methods: 7,622 individuals, including 83% at higher risk for developing myeloma—Black/African-Americans and those with a family history of hematological malignancies—were screened by quantitative mass spectrometry (MS) for monoclonal gammopathies (MG).Findings: Using SPEP/IFX, the MGUS prevalence was 6% in high-risk individuals ≥50. Using MS, we observed a >40% total MG prevalence in this group. We termed MGs below the current clinical IFX detection level (<0.2g/L) monoclonal gammopathy of indeterminate potential (MGIP), to differentiate them from higher-concentration MGs, termed MS-MGUS, which had a 13% prevalence by MS. MGIP was predominantly of IgM isotype, and its prevalence increased with age. MS-MGUS prevalence increased with age and was higher in males and high-risk individuals. Screen-detected MGs correlated with worse overall survival (HR, 1.55; 95% CI, 1.16-2.08, P=0.003), whereas MS-MGUS was associated with increased all-cause mortality (HR, 2.35; 95% CI, 1.59-3.47, P=0.001). All MGs were associated with an increased likelihood of comorbidities, including myocardial infarction (MGIP: OR, 1.33; 95% CI, 1.09-1.61, P=0.005; MGUS: OR, 1.39; 95% CI, 1.07-1.80, P=0.015).Interpretation: MS-based screening detected an unprecedented high prevalence of MGs, including age-associated MGIP and increased estimates of MS-MGUS. MGs were associated with worse overall survival and adverse clinical phenotypes. Clinical Trial Registration Details: (PROMISE, NCT03689595).Funding Information: This research was supported by the Multiple Myeloma Research Foundation and Stand Up to Cancer.Declaration of Interests: H.E.K., D.J.L., J-B.A., R.R., CJ.C-C., J.P., H.B., C.M., H.L., A.C., M.I.D., J.A., C.J.B., E.D.L., R.S-P., E.K., L.T., declare no conflicts of interest. D.S., D.B, M.C.P. are current employees of The Binding Site. S.H. is a current employee, member of the Board of Directors, and holds patent related to The Binding Site. C.C.M. is a consultant for Eli Lilly and Epizyme, is an advisory board member for BMS, has served as a consultant and advisory board member for GSK, has received honoraria Janssen, and received honoraria and served as an advisory board member for Karyopharm and Sanofi. G.G. receives research funds from IBM and Pharmacyclics, and is an inventor on patent applications related to MuTect, ABSOLUTE, MutSig, MSMuTect, MSMutSig, MSIDetect, POLYSOLVER and TensorQTL. G.G. is a founder, consultant and holds privately held equity in Scorpion Therapeutics. C.R.M. has serves as a consultant for JBF Legal and received research funding from GRAIL, Inc. I.M.G. has served as a consultant for AbbVie, Adaptive, Aptitude Health, BMS, Cellectar, CurioScience, Genetch, Janssen, Janssen Central American and Caribbean, Karyopharm, Medscape, Oncopeptides, Sanofi ,Takeda, The Binding Site, GNS, and GSK. I.M.G.'s spouse, William Savage MD, PhD, is CMO and equity holder of Disc Medicine.Ethics Approval Statement: This study was conducted upon approval of protocols 18-370 and 2021P001703 by the institutional review boards of Dana-Farber Cancer Institute and the Mass General Brigham, respectively.