The systemic cytokine environment is permanently altered in multiple myeloma

Abstract

Multiple myeloma (MM) is an incurable bone marrow malignancy of the B cell lineage. Utilizing multiplex Luminex technology we measured levels of 25 cytokines in the plasma of normal donors (n = 177), those with monoclonal gammopathy of undetermined significance (n = 8), and MM patients (n = 55) with either active disease, on treatment, or in remission. The cytokine levels were compared between normal donors and MM patients as well as between various phases of MM, and discriminant analysis was used to create a predictive classification model based on the differentially expressed cytokines. Evaluating age- and gender-dependence of cytokine expression, we determined that with age there is a shift toward a pro-inflammatory environment. Moreover, we observed a strong gender bias in cytokine expression. However, the profile of differentially expressed cytokines was heavily skewed toward an anti-inflammatory, pro-tumorigenic response in patients with MM. Significantly, our predictive model placed all patients in remission in the same category as those with active disease. Thus, our study demonstrates that the homeostasis of systemic cytokines is not restored when MM patients enter remission, suggesting that once an individual has cancer, the microenvironment is permanently altered and the system is primed for a relapse.

Figure 1. Expression of systemic cytokines in healthy donors changes with age.

Expression of 25 cytokines was measured utilizing multiplex technology of the Bio-Plex Luminex 100 XYP in the plasma of healthy adults, N1 and N2, ages 46±10 and 73±8 respectively. Differential expression of systemic cytokines between N1 and N2 populations of healthy donors (fold change >1.25 with p-values <0.05; exact values for all fold-changes and p-values are in Table 3). (A) pro-inflammatory cytokines (B) anti-inflammatory cytokines, (C) lymphocyte growth factor.

Figure 2. Multiple cytokines are elevated in healthy males compared to females: the difference that is lost with age.

Gender differences in the expression of systemic cytokines in each population of healthy donors (fold change >1.25 with p-values <0.05). (A) Male versus female expression of cytokines in N1 population, (B) Male versus female expression of cytokines in N2 population, (C) Gender differences in cytokines differentially expressed between N1 and N2 cohorts.

Figure 3. Patients with multiple myeloma present with a systemic cytokine profile consistent with an anti-inflammatory environment.

Differential expression of cytokines between N2, MGUS, and MM populations (fold change >1.25 with p-values <0.05; exact values for all fold-changes and p-values are in Table 3). MGUS* denotes those cytokines with significant differences in expression between MGUS and MM; MGUS** denotes those cytokines with significant differences in expression between MGUS and N2; (A) pro-inflammatory cytokines (B) anti-inflammatory cytokines, (C) lymphocyte growth factors.

Figure 4. Cytokine profile of patients entering remission from multiple myeloma is not restored to homeostasis.

(A) Levels of cytokines differentially expressed between N2 and MM were evaluated as a function of the disease stage (i.e. active disease (AD), treatment (Tx), and remission (Rmss)). Median expression values for each cytokine are plotted. All cytokines levels were statistically different (p<0.01) between N2 and MM, but not between the stages of MM (p>0.05). With the exception of sIL-2R, MCP-1, and MIG, all cytokines were differentially expressed between N2 and MGUS (<0.03). (B) Expression of IL-17, the only cytokine expression of which is restored after treatment, for each stage of MM. The solid line denotes the trend of IL-17 expression over the course of the disease. Statistical significance: N2 vs. MGUS, p>0.05; N2 vs. active disease, p<0.0001; N2 vs. treatment, p<0.0001; N2 vs. remission, p>0.05. (C) Dendogram depicting the results of hierarchical clustering of all plasma donors based on their placement into N1, N2, MGUS, and MM cohorts. The colored bar shows the placement of each donor within the study populations (N1, light blue; N2, dark blue; MGUS, orange; MM, red).

Figure 5. A model of possible regulation of MM-CSC by IL-3/GM-SCF and IL-7.

Based on our observation that MM, but not normal, plasma supported the growth of the MM clone and maintained dormancy of MM-CSC , we speculate that IL-3 and GM-CSF induce differentiation of MM-CSC (unpublished), and have to be downregulated in patients to prevent the loss of the MM-CSC due to differentiation, and thus, contribute to maintaining the pool of MM-CSCs that can be activated to initiate a relapse. Conversely, since IL-7 stimulates proliferation of the B cells, its expression has to be maintained at higher levels to ensure that the B cell population of the multiple myeloma clone, and thus, the MM-CSC, persists through treatment and remission.