The Spatial Heterogeneity in Newly Diagnosed Multiple Myeloma Patients - from Sub-Clonal Architecture to the Immune Microenvironment

Tumor heterogeneity plays a significant role in the development of therapy resistance in multiple myeloma (MM). Focal lesions (FLs), which are nodular accumulations of MM cells, have been shown to be hotspots of genetic spatial tumor heterogeneity, which is characterized by unique tumor sub-clones at different sites in the bone marrow (BM). However, little is known about the mechanisms leading to mutations in FLs, the architecture of the tumor microenvironment (ME) at these sites, and the link between FL sub-clones and relapse.

We applied whole genome sequencing (WGS) to CD138 ⁺ MM cells from paired FL and iliac crest random BM aspirates (RBMA) of 15 newly diagnosed MM (NDMM) patients. For 7 of these patients, single cell (sc) analyses were performed, including sc gene expression (scRNA) and T-cell receptor (TCR)-sequencing and sc assay for transposase-accessible chromatin (ATAC)-sequencing for paired BM CD138 ⁺ MM and CD138 ^- ME, as well as peripheral blood mononuclear cells (PBMC). WGS data was analyzed using inhouse pipelines. Mutations, copy-number-variations and mutational signatures were called using mpileup, ACESeq and mmsig. Neoantigen epitopes were predicted using NeoPredPipe. Sc data was generated using the 10X Genomics platform. Pre-processing and analysis of the sc data was performed with CellRanger and the R-packages Seurat, ArchR and inferCNV.

In 13/15 patients we found significant differences in chromosomal and mutational profiles between FLs and paired RBMAs, with major unshared mutations (mutation seen in > 60% cells) being enriched at the FL site (mean 310 vs. 123, p<0.05). Mutations in driver genes, such as KRAS, CYLD, CDKN2C and TP53, were site-unique or strongly enriched in FLs in 6/15 patients. To identify the mechanisms underlying heterogeneous mutations, we analyzed mutational signatures and found COSMIC signature SBS18 in these mutations, suggesting a role of reactive oxygen species. Combining WGS and sc sequencing, we observed between 3 and 6 sub-clones per patient. Sub-clones, which dominated in FLs, showed increased regulatory accessibility and expression of genes associated with disease aggressiveness and drug resistance such as CXCR4 and members of the NFKB- and interferon pathways, implying that FLs could play a significant role in the development of treatment resistance. Indeed, comparing sub-clones at baseline and at relapse after high-dose melphalan and autologous stem cell transplantation in one patient, we observed expansions of tumor cells at relapse, which were closely related to the main FL sub-clone at baseline.

On average, 23 (range 0-83) site-unique baseline mutations were predicted to be neoantigens. Thus, we hypothesized that spatial tumor heterogeneity could be associated with heterogeneity in the tumor ME. We did not observe expansion of site-unique T cell clones, but some of the clones were enriched up to 10-fold at one of the two sites. These clones were typically seen in the PB at low frequency. Expanded T-cells clones were almost exclusively found in the CD8 ⁺-compartment, with 65% and 27% of expanded T-cell clones being CD45RO ⁺/CD57 ⁺-memory- and CD69 ⁺-effector-T-cells, respectively. Besides differences in the T-cell clonality, we observed changes in proportions of other cell types, including a depletion of CD14+- and CD16+-macrophages in FLs (p<0.05). Furthermore, we observed gene expression differences between FL and RBMA macrophages, especially for genes involved in TNFα, IL-6 and JAK/STAT3 signaling. While CCL2, CD44, CXCL2/3, KLF2/4 and CCR1 were significantly higher expressed in FLs compared to RBMAs, BTG2, DUSP1 and HIF1A were down-regulated.

In conclusion, our results strengthen the concept of MM as a spatially heterogeneous disease, suggest that reactive oxygen species result in site-specific mutagenesis, and support the hypothesis that FLs are the origin of aggressive disease. We demonstrate spatial heterogeneity at single-cell level in the BM immune ME for the first time, which implies that understanding the complex biology of FLs could be important in the context of novel immune therapies such as bispecific antibodies and CAR-T-cells.