Altered cytokine and chemokine profiles in multiple myeloma and its precursor disease

Abstract

Currently, no reliable biomarkers are available to predict transformation from smoldering myeloma (SMM) to multiple myeloma (MM). Using an ultrasensitive enzyme-linked immunosorbent assay (ELISA) we assessed the levels of a broad range of cytokines and chemokines in the peripheral blood (PB) and bone marrow (BM) supernatant collected from 14 SMM and 38 MM patients and compared to healthy donors. We found significantly increased levels of key cytokines, in particular CXCL8 (IL-8), associated with progressive disease state (controls→SMM→MM). Cytokine profiles were found similar in PB and BM. Five of fourteen SMM patients (36%) progressed to MM. Our findings, although based on a limited number of patients, suggest that serum-based cytokines may have a future role as biomarkers for disease progression and could potentially be assessed as novel targets for treatment.

Keywords: CXCL8; Chemokines; Multiple myeloma.

Figure 1. Comparison of cytokine and chemokine levels in peripheral blood (PB) and bone marrow (BM) supernatant of SMM and MM patients. PB (serum)

(A) Values are expressed as log2. Level of IL-6 significantly elevated in MM patients (21 ± 9.5 pg/mL) compared to SMM (1.2 ± 0.7 pg/mL) compared to controls (1.0 ±0.4 pg/mL). *p= 0.006 and *p= 0.001 respectively. (B) CXCL8 (or IL-8) concentration elevated in SMM patients (13.9±2.4 pg/mL) and MM patients (133.2 ± 48 pg/mL) versus controls (6.2 ± 1.2 pg/mL. *p=0.008 and *p< 0.0001 respectively. (C) TNF-alpha level increased in SMM patients (8.5±2.9pg/mL) and MM (78±16.9 pg/mL) compared to controls (6.3±1.1 pg/mL). MM vs SMM:*p=0.0005). (D) Levels of IFN-gamma in SMM (7.0±1.1 pg/mL) and in MM (17.9±5.9 pg/mL) compared to controls (1.9±0.3pg/ml) *p=0.002 and 0.001 respectively. (E) Level of IL-10 significantly increased in MM patients (18.08 pg/mL±3.2) versus SMM (11.16±5.4) and controls (2.7±0.6 pg/mL). *p= 0.001 and 0.0003 respectively. BM supernatant. (F) Level of IL-6 increased in MM patients (1.66 ±0.3 pg/mL) compared to controls (0.3±0.09pg/ml) * p= 0.0003. (G) Level of CXCL8 increased in MM patients (11.3±1.6 pg/mL) compared to controls (2.8±0.4 pg/mL)*p=0.0001. (H) TNF-alpha elevated in MM patients (9.03±0.8 pg/mL) compared to controls (3.49±0.8 pg/mL) *p=0.0008. (I) Level of IL-2 elevated in MM patients (1.6±0.2 pg/mL) versus controls (0.6±0.1 pg/mL). *p=0.007. All samples were assayed in two ELISA multi-array experiments using an ultra-sensitive Human TH1/TH2 10-plex multi-spot plate for quantifying the levels of IL-1β, IL-2, IL-4, IL-5, CXCL-8, IL-10, IL-12 p70, IL-13, IFN-gamma, TNF-alpha; and an ultra-sensitive multi-array plate for IL-6 detection (Meso Scale Discovery®).

Figure 2

(A) Change of cytokine and chemokines levels in SMM progressed to MM versus SMM with stable disease (SD). CXCL8 is the most elevated chemokine within the five SMM patients (36%) that progressed to MM. (B) Transformation from normal PC to MM is associated with a decreased CXCL8 transcript level. Gene expression profile data including 15 normal PC, 25 SMM and 73 MM patients show that CXCL8 expression is significantly decreased in SMM and MM patients compared to PC controls (p=0.0002 and 0.00001 respectively). (C) Heatmap displaying a CXCL8 signature. A CXCL-8 signature was defined including the top ten genes most positively and negatively correlated with CXCL8 expression in using the above subset of samples. SMM patients show a profile more similar to MM patients than the normal PC. (D) List of gene sets that correlate with CXCL8 expression. By GSEA analysis we defined three main sets of genes negatively correlated with CXCL8 expression, including genes regulating cellular metabolism (Tricyclic Acid Cycle), RNA splicing and RNA trancsription.