Peripheral Cellular Immunome Reveals Heterogeneity Spanning Myeloma Spectrum Diseases

INTRODUCTION: Progression from precursor states, termed monoclonal gammopathy of undetermined significance (MGUS) and smoldering multiple myeloma (SMM), to multiple myeloma (MM) is facilitated by cross-talk between malignant plasma cells and cellular/soluble components of the immunosuppressive bone marrow milieu, which promotes angiogenesis, bone destruction and immune-impairment. In support of this view, the frequencies of several peripheral immune cell subsets have been shown to be affected by the disease state. We hypothesized that beyond numerical alterations, activation and differentiation state of circulating innate and adaptive immune cell subsets determines progression from pre-malignant to malignant stage.

METHODS: We compared multiple compartments of immune cell subsets in the peripheral blood samples of patients with MGUS (n=7), SMM (n=4) and MM (n=7) enrolled on an IRB-approved prospective biospecimen collection protocol of plasma cell disorders. Peripheral blood mononuclear cells obtained from each subject were immunophenotyped using two 14-color flow cytometry panels. Broadly, immune subsets surveyed included NK cell (mature and immature), NK-T cell, γδ T cell (δ9+γ2- and δ9-γ2- subsets), conventional CD4 and CD8 T cell (effector, effector memory and central memory) and regulatory T cell. Expression of activation, inhibition and maturation markers including NKG2A, NKG2D, KIR3D4, KIR2DS4, CD25, CD38, PD1, and Tim3 was analyzed in all relevant subsets. In total 62 immune variables (cell lineage and functional markers) were generated. For comparisons between MGUS, SMM and MM, one-way ANOVA was used. Unsupervised hierarchical cluster analysis was then applied to all samples with selected variables that were differentially expressed among three groups (p<0.1 by ANOVA) using WPGMA and Euclidean distances. Pair-wise comparisons between MGUS+SMM versus MM or MGUS versus SMM+MM were performed using Welch's t-test.

RESULTS: Overall, distribution and functional state of three main immune cell lineages - NK cell, CD4+ T and CD8+ T cell - were altered across MGUS, SMM and MM. Compared to subjects with MGUS and SMM, MM patients had fewer circulating NK cells (p=0.007) with a noticeable shift in NK maturation (Immature/mature NK ratio approximately 3 to 1 for MGUS and SMM vs. 1 to 1 for MM, p=0.015). Mature NK phenotype was confirmed by higher expression of NKG2D on NK cell in MM patients (p=0.041). MGUS and SMM subjects shared an increased expression of immune checkpoints Tim3 (p=0.005) on peripheral CD4+ T effector cells and PD1 (p= 0.012) on functional memory cells, compared with MM patients. Levels of circulating activated CD8 T effector cells were increased in SMM and MM patents compared with MGUS subjects (p<0.001). Unsupervised hierarchical clustering with ten immune variables that were differentially expressed among three groups separated 7/7 MM patients and 9/11 MGUS and SMM subjects (Figure).

CONCLUSION: Transition from MGUS and SMM to MM is associated with systemic immune alterations in the functional state of innate (NK) and adaptive (CD4 and CD8 effectors) immune cell subsets. This proof of concept study suggests a role of the peripheral cellular immunome as a possible biomarker that might allow for identification of a subset of MGUS and SMM subjects with a high risk of progression to active MM. We are evaluating this approach prospectively in a larger cohort of patients, including the clonality of the T cell repertoire.

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