Comparative evaluation of involved free light chain and monoclonal spike as markers for progression from monoclonal gammopathy of undetermined significance to multiple myeloma

Abstract Monoclonal gammopathy of undetermined significance (MGUS) is a premalignant clonal plasma cell disorder, with a 1% yearly risk of progression to multiple myeloma (MM). Evolution of M‐spike and serum free light chain (sFLC) during follow‐up could identify patients at high risk of progression. In this region‐wide study, including 4756 individuals, 987 patients with MGUS were identified, and baseline factors as well as evolving involved FLC (iFLC) were evaluated as potential markers for risk of progression from MGUS to MM. Furthermore, evolving iFLC and M‐spike were assessed quarterly for a median of 5 years. At baseline, patients that progressed had significantly higher iFLC compared to non‐progressors. The risk factors of M‐spike >1.5 g/dL, age >65 years and iFLC >100 mg/L were all independently associated with increased risk of MGUS to MM progression. For patients that had any two or three risk factors, the 5‐year cumulative probability of progression was significantly higher (31%) compared to no risk factors (2%). Evolving iFLC >100 mg/L during follow‐up was consistently associated with increased risk of progression. Based on our observations, we propose to include iFLC as a monitoring tool for all MGUS patients. Furthermore, we recommend a quarterly monitoring in all high‐risk patients. Finally, we suggest that the risk of MGUS progression should be stratified with age, M‐spike, and iFLC at baseline.

(IMWG) risk criteria. 2 Evolving M-protein (increasing levels over time) has been suggested as a risk factor in smoldering multiple myeloma (SMM) in smaller retrospective studies. [7][8][9] Furthermore, it has been reported that MGUS patients that progress to MM convert from low/intermediate-risk to high-risk MGUS prior to the MM diagnosis. 10 Abnormal FLCr are detected in more than 95% of MM but only in 30%-47% of MGUS cases. 4,6,[11][12][13] It can be hypothesized that evolving sFLC could be a predictor for progression from MGUS to MM. The current IMWG diagnosis criteria recommend measuring sFLC in combination with serum protein electrophoresis (sPEP) and immunofixation when screening for plasma cell dyscrasia and for risk classifications. However, sFLC measurements are not currently included in MGUS monitoring guidelines. 2 It is of importance to identify high-risk MGUS groups, both for better risk classification at diagnosis in conjunction with a more optimal monitoring during follow-up. Furthermore, high-risk MGUS patients may potentially be considered for preventive measures to limit the progression to MM. As the percentage of patients with detectable iFLC are, and becoming higher in MM compared to MGUS, we hypothesized that evolving iFLC during monitoring may be an essential risk factor for progression. We therefore analyzed a unique dataset from hematology clinics and primary care units in Sweden, with up to 46 years of follow-up.  Figure S1). All MGUS patients were required to have no CRAB symptoms (hypercalcemia, renal failure, anemia and bone lesions) and a M-protein value equal to or below 3.0 g/dL. 2 Patients with high risk MGUS or symptoms of bone lesions were referred to imaging according to local guidelines. In patients with a M-protein equal to or below 1.5 g/dL (n = 836) a confirmatory bone marrow was not required for MGUS diagnosis. In the remaining 151 patients, a bone marrow confirmation of the MGUS diagnosis were only obtained in 73 patients.
For each individual, besides sFLC, routine laboratory and clinical parameters were collected and compiled from all available sampling occasions during the study period (Table 1). Regarding s/uPEP; M-protein of heavy and light chain types, Mprotein size, total immunoglobulin levels, urine kappa and lambda levels, were included in the database. Serum FLC assays were conducted with latex-enhanced immunonephelometric assay (Siemens Healthcare GmbH, Erlangen, Germany). Total serum immunoglobulin (IgG, IgA, and IgM) concentrations were analyzed using immunoturbidimetric assay (Roche Diagnostics GmbH, Mannheim, Germany). Urine light chains (kappa and lambda) concentrations were analyzed using immunonephelometric assay (Siemens Healthcare GmbH, Erlangen, Germany). S/uPEP and immunofixation were performed with agarose gels on the Hydrasys/Hydrasys 2 platform (Sebia, Lisses, France). Sequential data were evaluated in a subgroup of the main MGUS cohort, where at least two serial samples, for example, a baseline sample and one additional sample, with a minimum of 3 months between, per patient were available during the study period and before the progression to MM, and where the sample nearest to MM diagnosis was stipulated to be a minimum of 6 months prior to the date of the diag-

| Statistical analyses
The endpoint was time from first sample available at MGUS diagnosis (baseline sample) to progression of MM, either smoldering or treatment demanding MM as defined by IMWG. 14 The effects of prognostic factors were estimated by univariate Cox regression, where the hazard ratios (HR) (CI threshold: 95% and P value threshold < .05) were reported. Log-rank tests were performed for intergroup comparisons. The Kaplan-Meier method was used to calculate medians at 60 months and their associated 95% confidence intervals.    (Table 1).
In the sequential cohort, M-spike >1.5 g/dL and abnormal FLCr were observed more frequently in MGUS-MM (P < .001) compared to MGUS-NP (P < .001). However, no differences were observed for heavy chain type between MGUS-MM and MGUS-NP (Table S1).

| Risk of progression
The five-year cumulative probability of progression was 5% for the whole MGUS cohort ( Figure S2) which is in line with previously published reports. 5,17

| By IMWG classification
Previously reported factors associated with progression from MGUS to MM are shown in Table 1. Of these risk factors, abnormal FLCr (P < .001) and serum M-spike >1.5 g/dL (P < .001) were associated with increased risk of progression to MM, whereas non-IgG was not associated with any increased risk.

| Proposed additional factors at diagnosis
Age was a major correlate to progression, both as a continuous variable and with cut-offs of >65 years and >70 years. Another factor that was significant in univariate/multivariate analyses was hemoglobin. However, when investigating the differences between MGUS-NP and MGUS-MM using a chi-square test (Table 1), no significant differences between the median hemoglobin levels in MGUS-NP (median 129 g/L, range 118-140 g/L) and MGUS-MM (median 128 g/L, range 115-137 g/L) can be found (P = .36). Therefore, we cannot deduce that hemoglobin is a risk factor for this cohort.

| Cumulative probability of progression
To further investigate the impact of the proposed risk factors, M-spike >1.5 g/dL, age >65 years, and iFLC >100 mg/L, we stratified the patients according to the risk factors. As there was no significant difference, and the TTP-KM curves were superimposed, between the groups with two and three risk factors, potentially due to the low number of patients with all the three risk factors (n = 21, 2%), these groups were merged. Patients were assessed as low risk (no risk factor, n = 301, 30%), intermediate-risk (any one of the risk factors, n = 585, 59%), and high risk (two or more risk factors, n = 101, 10%).  Figure 2C).
An increase in iFLC of >100 mg/L, from baseline value, was consistently associated with a significantly increased risk of developing MM, 6 months from baseline, and up until 6.25 years, ( Figure 2B). An increase in iFLC of >100 mg/L, regardless of baseline values, was significantly associated with higher risk of progression from baseline and up until 7 years, ( Figure 2D).
To assess the performance of the evolving biomarkers as risk fac-   Beside the already established risk factors recommended by the IMWG, other groups have suggested that immunoparesis, 10,12,13,18 aberrant plasma cells in bone marrow plasma cell compartment 18  Patients with low risk (30%) had a very low five-year risk to progression (2%). Only eight (3%) of the patients without any risk factor progressed during follow-up in our cohort. Thus, we suggest that patients with low risk of progression could be emitted from follow-up.

| DISCUSSION
The five-year risk of progression observed in intermediate-risk was 11% compared to 31% in high-risk patients. On the contrary to lowrisk patients, the increased risk observed in both intermediate and high-risk patients supports these patients' regular monitoring. As evolving iFLC seems to be a significant factor during follow-up, we suggest every 6-12 months, for intermediate-risk, and quarterly, for high risk, monitoring with iFLC during the first 5 years.
The main goals of this study were to incorporate the iFLC into prognostic models and the monitoring of MGUS patients. The presence of evolving iFLC is a clinically significant risk factor of MGUS progression. Based on the data reported here we propose to monitor iFLC for patients with evolving iFLC values >100 mg/L quarterly. We further recommend that incorporation of M-spike >1.5 g/dL, age >65 and iFLC >100 mg/L as risk factors in prediction models.

ACKNOWLEDGEMENTS
This study was funded by Cancerfonden, grant number 190190 Pj01.

AUTHOR CONTRIBUTIONS
Charlotte Gran and Hareth Nahi conceived the study and oversaw overall direction and planning. Charlotte Gran and Hareth Nahi wrote the manuscript with input from all authors. Johan Liwing, Andre Verhoek and Ana Gezin analyzed the data. Evren Alici and Hareth Nahi supervised the project. All authors critically revised the manuscript and approved the final version.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available upon reasonable request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.