Introduction A patient's risk of progression from Monoclonal Gammopathy of Undetermined Significance (MGUS) and Smoldering Multiple Myeloma (SMM) to full-blown Multiple Myeloma (MM) is the result of complex interactions between the tumor burden, alterations of tumor -omics, and tumor-extrinsic factors such as immune dysregulation. Prior studies have suggested that immune dysregulation can be observed as early as at the MGUS stage, but immune alterations in patients with MGUS have not been comprehensively studied at scale. Furthermore, it is unclear which of the immune cell populations that are altered in disease may directly interact with the tumor, as opposed to responding to general changes in the milieu.
Methods Here, we present results from the largest single-cell RNA-sequencing cohort (n=370) of bone marrow CD138-negative (n=243) and peripheral blood mononuclear cell samples (n=127) from patients with MGUS and SMM. Specifically, we profiled 51 samples from patients with MGUS, 226 samples from patients with SMM, 28 samples from patients with MM, and 61 samples from healthy donors. Bone marrow samples were subjected to magnetic bead enrichment for CD138 (Miltenyi Biotec) prior to library preparation. Libraries were prepared with the Chromium Single-cell 5' kit by 10X Genomics and sequenced on NovaSeq S4 flow cells at the Genomics Platform of the Broad Institute of MIT and Harvard (Cambridge, MA). CellRanger v.6.0.1. was used to extract FASTQ files and produce count matrices. Droplets with more than 15% mitochondrial gene expression or fewer than 200 genes per cell were filtered out, doublets were identified using Scrublet, SCDS, and scDblFinder, and samples were integrated using Harmony. Excluding plasma cells, immune cells isolated from both the CD138-positive and CD138-negative fractions were included in this analysis.
Results Overall, we profiled 2,327,008 immune cells, including T-cells, NK cells, B-cells, Monocytes, Dendritic cells, megakaryocytes, and progenitor cells. To identify immune cell populations that are more likely to interact with tumor cells, we compared the abundance of populations between matched bone marrow CD138-positive and CD138-negative fractions in patients and healthy donors. In healthy donors, we observed a significant enrichment of CD14+ monocyte subpopulations and CD168+ macrophages expressing complement factor C1q in the CD138-positive fractions, which is in line with their role in promoting and maintaining plasma cell differentiation. In patients, where decreased abundance of CD14+ monocytes is observed, this interaction was no longer significant, although macrophages were still enriched in the CD138-positive fraction. Consistent with prior publications, megakaryocytes were enriched in the CD138-positive fraction of patients, an interaction that has been shown to promote myeloma progression in vitro and in vivo. Of note, pro-B-cells, pre-B-cells, and memory B-cells were enriched in the CD138-positive fraction of patients, raising questions about their potential involvement in the clonal population. Lastly, Granzyme K-expressing CD8+ T-cells were significantly enriched in the CD138-positive fraction of patients, suggesting an interaction between this population and tumor cells.
Remarkably, despite their early stage, patients with MGUS had on average fewer pDCs, progenitor cells, and CD14+ Monocytes, and more CD4+ T-cells (including Tregs), Granzyme B-expressing CD8+ T-cells and CD56dim NK cells, compared to healthy donors. These results suggest that bone marrow immune cell composition can be significantly altered at the MGUS stage.
Discussion In the largest to date single-cell RNA-sequencing study of bone marrow and peripheral blood immune cells from patients with MGUS and SMM, we have comprehensively mapped alterations in bone marrow immune cell composition in patients with MGUS, and identified immune cell populations that are more likely to interact directly with tumor cells in the bone marrow microenvironment.
Haradhvala:MorphoSys: Consultancy. Soekojo:Pfizer: Other: advisory board. Aguet:Illumina: Current Employment. Getz:SignatureAnalyzer-GPU: Patents & Royalties; MSMuTect: Patents & Royalties; MSMutSig: Patents & Royalties; Pharmacyclics: Research Funding; IBM: Research Funding; Scorpion Therapeutics: Consultancy, Current equity holder in publicly-traded company, Other: Founder; MSIDetect: Patents & Royalties; POLYSOLVER: Patents & Royalties. Ghobrial:Takeda: Honoraria; Janssen: Honoraria; Adaptive: Honoraria; Celgene: Research Funding; GSK: Honoraria; The Binding Site: Honoraria; Vor Biopharma: Honoraria; Huron Consulting: Honoraria; Novartis: Research Funding; Oncopeptides: Honoraria; Menarini Silicon Biosystems: Honoraria; Pfizer: Honoraria; Sanofi: Honoraria; Sognef: Honoraria; Bristol Myers Squibb: Honoraria; Aptitude Health: Honoraria; Amgen: Honoraria; AbbVie: Honoraria; Veeva Systems: Honoraria; Window Therapeutics: Other: Advisory board participation.
Asterisk with author names denotes non-ASH members.