Plasma Circulating Proteasomes As Biomarkers Along Natural History Of Asymptomatic Monoclonal Gammopathies
Authors: Larrea Carlos Fernández de , Zingone Adriana , Manasanch Elisabet E. , Korde Neha , Wu Peter , Costello Rene , Zuchlinski Diamond , Mulquin Marcia , Burton Debra , Maric Irina , Calvo Katherine R , Stetler-Stevenson Maryalice , Yuan Constance , Roschewski Mark , Landgren Ola 
Published: October 2019

Abstract

Background
Monoclonal gammopathy of undetermined significance (MGUS) and smoldering multiple myeloma (SMM) are asymptomatic plasma cell dyscrasias with a heterogeneous probability to progress to symptomatic multiple myeloma (MM). Reliable markers for progression to MM are vital to advance the understanding of myeloma precursor disease and for the development of intervention trials designed to delay/prevent MM. The Mayo Clinic and Spanish PETHEMA have proposed models to stratify patient risk based on clinical parameters. At the current time, no molecular biomarkers have been established to determine risk of transformation. Based on the fact that MM tumor cells are highly sensitive to proteasome inhibition and that circulating proteasomes (cProt) have been detected in the blood of MM patients, we conducted a prospective clinical study designed to characterize patterns of cProt in peripheral blood from MGUS, SMM and MM patients.

Patients and Methods
Ninety two patients diagnosed with asymptomatic monoclonal gammopathies (39 MGUS and 53 SMM; median age 63 years; 46M/47F) were studied. This group was compared to normal sera from healthy donors (n=6) and untreated patients with recent diagnosed MM (n=38). Initial baseline demographics, clinical and laboratory data were collected. MGUS patients were classified according to Mayo Clinic risk score (M-protein, monoclonal isotype and serum FLC), while SMM could be stratified according to PETHEMA (malignant bone marrow plasma cell (BMPC) percentage and immunoparesis) and Mayo system (BMPC infiltration, serum M-protein and serum FLC). Plasma and bone marrow supernatant samples were collected at diagnosis and frozen to -80ºC. In 58 MGUS and SMM cases, sequential plasma samples at 6 months and 1 year were also analyzed. Chymotrypsin-like, caspase-like, and trypsin-like activities from cProt were assayed by continuously monitoring the production of 7-amino-4-methylcoumarin (AMC) from fluorogenic peptides by plasma. Briefly, samples were activated with SDS (for chymotrypsin-like and caspase-like) or 10% Tween-20 (for trypsin-like). The reaction wells contained 30 μL assay buffer (25 mmol/L HEPES), 10 μL activated sample, and 10 μL of the prospective fluorogenic peptide-AMC substrate. To measure the fluorescence release of free AMC with time, the SpectraMax M5 (Molecular Devices) instrument was used with a read interval of 1 min during 30 min at 37ºC. All samples were performed by triplicate. Enzymatic activities were quantified (pmol AMC/s/mL plasma) by generating a standard curve of AMC.

Results
MGUS patients had zero (38.5%), one (41%) or two risk factors (20.5%) according to the Mayo Clinic model. In contrast, 49% of the patients with SMM were classified as high-risk according to the PETHEMA model, versus 69.8% with 2 or 3 risk factors in the Mayo Clinic model. Chymotrypsin activity levels in plasma were statistically correlated with serum M-protein concentration and total IgG concentration (p<0.001). Chymotrypsin-like activity was differentially expressed in plasma across the different groups of patients (p=0.009; Figure 1). Particularly, SMM and MM showed higher levels than healthy controls and MGUS patients. In SMM, patients with highest-risk of transformation showed a higher levels of this chymotrypsin-like activity than the other groups (p=0.02). When only IgG SMM and MGUS patients were considered, a correlation with immunoparesis (reduction of IgM and IgA), BMPC infiltration, relative lower hemoglobin levels and higher FLC ratio (p<0.05) was observed. Caspase-like activity was also associated with diagnosis, showing higher levels in symptomatic and SMM patients than healthy donors and MGUS (p=0.016) (Figure 2) and correlated with IgG and serum M-protein (p=0.01 and p=0.006). In contrast, trypsin-like levels were negatively correlated along the spectrum of tumoral mass in the four groups (p=0.004) (Figure 3). Bone marrow supernatant chymotrypsin activity was higher in symptomatic MM than MGUS patients (p=0.004), with a trend for caspase.

Conclusion
Chymotrypsin-and caspase-like activity of circulating proteasome in asymptomatic gammopathies is related to tumoral mass and immunoparesis degree. MGUS patients are close to healthy individuals, with SMM not so different than symptomatic patients. Prognostic significance of these findings after longer follow-up is warranted.

Disclosures:
No relevant conflicts of interest to declare.