Intra-sample flow cytometry analysis identifies upregulated interleukin-6 receptor (CD126) as a potential therapeutic target in gain(1q) myeloma

Abstract

Abstract Introduction: Interleukin-6 (IL6) is a proliferative factor in multiple myeloma (MM) and important for plasma cell differentiation, with higher IL6 levels associated with worse outcomes. IL6 is also linked to increased osteoclast recruitment and activation, as well as inhibition of osteoblast differentiation, leading to bone loss. The IL6 receptor α-chain (CD126) has recently been shown to be overexpressed in malignant plasma cells from MM patients compared to plasma cells from normal individuals. However, this has not been demonstrated by intra-sample analysis using a single patient bone marrow sample that compares CD126 expression on the patient's myeloma cells as against their residual normal plasma cells. Gain(1q) is the most common adverse genetic finding in MM, affecting ~40% of newly diagnosed patients. CD126 is encoded within the 1q21 region, and is overexpressed on malignant plasma cells taken from patients with gain(1q) disease. Gain(1q) has also been linked to lower CD38 expression on malignant plasma cells, potentially affecting treatment responses to anti-CD38 therapies such as daratumumab. IL6 has been shown to downregulate CD38 expression in Gain(1q) human myeloma cell lines. Methods: Bone marrow aspirate samples from 37 myeloma patients were investigated using flow cytometry to determine expression of CD126, CD38 and CD138 on both malignant plasma cells and on residual normal plasma cells. Gates were applied to analyse myeloma cells (CD38+, CD56+, CD19-) and normal plasma cells (CD38+, CD56-, CD19+), with a minimum threshold of 25 events for analysis. CD126 expression was compared for each cell population allowing intra-sample analysis for each patient (n=20), as well as pooled data (n=37). Results were analysed against cytogenetic abnormalities, focusing on gain(1q) which was prevalent in our population (13/28 patients with FISH results available), as well as lytic bone disease, R-ISS score, albumin, LDH and beta-2 microglobulin levels. Non-parametric tests (Mann-Whitney, Wilcoxon signed rank and Spearman's rank) were used throughout due to non-normally distributed data. Results: Median CD126 expression, as measured by geometric mean (geomean) of fluorescence intensity, was higher in malignant myeloma cells compared to normal plasma cells by intra-sample analysis for individual patients (N=20 patients, median 281.5 vs 11, p<0.0001, range 3-3678). CD126 expression was also higher on malignant myeloma cells vs normal plasma cells using pooled data (n=37 patients, median 212.5 vs 11, p<0.005). CD126 expression on malignant plasma cells was higher in gain(1q) compared to non-gain(1q) patients (n=13 gain(1q), n=15 non-gain(1q), 501 vs 103, p<0.005). There was a trend towards significance for increased malignant:normal plasma cell CD126 expression ratio in gain(1q) patients (p=0.055). CD126 expression on malignant plasma cells did not correlate with IGH abnormalities, bone disease, albumin, beta-2 microglobulin levels, LDH or r-ISS stage. Other high-risk lesions, including t(4;14) (n=1) were present in too few patients for assessment. Both CD38 and CD138 expression were higher on malignant plasma cells compared to normal plasma cells on intra-sample analysis for individual patients (n=20, 11143 vs 7328, p<0.0001 and n=12, 4880 vs 28, p<0.001 respectively). CD138 expression correlated with CD126 expression (n=30 patients, Spearman's rank 0.439, p=0.015), but CD38 expression did not (n=36 patients, Spearman's rank –0.283, p=0.095). Gain1q was significantly associated with higher CD138 (n=11 gain1q+, 12 gain1q-, 16392 vs 2896, p<0.05), but not with CD38. Conclusions: We find that CD126 is overexpressed on myeloma cells, compared to healthy plasma cells, particularly in patients with gain1q. This data supports the hypothesis that up-regulation of the IL6-receptor is relevant to the biology of myeloma and potentially its microenvironment in the bone marrow niche. We propose that identifies CD126 as a potential therapeutic target in myeloma. CD126 blockade is used in MM, but to counteract side effects of therapy, such as cytokine release syndrome. Using such therapies alongside current treatment could therefore be considered, including those resistant to anti-CD38 monoclonal antibodies.