Characterization of monoclonal gammopathy of undetermined significance by calorimetric analysis of blood serum proteome

Abstract

Monoclonal gammopathy of undetermined significance (MGUS) is a premalignant proliferative disorder that may progress to multiple myeloma, a malignant plasma cell neoplasia. We evaluated differential scanning calorimetry (DSC) as an experimental tool for differentiating serum samples of MGUS patients from healthy individuals. DSC thermograms can be used for monitoring changes in the serum proteome associated with MGUS. MGUS patients showed great variability in serum thermogram characteristics, which depended on the IgG, IgA or IgM isotypes and/or the κ or λ light chains. Thermogram feature parameters distinguished patients with MGUS from healthy people. Serum samples, named as non-MGUS, were also collected from patients with subjacent immunological pathologies who were discarded of having MGUS through serum immunofixation. They were used to verify the sensitivity of DSC for discriminating MGUS from related blood dyscrasias. Only some DSC thermogram feature parameters differentiated, to a lesser extent, between MGUS and non-MGUS individuals. We contemplate DSC as a tool for early diagnosis and monitoring of MGUS.

Fig 1. DSC thermograms of blood serum samples stored at 4°C for different periods of time.

(A) Thermograms of healthy serum samples analyzed immediately (0 days), and after 2, or 7 days. (B) MGUS-κ serum samples (experimental group based on the κ light chain that is associated with different monoclonal immunoglobulin heavy chains) analyzed after 0, 2, and 7 days.

Fig 2. DSC thermograms of serum samples from healthy individuals and patients with MGUS-κ.

Samples were gathered as MGUS-κ based on the κ light chain that was found associated with different monoclonal immunoglobulin heavy chains. (A) A set of thermograms obtained from six healthy (control) serum samples. The panel shows an average healthy control plot (dashed brown line) and the standard deviation (shadow). (B) A set of thermograms of serum samples obtained from MGUS patients with IgG κ subtype. (C) Another set of themograms obtained from individuals with IgG κ subtype. For the ease of visualization, thermograms were clustered in either panel B or C based on the similarity of their DSC profiles. (D) Thermograms from patients having IgG1-κ subclass. (E) Thermograms from patients having IgA κ (black) or IgM κ (red) subtypes. For the sake of comparison, the average healthy (control) thermogram (dashed brown line) is presented in all the panels.

Fig 3. DSC thermograms of serum samples from patients with MGUS-λ.

Samples were gathered as MGUS-λ based on the λ light chain that was found associated with the different monoclonal immunoglobulin heavy chains. (A) A set of thermograms of serum samples obtained from patients with IgG λ subtype. (B) A set of thermograms from individuals with MGUS IgG λ subtype (black) or IgM λ subtype (red) grouped together to highlight their similar shape. (C) Several thermograms from patients having IgA λ (continuous black and red plots, and dashed black plot) or IgM λ (dashed red plot) subtypes grouped together because they show similar shapes. For the sake of comparison, the average healthy (control) thermogram is presented in all the panels.

Fig 4. DSC thermograms of serum samples from non-MGUS individuals.

Non-MGUS refers to serum samples from individuals with subjacent immunological pathologies who were ruled out of having MGUS through serum immunofixation. (A), (B) Two sets of themograms obtained from non-MGUS individuals. For the ease of visualization, the experimental thermograms were clustered in either panel based on the similarity of the shapes of their DSC profiles. For the sake of comparison, the average healthy (control) thermogram is presented in all the panels.

Fig 5. Box chart representations of thermogram feature parameters.

For each panel, boxes from left to right represent serum samples from (a) healthy controls, (b) MGUS-κ, (c) MGUS-λ, (d) All-MGUS (which compiles MGUS-κ and MGUS-λ samples together) and (e) non-MGUS. The bottom and top box edges indicate the 25^th and 75^th percentiles. Within the box, the median value is indicated by the horizontal line, and the mean by the cross inside the box. The whiskers extend from the ends of the boxes to the minimum and maximum experimental values.

Fig 6. Differences in thermogram feature parameters across the serum sample groups.

The thermogram feature parameters are identified at the top of each panel. The analysis of the data by a non-parametric Kruskal-Wallis test revealed significant differences in the six thermogram parameters displayed in the figure (Area, p = 9.82E-3; T_max, p = 8.73E-3; Cp1^ex, p = 5.59E-3; Cp2^ex, p = 4.26E-4; Cp1^ex/ Cp2^ex, p = 5.92E-4; T_FM, p = 2.16E-3), indicating they can be used to distinguish the different serum groups, and also validated the utilization of post-hoc pairwise comparisons. Differences between the serum sample groups were examined for every thermogram parameter by the unpaired Mann-Whitney U test. The figure panels indicate “yes” or “no” to specify whether statistically significant differences occurred (the actual p values are shown in the panels).