Whole Exome Sequencing of Paired MGUS/SMM to MM Patients Reveals Novel Subclonal Tumour Evolution Models in Disease Progression of Multiple Myeloma

Introduction: Next Generation Sequencing studies in Multiple Myeloma (MM) have demonstrated that genetic heterogeneity is characteristic of MM at presentation. However, while intraclonal heterogeneity is now an established feature of MM, the subclonal tumour evolution associated with disease progression is not well understood. Here we present the first whole exome sequencing (WES) analysis of 10 paired MGUS-MM or SMM-MM patient samples, providing new insights into the genomic complexity, key molecular mechanisms and subclonal tumour evolution underlying the progression from MGUS/SMM to symptomatic MM.

Methods: Fluorescence-activated cell sorting was used to purify CD138⁺CD38⁺⁺ plasma cells and matched CD138^-38^- normal cells from longitudinal MGUS-MM (n = 5) and SMM-MM (n = 5) patient samples, where bone marrow samples were isolated from patients when they were first diagnosed with MGUS/SMM, and then subsequently when they developed MM. Exome libraries were generated using the Nimblegen Hyper Library Prep kit followed by the Agilent Sure SelectXT Clinical Research Exome capture kit before WES using the Illumina HiSeq4000. Bioinformatics analysis was performed using the GATK best practices MuTect2 pipeline to identify the somatic variants, custom in house package to identify copy number changes and PhyloWGS was used to integrate SNVs/CNVs to infer the subclonal evolution associated with MM progression.

Results: WES was performed to a minimum depth of 140x and identified a total 4997 somatic non-synonymous single nucleotide variants (NS-SNVs) in the MGUS/SMM samples (range 230-796), with a median 456 per patient. Interestingly, in the MM samples, we identify a total 4127 somatic NS-SNVs (range 221-609), with a median 344 per patient. We observe widespread copy number variations (CNVs), with a total of 82 genes gained or lost with progression across all patients. While we observe some previously identified known "drivers" of MM, we find that the driving events involved in progression are complex and not limited to the known SNVs or CNVs. The RAS/MAPK pathway was found to be the most frequently deregulated pathway, with KRAS and NRAS mutations observed in 40% of patients at MGUS/SMM and 70% of patients at MM. These findings highlight that "driver" mutations can be attained at both the early stages of MM and be maintained with transformation, or attained only at the later symptomatic MM stage.

Subclonal reconstruction of the tumour evolution process was carried out for 8 paired patients, to identify both the patterns of evolution and the key genetic changes that occur with MM progression. Our analysis revealed two models of subclonal evolution; firstly, a dominant model (3/8 patients) in which the outgrowth of subclones from MGUS/SMM to MM was observed, and secondly, a maintenance model (5/8 patients) in which subclones that were present at MGUS/SMM are retained at the MM stage. Notably, we observed a decrease in the average number of clones present in MGUS vs. SMM patients, indicative of a reduction in clonal complexity through dominant clonal outgrowth and/or extinction of indolent clones with advancement of disease. The survival of subclonal branches to MM was determined by their clonal fitness, either through their emergence with the acquisition of candidate "driver" gene mutations or outcompeting other subclones that were present. Finally, we describe potential candidate driving events of clonal progression in a range of loci, including ICAM5, DUSP27, HERPUD1, NOD2 and TOP2A.

Conclusion: Our genomic analysis of longitudinal MGUS/SMM to MM samples has revealed new insights of the subclonal tumour evolution and identified candidate mutated genes associated with MM transformation. Our analysis has revealed two models of tumour evolution involved in MGUS/SMM to MM transformation; namely the dominant subclonal tumour evolution model, and the maintenance subclonal tumour evolution model. We identified the existence of multiple subclones at the MGUS/SMM stages that are associated with MM progression. Our study suggests that in both circumstances, the subclonal populations involved in MM transformation are already present at the stages of MGUS/SMM diagnosis. Defining potential candidate genes associated with MM disease progression will assist in treatment approaches to arrest MM at the asymptomatic stages.