Severe Isotype-Matched Immunosuppression (IMI) as a Potential Risk Factor for Progression of MGUS Patients

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1 Severe Isotype-Matched Immunosuppression (IMI) as a Potential Risk Factor for Progression of MGUS Patients Juana J. Jiménez, 1 * Tiago M. Pais, 2 Nuno Barbosa, 2 Maria Luisa Campos, 2 Maria Antonia Peñalver Díaz, 3 and Carmen H. de Larramendi 1 Background: Monoclonal gammopathy of undetermined significance (MGUS) precedes multiple myeloma in virtually every case. However, only a small percentage will progress and at very different rates. In addition, recent data have suggested that MGUS is associated with other comorbidities including infections, suggesting impaired immune function in some MGUS patients. Therefore, we aimed at assessing the value of isotype-matched immunosuppression (IMI; e.g., suppression of an IgAκ in an IgAλ patient), a type of immunosuppression more specific than classical immunoparesis (IP; e.g., IgG and/or IgM suppression in an IgA patient), as a prognostic marker for MGUS progression. Methods: The Hevylite assay was used to assess IMI and immunoglobulin ratios in 307 serum samples from a cohort of 248 MGUS patients. Follow-up clinical records were available for 154 individuals. Results: A greater incidence of IMI (51%) over classical IP (37%) was observed, although both show a progressive increase with higher risk groups. Survival analysis of 154 patients showed that severe IMI (>50% suppression) differentiates 2 groups with significantly different time to progression (P = 0.024) while severe IP does not (P = 0.48). Also, a combination of severe IMI and involved monoclonal immunoglobulin >1.5g/dL by Hevylite (both variables found to be independent prognostic markers in multivariate analysis) identified a group of patients with a median time to progression 6-fold shorter than the remaining group (P < ). Conclusions: These findings indicate a possible role for IMI in the malignant transformation of MGUS patients and a potential utility as a new risk factor. IMPACT STATEMENT Patients diagnosed with MGUS are at a greater risk of developing multiple myeloma or a related disease. Importantly, the conversion rate is highly heterogeneous, and hematologists rely on biomarkers to estimate the risk of progression and to decide on the clinical approach. We describe the role of a new type of immunosuppression as an independent indicator of early progression, contributing to a better characterization of the condition. This immunosuppression is characterized by decreased levels of the immunoglobulins of the same type of the tumor B-cells but of the alternative light chain, and is easily measured by a simple serum test called Hevylite. 1 Department of Clinical Chemistry, University Hospital Severo Ochoa, Madrid, Spain; 2 Scientific Department, The Binding Site Iberia, Barcelona, Spain; 3 Department of Haematology, University Hospital Severo Ochoa, Madrid, Spain. *Address correspondence to this author at: Department of Clinical Chemistry, University Hospital Severo Ochoa Av. de Orellana, Leganés, Leganés, Madrid, Spain. Fax ; mandalay1954@gmail.com. 700 JALM :05 March 2018

2 Monoclonal gammopathy of unknown or undetermined significance (MGUS) 4 is an asymptomatic plasma cell dyscrasia that is present in >3% of the population over the age of 50 (1) and can evolve to multiple myeloma. MGUS is defined as the presence of an M-protein <3 g/dl with <10% plasma cells in the bone marrow and no evidence of endorgan damage (i.e., no hypercalcemia, renal insufficiency, or anemia or bone lesions). The etiology of the disease remains unclear, but race (prevalence of MGUS is 2- to 3-fold higher in African-Americans and blacks from Africa than in Caucasians), older age, gender, exposure to pesticides, and family history of MGUS or multiple myeloma (MM) were previously associated with greater risk of having MGUS (2, 3, 4, 5). Studies have demonstrated that MGUS is the precursor state of virtually all MMs (6, 7). However, the average transformation rate is highly variable, ranging from 0.25% to nearly 3% per year for the lifetime of the patient (or 0.3% 1.3% when adjusting for deaths as a competing risk factor) (8, 9). Proper characterization of these patients is important to allow optimized monitoring and prevention of secondary complications associated not only with the progression but also with the premalignant phase. A recent study suggests that MGUS itself is associated with a number of comorbidities including osteoporosis and fractures, thrombosis, and infections (10, 11, 12, 13). The presence of infections suggests a compromised immune system including reduced levels of uninvolved immunoglobulins. The challenge is on how to identify these patients that are at an imminent risk of developing symptomatic disease or severe comorbidities. Currently, there are several biomarkers that were observed to be associated with a greater risk of transformation, making the base of 2 major models on risk assessment of MGUS patients at diagnosis. These models are commonly known as the Mayo Clinic Model (8, 14) and the Spanish Myeloma Group model (15). Both models rely on different risk factors; while the former is based on serum biomarkers only, the latter includes information from plasma cells on bone marrow (DNA aneuploidy and percentage of aberrant plasma cells), allowing the identification of higher-risk patients (9.8% annually). However, determining the percentage of phenotypically aberrant plasma cells in patients with generally low levels of bone marrow infiltration is not always straightforward, especially in nonspecialized centers. Furthermore, repetition of bone marrow analysis on routine follow-up would be difficult. Therefore, the identification of additional biomarkers with prognostic value that are easily determined could represent an important contribution for the management of MGUS patients. Systemic immunoparesis (IP) of the unaffected immunoglobulins (i.e., suppression of IgA and/or IgM in a patient with IgG monoclonal gammopathy) is generally associated with poorer outcomes in monoclonal gammopathies, but for MGUS patients, results have been inconsistent (16, 17, 15). The Hevylite assay developed in 2009 (18) allowed, for the first time, to easily quantify the uninvolved immunoglobulin associated with the isotype of the immunoglobulin produced by the tumor, but of the alternative light chain (e.g., the IgGκ in a IgGλ producing tumor) and, therefore, study the impact of a new type of IP: the isotype-matched immunosuppression (IMI). The aim of this study was to investigate the correlation of this new parameter with the different MGUS risk groups and to verify its significance as a prognostic factor of MGUS transformation. DOI: /jalm American Association for Clinical Chemistry 4 Nonstandard abbreviations: MGUS, monoclonal gammopathy of undetermined significance; IMI, isotype-matched immunosuppression; IP, immunoparesis; IMWG, International Myeloma Working Group; I/U, involved-to-uninvolved Hevylite ratio; invhlc, involved Hevylite pair; TTP, time to progression.... March : JALM 701

3 MATERIALS AND METHODS Study population The study group was composed by 309 stored serum samples from 248 MGUS patients attending our hospital. The diagnosis sample was considered the earliest available stored sample obtained in the context of the physician visit without a defined time-limit; subsequent available samples of the same patient were regarded as follow-up samples. Light chain only MGUS patients were not included in the analysis. Patients were risk stratified on the basis of the results obtained from the earliest available stored sample and according to the risk factors recommended by 2010 International Myeloma Working Group (IMWG) guidelines (14); in short, serum M- protein levels above 1.5 g/dl, abnormal serum free light chain ratio (<0.26 or >1.65), and non-igg monoclonal immunoglobulin are the independent risk factors for MGUS transformation. METHODOLOGY Immunoglobulin heavy + light chain pairs were determined by the Hevylite assay (The Binding Site) in stored samples and according to the isotype of the monoclonal immunoglobulin. That is, samples of IgG MGUS patients were analyzed with Hevylite IgGκ and Hevylite IgGλ, samples of IgA MGUS were analyzed with Hevylite IgAκ and Hevylite IgAλ, and, finally, samples of IgM MGUS were analyzed with Hevylite IgMκ and IgMλ. All Hevylite determinations were carried out on a BNII analyzer (Siemens Healthcare). The normal Hevylite ranges used for each immunoglobulin were those provided by the manufacturer (95th percentile): IgGκ = g/l, IgGλ = g/l, IgAκ = g/l, IgAλ = g/l, IgMκ = g/l, and IgMλ = g/l. Values determined by Hevylite below the normal ranges of the immunoglobulins were defined as IMI. In contrast to classical IP, the IMI corresponds to suppression of the immunoglobulins of the same heavy chain isotype as the one produced by the tumor, but of the alternative light chain, e.g., suppression of IgAκ in an IgAλ MGUS patient. Severe Hevylite IMI or severe IP were defined by values 50% below the respective lower limit of the normal range. For the Hevylite Ig κ/ig λ ratios, the normal ranges used were also those provided by the manufacturer: for IgGκ/IgGλ, for IgAκ/IgAλ, and for IgMκ/IgMλ. Samples with ratios outside these ranges were considered abnormal. Serum protein electrophoresis and immunofixation were carried out by use of Sebia gels on Hydrasys 2 scan automated (Sebia). Serum free light chains were measured with use of the Freelite κ and Freelite λ assays (The Binding Site) in a BN prospec nephelometer. Total immunoglobulin levels (Siemens Healthcare) were also recovered to study the frequency of classical IP (normal ranges: IgG = g/L, IgA = g/L, and IgM = g/L). Statistical significance of group differences were assessed by one-way ANOVA, chi-square, and Student t-tests for α = 0.05 (95% CI). Progression curves were generated by the Kaplan Meier method in which patients who died were censored and curves were compared by the log-rank test. Multivariate analysis was performed by Cox regression with use of only significant variables from univariate analysis. The primary end point was progression to MM or related disorders or to smoldering Myeloma confirmed by clonal bone marrow plasma cells >10%. SPSS and GraphPad Prism v.5 were used for statistical analysis. RESULTS Characterization of MGUS patients Patients' characteristics are described in Table 1. Briefly, the population is well balanced in terms 702 JALM :05 March 2018

4 Table 1. Characterization of MGUS patients at the time of first Hevylite determination. MGUS isotype IgG IgA IgM All N, (% of All) 164 (66) 44 (18) 40 (16) 248 Light chain Kappa, % Lambda, % Gender Male, % Female, % Age, years Median (range) 74 (29 100) 71 (39 92) 73 (42 93) 73 (29 100) M-protein Median (range), g/dl 0.7 (0.1 3) 0.4 ( ) 0.6 ( ) 0.6 (0.1 3) Free light chain ratio Normal ( ) 45% 32% 45% 43% Hevylite ratio Abnormal 87% 100% 92% 90% Risk group (IMWG) Low, % Low-intermediate, % Intermediate-high, % High, % of gender (1.07 female to male proportion), and the median age of diagnosis (73 years) and isotype MGUS distribution (66% for IgG, 18% for IgA, and 16% for IgM) are in fair agreement to what was previously reported by recent studies on MGUS populations (9, 19). The distribution of MGUS risk groups (Table 1) according to the IMWG risk stratification model shows a moderate overall underrepresentation of the lowest risk group (28%) in favor of the intermediate-high group (29%) when comparing to data published by the Mayo Clinic based on a cohort of Minnesota residents (8). Immunoglobulin heavy + light chain analysis by Hevylite Of the total number of patients analyzed with Hevylite, 90% showed an abnormal Hevylite Ig κ/ Ig λ ratio at baseline, with the greatest percentage of altered ratios observed in IgA and IgM patients (Table 1). In addition, samples from patients of higher risk MGUS groups were observed to have greater imbalance of involved-to-uninvolved Hevylite ratios (I/U; i.e., proportion of the monoclonal to polyclonal Hevylite immunoglobulin pair), with median I/U values of 3.7, 7.8, 37.6, and 173 for patients with 0, 1, 2, and 3 risk factors, respectively (P < ). The I/U Hevylite ratio shows a weak correlation with the age of the patients (P = , r = 0.174) and with the concentration of monoclonal component by serum protein electrophoresis (P = , r = 0.159). Isotype-matched immunosuppression The specific uninvolved heavy + light chain combination corresponding to the monoclonal isotype could be determined in 307 out of the 308 available serum samples. Insufficient volume of the stored sample prevented determination of the whole set. Considering baseline samples only, IMI... March : JALM 703

5 the noninvolved isotypes (e.g., suppression of IgG and/or IgM in an IgA-MGUS). This value is significantly lower than the result obtained for the frequency of IMI (P < 0.002). In addition, a tendency to a greater number of patients with IP is observed with increasing MGUS risk group (Fig. 1). Outcomes Fig. 1. Patients with suppressed immunoglobulin levels divided by MGUS risk stratification. Risk stratification was based on the recommendations of the IMWG. Risk factors: monoclonal component >1.5g/ dl; non-igg monoclonal isotype; abnormal serum free light chain ratio. Patients with 0, 1, 2, or 3 risk factors are low risk, low-intermediate risk, intermediate-high risk, and high risk, respectively. Isotype-matched immunosuppression (IMI). was observed in 51% of the patients (n = 121), and severe IMI was observed in 17% (n = 41). Analyzing the frequency of IMI as a function of monoclonal isotype shows that IgM patients (18% IMI) are less affected than IgG (57%) and IgA (58%) patients (P < ). The presence of IMI by Hevylite showed a tendency to be more frequent in patients of higher risk MGUS groups (Fig. 1), although not reaching a statistical difference (P = 0.1). Analysis of the uninvolved IgGλ concentrations of IgGκ MGUS patients showed a significant trend (P = ) toward lower levels (median values) as the risk group increases (Fig. 2). The same correlation was observed for the uninvolved IgGκ levels in IgGλ MGUS patients (P = ). No correlation was observed between levels of uninvolved Hevylite immunoglobulins and parameters such as β 2 -microglobulin levels, monoclonal component concentration by serum protein electrophoresis, free light chain involved/uninvolved ratios, or age of the patient (data not shown). Regarding classical IP, 37% of the baseline samples showed suppression of at least one of Of the 248 MGUS patients, there were follow-up clinical records available for 154 individuals (107 IgG, 20 IgA, and 27 IgM), representing 12 progressions to MM or related disorder, and 4 progressions to smoldering myeloma, and an estimated median time to progression (TTP) of 100 months. The distribution according IMWG risk group was similar to the initial patient cohort: 31% low risk, 41% low-intermediate, 27% intermediate-high, and 1% high risk. The effects of Hevylite severe IMI, Hevylite Ig κ/ig λ ratio, severe classical IP, and abnormal κ/λ free light chain ratios were studied for their significance as prognostic factors of MGUS progression to symptomatic disease or to a smoldering state. Severe IMI was present in 21 out of 154 patients (14%) with follow-up, close to the incidence observed for the complete cohort (17%), and was able to differentiate 2 groups of patients with significantly different median TTP (P = 0.024; HR = 3.2; 95% CI, ), while severe classical IP of either one of the noninvolved immunoglobulins (e.g., IgA or IgM in an IgG patient) was not associated with a statistically significant shorter TTP (P = 0.48) (Fig. 3). Importantly, there were almost twice as many patients with severe IMI (n = 21) than with severe classical IP (n = 11). The median Hevylite Ig κ /Ig λ ratios calculated for each monoclonal isotype group were as follows: IgGκ = 7.2, IgGλ = 0.41, IgAκ = 17.3, IgAλ = 0.09, IgMκ = 30.4, and IgMλ = Patients with Hevylite ratios more extreme than the median value for the respective MGUS isotype showed only a tendency toward a shorter TTP (P = 0.089), 704 JALM :05 March 2018

6 Fig. 2. Results of the levels of uninvolved immunoglobulins determined by Hevylite, divided by risk group and by heavy/light chain pair. Risk groups according to the IMWG recommendations on MGUS stratification. Horizontal lines represent median values for each risk group. Only IgG patients show statistical differences between groups (P < 0.001). while an involved Hevylite pair (invhlc) or an M-spike >1.5g/dL both significantly correlated with shorter TTP (Table 2). An abnormal κ/λ free light chain ratio was found to significantly correlate with a shorter TTP only for IgG MGUS patients (P = 0.045; HR = 3.2; 95% CI, ). Multivariate analysis by Cox regression (Table 2) including the Hevylite variables that were significant in the univariate analysis (i.e., severe IMI and invhlc >1.5g/dL) retrieved a hazard ratio of 3.1 (95% CI, ; P = 0.046) for severe IMI and a hazard ratio of 5.9 (95% CI, ; P = 0.001) for invhlc >1.5g/dL. Eight patients had both an invhlc >1.5g/dL and severe IMI, showing an estimated median TTP of 17 months (4 progressions to MM and 1 to smoldering myeloma) in contrast with the 100 months (Fig. 4) estimated for the remaining group (P < ).... March : JALM 705

7 DISCUSSION Fig. 3. Progression-free survival in 154 MGUS patients according to the severity of the isotype-matched immunosuppression and of the classical immunoparesis. (A), The graph illustrates the effect of severe isotypematched immunosuppression (IMI), i.e., >50% reduction of the levels of the uninvolved immunoglobulin determined by Hevylite, on progression-free survival estimated by the Kaplan Meier method (P = 0.024). Severe IMI was observed in 21 patients of this subgroup. (B), The graph illustrates the effect of severe immunoparesis (IP), i.e., >50% reduction of the levels of the immunoglobulins of a different isotype than that produced by the tumor (e.g., suppression of IgA and/or IgM in an IgG MGUS patient), on progression-free survival (P = 0.48). Finally, out of the 154 patients used in the survival analysis, 18 had >1 Hevylite determination: 6 had an estimated negative variation of the uninvolved Hevylite pair of >20% per year and were positively associated with a shorter TTP when comparing with the other 12 who did not present with a similar trend, with 5 years vs 8 years of median time to progression, respectively (P = ; HR = 27.5; 95% CI, ). To investigate the potential utility of the Hevylite assay and in particular the contribution of IMI to the risk assessment of progression for MGUS patients, 2 approaches were designed for this study. First, the results of the assay were compared with the risk stratification obtained by using the previously validated risk factors of the Mayo Clinic Model; second, a progression-free survival analysis was performed to determine the prognostic significance of IMI measured by Hevylite in predicting TTP. The first approach showed that both the frequency of Hevylite IMI and the frequency of classical IP correlated with the MGUS risk group, although significantly more patients had IMI than classical IP. The result obtained in this study with IMI (51%) is much higher than the value of 27% observed by Katzmann and coworkers (9) and closer to what Pika et al. had (20). The difference might be related to the already mentioned underrepresentation of the lowest risk group when compared to the Minnesota cohort. On the other hand, the levels of the uninvolved Hevylite pair were also observed to progressively decrease with increasing risk group, indicating that immunosuppression is very likely related with a greater risk of progression. Similar observations were reported in previous studies (20, 9), highlighting the relevance of a compromised host immune system in myelomagenesis. In the study of Pika et al. (20), it is also apparent that there is a greater incidence of IMI over classical IP, although in the high-risk group the differences are only marginal (a result also seen in our study). These observations may simply be the result of the greater sensitivity of the Hevylite assay, or they could be related to the mechanism of isotype-specific feedback involved in the regulation of circulating IgG levels, first reported for monoclonal gammopathy patients by Wang and Young in 2001 (21). The authors speculate that the absence of correlation observed between the 706 JALM :05 March 2018

8 Table 2. Univariate and multivariate analyses of analytical serum parameters. Univariate analysis Multivariate analysis HR (95%CI) p HR (95%CI) p HR (95%CI) p Abnormal FLC a ratio 2.2 ( ) Non-IgG MGUS 0.5 ( ) M-Ig > 1.5g/dL 8.3 ( ) < ( ) HLC Ig'κ /Ig'λ 2.4 ( ) ihlc > 1.5g/dL 8.7 (2.7 28) ( ) IMI b 1.1 ( ) Severe IMI 3.2 ( ) ( ) ( ) Severe IP c 1.8 ( ) Bold p values represent statistically significant results. a FLC stands for free light chain. b IMI stands for isotype-matched immunosuppression. c IP stands for classical immunoparesis. Fig. 4. Progression-free survival as a function of the number of risk factors. The risk factors considered were involved Hevylite (invhlc) >1.5g/dL and isotype-matched immunosuppression (IMI) >50%. Blue line: 0 risk factors; black line: any 1 risk factor; red line: both risk factors present. P < levels of polyclonal immunoglobulins (i.e., classical IP) and M-proteins precludes a mechanism of suppression solely dependent on the M-protein levels. Instead, a mechanism of isotype-specific suppression dependent on increased numbers of T cells bearing surface receptors of the respective monoclonal isotype might be more relevant, as observed in mouse models with plasmacytomas (22, 23). More recently, several publications have shown that functional regulatory T cells (T-regs) were increased in the peripheral blood of MGUS and MM patients, and it is proposed that this could be a consequence of the malignant transformation of the clone (24, 25). If indeed clone expansion stimulates the production of isotype-specific T cells with the consequent suppression of the isotypematched B-cells, severe IMI could be a more specific and precocious marker of disease progression than classical IP. This could be perceived as a very simplistic interpretation, as the network of interactions between plasma cells (malignant and nonmalignant), natural killer cells, T cells, and other components of the immune system are tremendously complex (26). Indeed, additional suppression mechanisms must be considered, such as those dependent on high levels of BCMA (B-cell maturation antigen) expressed by tumor cells with the consequent harvest of circulating BAFF (B-cell activating factor), which leads to a general reduction of polyclonal immunoglobulins (27). More studies would be necessary to further elucidate the underlying mechanism. The second approach of this study, as stated before, addresses the potential relation between severe immunosuppressed MGUS patients and a shorter TTP to overt MM or to a smoldering state. Using a subset of patients for which follow-up records were available, we observed that only severe... March : JALM 707

9 IMI, and not severe IP or very abnormal Hevylite ratios, could significantly predict a shorter TTP of MGUS patients. The lack of statistical significance of IP may be a question of low patient number, but it may also reflect the greater specificity of IMI over IP as a prognostic biomarker, which might be expected if an isotype-specific mechanism of immunosuppression plays a role in disease progression. These results are in line with a study by Katzmann and colleagues (9), in which, using a large population from southeastern Minnesota, the authors found that Hevylite pair suppression (i.e., IMI below the reference range), but not classical IP, was an independent risk factor for early progression validated by a multivariate analysis including M- protein size and free light chain ratios as additional variables. In our population, multivariate analysis confirmed that severe IMI and involved Hevylite >1.5g/dL were independent prognostic factors and that the group of patients presenting both risk factors showed a median TTP about 6-fold shorter than the remaining group. It is important to stress, however, that the absolute values presented in this study for TTP have to be interpreted with caution because the subset of MGUS patients used for the analysis is a skewed population as evidenced by the median progression-free survival of only 100 months and an overrepresentation of the higher risk groups: 30% for the intermediate-highrisk and high-risk groups together, as compared with the 20% observed for the same 2 risk groups in the Minnesota cohort (8). This is most likely due to the fact that patients with more risk factors are more closely followed than those with a lower risk. More recently, Magnano et al. (28) commented that an evolving pattern of Hevylite suppression was observed in 3 out of 4 patients with an evolving M-spike. An evolving M-spike has been shown to be a marker of progression of MGUS and smoldering myeloma patients (29, 30, 7). In the present study, we found a correlation between an evolving immunosuppression pattern and a shorter TTP, which could further add to the potential role of Hevylite pair suppression as a marker for MGUS transformation. However, the fact that only 18 MGUS patients had >1 Hevylite follow-up result precludes any real assumptions about the results of the evolving immunosuppression pattern. Finally, 2 recent works approached the role of IMI in MM patients (31, 32) and found that the levels of the uninvolved Hevylite pair correlated with response status, progression-free survival, and overall survival of newly diagnosed and relapsed/ refractory patients. Earlier, Bradwell et al. (33) had also found that myeloma patients with lower uninvolved immunoglobulin concentrations had shorter progression-free survival. These observations suggest that the role of IMI could remain relevant throughout the entire disease course. In summary, our findings further contribute toward strengthening the potential utility of IMI as a new and independent risk factor for malignant transformation in MGUS patients, prognostic of shorter time to disease. Inclusion of this parameter in the risk stratification model of MGUS patients could contribute to more accurately predict malignant transformation and help identify patients at imminent risk of transformation who could, eventually, benefit from early treatment. 708 JALM :05 March 2018

10 Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 4 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; (c) final approval of the published article; and (d) agreement to be accountable for all aspects of the article thus ensuring that questions related to the accuracy or integrity of any part of the article are appropriately investigated and resolved. J.J. Jiménez, C.H. Larramendi and M.A.P. Díaz performed the research. J.J. Jiménez, T.M. Pais, N. Barbosa, and M.L. Campos analyzed the data and wrote the paper. M.A.P. Díaz and C.H. Larramendi reviewed the paper. Authors Disclosures or Potential Conflicts of Interest: Upon manuscript submission, all authors completed the author disclosure form. Employment or Leadership: T.M Pais, N.M. Barbosa and M.L. Campos, The Binding Site, Spain. Consultant or Advisory Role: None declared. Stock Ownership: None declared. Honoraria: None declared. Research Funding: None declared. Expert Testimony: None declared. Patents: None declared. Role of Sponsor: The sponsor played a direct role in the preparation and final approval of the manuscript and the choice of enrolled patients. REFERENCES 1. Kyle RA, Rajkumar SV. Monoclonal gammopathy of undetermined significance. Br J Haematol 2006;134: Singh J, Dudley AW Jr., Kulig KA. Increased incidence of monoclonal gammopathy of undetermined significance in blacks and its age-related differences with whites on the basis of a study of 397 men and one woman in a hospital setting. J Lab Clin Med 1990;116: Landgren O, Kristinsson SY, Goldin LR, Caporaso NE, Blimark C, Mellqvist UH, et al. Risk of plasma cell and lymphoproliferative disorders among first-degree relatives of 4458 patients with monoclonal gammopathy of undetermined significance in Sweden. Blood 2009; 114: Landgren O, Kyle RA, Hoppin JA, Beane Freeman LE, Cerhan JR, Katzmann JA, et al. Pesticide exposure and risk of monoclonal gammopathy of undetermined significance in the Agricultural Health Study. Blood 2009; 113: Vachon CM, Kyle RA, Therneau TM, Foreman BJ, Larson DR, Colby CL, et al. Increased risk of monoclonal gammopathy in first-degree relatives of patients with multiple myeloma or monoclonal gammopathy of undetermined significance. Blood 2009;23: Landgren O, Kyle RA, Pfeiffer RM, Katzmann JA, Caporaso NE, Hayes RB, et al. Monoclonal gammopathy of undetermined significance (MGUS) consistently precedes multiple myeloma: a prospective study. Blood 2009;113: Weiss BM, Abadie J, Verma P, Howard RS, Kuehl WM. A monoclonal gammopathy precedes multiple myeloma in most patients. Blood 2009;113: Rajkumar SV, Kyle RA, Therneau TM, Melton LJ 3rd, Bradwell AR, Clark RJ, et al. Serum free light chain ratio is an independent risk factor for progression in monoclonal gammopathy of undetermined significance. Blood 2005;106: Katzmann JA, Clark R, Kyle RA, Larson DR, Therneau TM, Melton LJ 3rd, et al. Suppression of uninvolved immunoglobulins defined by heavy/light chain pair suppression is a risk factor for progression of MGUS. Leukemia 2013;27: Bida JP, Kyle RA, Therneau TM, Melton LJ 3rd, Plevak MF, Larson DR, et al. Disease associations with monoclonal gammopathy of undetermined significance: a population-based study of 17,398 patients. Mayo Clin Proc 2009;84: Gregersen H, Jensen P, Gislum M, Jørgensen B, Sørensen HT, Nørgaard M. Fracture risk in patients with monoclonal gammopathy of undetermined significance. Br J Haematol 2006;135: Kristinsson SY, Fears TR, Gridley G, Turesson I, Mellqvist UH, Björkholm M, Landgren O. Deep vein thrombosis after monoclonal gammopathy of undetermined significance and multiple myeloma. Blood 2008;112: Kristinsson SY, Björkholm M, Andersson TM, Eloranta S, Dickman PW, Goldin LR, et al. Patterns of survival and causes of death following a diagnosis of monoclonal gammopathy of undetermined significance: a population-based study. Haematologica 2009;94: Kyle RA, Durie BG, Rajkumar SV, Landgren O, Bladé J, Merlini G, et al. Monoclonal gammopathy of undetermined significance (MGUS) and smoldering (asymptomatic) multiple myeloma: IMWG consensus perspectives risk factors for progression and guidelines for monitoring and management. Leukemia 2010;24: Pérez-Persona E, Vidriales MB, Mateo G, García-Sanz R, Mateos MV, de Coca AG, et al. New criteria to identify risk of progression in monoclonal gammopathy of uncertain significance and smoldering multiple myeloma based on multiparameter flow cytometry analysis of bone marrow plasma cells. Blood 2007;110: Kyle RA, Therneau TM, Rajkumar SV, Offord JR, Larson DR, Plevak MF, Melton LJ 3rd. A long-term study of prognosis in monoclonal gammopathy of undetermined significance. N Engl J Med 2002;346: March : JALM 709

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