Whole-Genome Sequencing Reveals Evidence of Two Biologically and Clinically Distinct Entities: Progressive Versus Stable Myeloma Precursor Disease

Introduction

Multiple myeloma (MM) is consistently preceded by an asymptomatic expansion of clonal plasma cells, clinically recognized as monoclonal gammopathy of undetermined significance (MGUS) or smoldering multiple myeloma (SMM). Here, we present the first comprehensive whole-genome sequencing (WGS) analysis of patients with MGUS and SMM.

Methods

To characterize the genomic landscape of myeloma precursor disease (i.e. SMM and MGUS) we performed WGS of CD138-positive bone marrow mononuclear samples from 32 patients with MGUS (N=18) and SMM (N=14), respectively. For cases with low cellularity resulting in low amounts of extracted DNA (N=15), we used the low-input enzymatic fragmentation-based library preparation method (Lee-Six et al, Nature 2019). Myeloma precursor disease samples were compared with 80 WGS of patients with MM. All WGSs (N=112) were investigated using computational tools available at the Wellcome Sanger Institute.

Results

After a median follow up of 29 months (range: 2-177), 17 (53%) patients with myeloma precursor disease progressed to MM (13 SMM and 4 MGUS). To interrogate the genomic differences between progressive versus stable myeloma precursor disease we first characterized the single base substitution (SBS) signature landscape. Across the entire cohort of plasma cell disorders, all main MM mutational signatures were identified: aging (SBS1 and SBS5), AID (SBS9), SBS8, SBS18, and APOBEC (SBS2 and SBS13). Interestingly, only 2/15 (13%) stable myeloma precursor disease cases showed evidence of APOBEC activity, while 14/17 (82%) and 68/80 (85%) patients with progressive myeloma precursor disease (p=0.0058) and MM (p=0.004), respectively, had APOBEC mutational activity. The two stable cases with detectable APOBEC were characterized by a high APOBEC3A:3B ratio, a feature which defines a group of MAF-translocated MM patients whose pathogenesis is characterized by intense and early APOBEC activity (Rustad et al Nat Comm 2020) and is distinct from the canonical ~1:1 APOBEC3A:3B mutational activity observed in most cases.

When exploring the cytogenetic landscape, no differences were found between progressive myeloma precursor disease and MM cases. Compared to progressors and to MM, patients with stable myeloma precursor disease were characterized by a significantly lower prevalence of known recurrent MM aneuploidies (i.e. gain1q, del6q, del8p, gain 8q24, del16q) (p<0.001). This observation was validated using SNP array copy number data from 78 and 161 stable myeloma precursor disease and MM patients, respectively. To further characterize differences between progressive versus stable myeloma precursor disease, we leveraged the comprehensive WGS resolution to explore the distribution and prevalence of structural variants (SV). Interestingly, stable cases were characterized by low prevalence of SV, SV hotspots, and complex events, in particular chromothripsis and templated insertions (both p<0.01). In contrast, progressors showed a genome wide distribution and high prevalence of SV and complex events similar to the one observed in MM. To rule out that the absence of key WGS-MM defining events among stable cases would reflect a sample collection time bias, we leveraged our recently developed molecular-clock approach (Rustad et al. Nat Comm 2020). Notably, this approach is based on pre- and post-chromosomal gain SBS5 and SBS1 mutational burden, designed to estimate the time of cancer initiation. Stable myeloma precursor disease showed a significantly different temporal pattern, where multi-gain events were acquired later in life compared to progressive myeloma precursor disease and MM cases.

Conclusions

In summary, we were able to comprehensively interrogate for the first time the whole genome landscape of myeloma precursor disease. We provide novel evidence of two biologically and clinically distinct entities: (1) progressive myeloma precursor disease, which represents a clonal entity where most of the genomic drivers have been already acquired, conferring an extremely high risk of progression to MM; and (2) stable myeloma precursor disease, which does not harbor most of the key genomic MM hallmarks and follows an indolent clinical outcome.