Single-Cell Analyses Unravel Unique Features of Plasma Cell Clones in POEMS Syndrome

[Introduction] POEMS syndrome is a rare monoclonal plasma cell disorder characterized by multiple symptoms and VEGF elevation. Previous studies indicated that genetic and transcriptional features of plasma cells in POEMS syndrome are different from those in other plasma cell disorders such as multiple myeloma (MM) and monoclonal gammopathy of undetermined significance (MGUS). However, none of the studies have identified plasma cell clones in POEMS syndrome (“POEMS clones”), because they usually represent minor populations among bone marrow (BM) plasma cells, leaving their characteristics largely unknown.

[Aims] In the present study, we aimed to identify POEMS clones and unveil their transcriptional signatures by single-cell RNA-sequencing (scRNA-seq) using BM plasma cells obtained from patients with POEMS syndrome and other plasma cell disorders as references.

[Methods] Ten newly diagnosed or relapsed patients with POEMS syndrome, two newly diagnosed patients with MM and MGUS, respectively, and two patients with early stage B cell lymphoma without BM invasion as normal controls were enrolled in this study. CD138⁺ plasma cells were purified by FACS after MACS CD138 positive selection from BM aspiration samples. Single cell isolation and library preparation were performed by Fluidigm C1, and then the samples were read by paired-end sequencing. Plasma cells with the same V-(D)-J and CDR3 sequences in both IGL and IGHgenes were defined as “clonal” plasma cells.

[Results] We identified POEMS clones in 5 out of 10 POEMS cases, which had the same IGL sequences specific to POEMS syndrome, such as IGLV1-36, 40, 44 and 47 with IGLJ3*02. Proportion of POEMS clones in total BM plasma cells were much smaller (median: 14.7%, range: 7.3% - 32.5%) than MM (93 and 100%) and MGUS cases (57 and 81%). We confirmed highly conserved amino acid changes at position 38 IGLV1-44 (T to P/A), which had been previously reported (Bender et al. Blood. 2020). In addition, we identified amino acid changes at positions 38 and 40 of IGLV1-47 (Y to P and Y to N, respectively). Even in patients without detectable POEMS clones in scRNA-seq analysis, IGL sequences specific to POEMS syndrome were detected by RNA-seq of bulk plasma cells at very low frequencies at around 1%. scRNA-seq analysis of one of such patients detected a single plasma cell with POEMS-specific IGLV1-40 with amino acid changes at positions 38 and 40 (D to G and H to N, respectively), which had been conserved among patients with POEMS syndrome (Bender et al. Blood. 2020), indicating the presence of very small POEMS clones. We next compared transcriptome of POEMS clones with MM or MGUS clones as well as normal controls and non-clonal plasma cells in POEMS syndrome. T-distributed stochastic neighbor embedding (t-SNE) revealed that POEMS clones had different expression patterns from plasma cell clones in MM and MGUS, and their expression patterns were close to that of normal controls. Gene set enrichment analysis revealed that ribosomal protein gene sets, which indicate active protein synthesis, were positively enriched in POEMS clones compared with MGUS clones, but negatively enriched compared with MM clones. POEMS clones were also unique in significantly lower expression of c-MYC and CCND1 than MM and MGUS. Gene expression of cell surface markers indicated that POEMS clones were CD19 negative and CD138 positive as MM and MGUS clones. MHC class II but not class I genes were downregulated in POEMS clones, which might be associated with the ability of POEMS clones to escape immune surveillance. We also found that POEMS clones expressed several genes at higher levels than MM and MGUS, such as SERPING1, CFI, C2, MSLN, MUC1 and SPP1. We confirmed that the serum levels of osteopontin, encoded by SPP1, in patients with POEMS syndrome were significantly higher than those in healthy donors, and were significantly correlated with the levels of VEGF. Of note, VEGF mRNA levels were not upregulated in both POEMS clones and non-clonal plasma cells in patients with POEMS syndrome, suggesting that VEGF is produced by cells other than plasma cells.

[Conclusion] We identified POEMS clones by single plasma cell RNA-seq analysis and identified POEMS clone-specific gene expression signature. Our findings may have a great impact to understand the pathogenesis of POEMS syndrome.