P-049: The spatial sub-clonal architecture in newly diagnosed myeloma patients revealed by whole genome and single-cell sequencing

Abstract

<h3>Introduciton</h3> Tumor heterogeneity plays a significant role in the development of therapy resistance in multiple myeloma (MM). Focal accumulations of myeloma cells called focal lesions (FLs) have been shown to be the hotspots of 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 sub-clonal architecture in FLs, the mechanisms leading to site-unique mutations, and the link between sub-clones in FLs and treatment-resistance. <h3>Methods</h3> To fill this gap in knowledge, we used a comprehensive approach to characterize CD138-sorted tumor cells from FLs and random BM aspirates (RBMA) in 12 newly diagnosed MM patients, applying whole genome sequencing (WGS), single-cell(sc)-RNA-sequencing and sc assay for transposase-accessible chromatin (ATAC)-sequencing. Sc data was generated using the 10X Genomics platform. Pre-processing of the sc data was performed with CellRanger and the R-packages Seurat, ArchR and inferCNV were used for downstream analysis. 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. <h3>Results</h3> In the majority of patients (n=10/12) we found significant differences in the chromosomal and mutational profiles between FLs and paired RBMAs from the iliac crest. To identify the mechanisms underlying heterogenous mutations, we performed mutational signature analysis and found COSMIC signature SBS18 to be enriched in these mutations, suggesting that they were caused by reactive oxygen species. Interestingly several site-unique mutations were predicted as potential neoantigens. Driver gene mutations associated with relapse such as KRAS, CYLD, CDKN2C and TP53, were more often seen in FL sub-clones. Using a combination of WGS, sc-RNA and sc-ATAC-sequencing to characterize these sub-clones in more detail, we found 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. The latter implies that FLs could play a significant role in the development of treatment resistance. Indeed, comparing sub-clones at baseline and after high-dose melphalan and autologous stem cell transplantation in one patient, we demonstrate expansion of a single tumor cell, which was closely related to the main sub-clone from the baseline FL. <h3>Conclusion</h3> In conclusion, our data provides novel insights into the mechanisms underlying site-unique mutations and the sub-clonal architecture at different sites in the BM. The combination of bulk and sc-techniques showed that FLs are enriched for sub-clones with genetic, transcriptional and regulatory markers characteristic for aggressive disease and can be the source of relapse in MM-patients. This implies that targeting FLs is essential for achieving a cure of MM.