Whole Genome Sequencing of Extramedullary Myeloma Autopsy Tumors Reveals a Genomic Portrait at Culmination of Clonal Convergence

Introduction

Multiple Myeloma (MM) pathogenesis is characterised by extensive genetic and clonal evolution with frequent on-treatment progression. To date, most studies have focused on single diagnostic or paired diagnostic relapse biopsies, and the molecular mechanisms eventually resulting in treatment failure are poorly understood. To determine the molecular underpinnings of disease in its most advanced stage, we performed comprehensive genome profiling of 4 patients with extra-medullary metastatic disease.

Methods

A total of 8 patients with extramedullary myeloma with 188 (median = 22) distinct metastatic lesions were enrolled as part of the medical donation program at MSK. Here, we present results from 4 patients. Patients 1 and 2 had an indolent disease with a total survival of ~10 years whereas patients 3 and 4 had very aggressive disease and 2-3 years survival. All patients had received a sequence of multi-modal myeloma treatments. Targeted gene sequencing using a myeloma specific targeted panel myTYPE was performed in 28 samples from all 4 patients to a median coverage of 667x. Additionally, 6 tumors from patients 1 and 2 were subject to WGS to a median coverage 92x.

Results

Driver events:

Aberrations across all 28 samples sequenced using myTYPE were examined. We found t(4;14) in Patients 3 and 4 across all the metastatic lesions consistent with previous knowledge that these are early initiating events. Besides IGH translocations, we found copy number changes involving 1p-, 1q+ and 13q-. Patient 1 and 2 had 17p- and 8p- shared across all the metastatic lesions. All patients had RAS/RAF pathway mutations and additional mutations were found in FAM46C, TP53 and BIRC3. In WGS, we observed a median SNV, Indel & SV burden of 12150, 1196 & 70.5 respectively. This mutational load is greater than two-fold higher to previously published estimates derived from primary diagnostic samples.

Clonal structure using WGS:

Clonal phylogeny was constructed using nDirichlet process clustering. Evaluation of mutation and clonal spectra showed evidence of clonal diversification amongst sites but within each sample all mutations had fully clonal cancer cell fractions, i.e. there were no subclones. For Patient 1, the phylogenetic tree was dominated by 9,099 truncal mutations, and 150-462 site specific yet clonal mutations. For Patient 2, the tree was dominated by 8,540 truncal mutations and site specific clonal mutations (n= 356; 1,186). However, evaluation of copy number alterations showed evidence of subclonal emergence of copy number aberrations implicating chromosomes 5, 8,16,18, 20, 21. This suggests that in these patients late stage tumor development and metastatic dissemination is further shaped by accrual of CNAs.

Mutational processes using WGS:

Signature analysis was performed by deconvolution of observed WGS mutations on the set of mutational signatures reported by Alexandrov et al. Consistent with previous reports, Signature 9 was identified as the dominant mutation signature, contributing to a median of 27% of all mutations in our cohort. Signature 9 is related to AID and has been previously implicated in early myeloma pathogenesis. Whilst canonical IGH translocations were not identified in Patient 1 or 2, Patient 1 showed evidence for chromoplexy with closed chain translocations having breakpoints spanning chromosomes 1, 4, 11, 16, 17, 19 across all 4 sites. Patient 2 presented with localised hypermutation on chromosomes 1, 5 and 22 which are shared between the 2 sites. These results suggest that subsequent clonal sweeps have acted upon the genome since disease initiation.

Conclusion

Preliminary data from multi-region WGS of the evolutionary end-stage in MM shows a single dominant clone with known driver events in each patient. This is in contrast to the subclonal heterogeneity characteristic of early disease, and presents opportunities for targeted therapies. Our observations are consistent with convergent evolution, where selective pressure from many years of therapy results in a relatively homogenous genomic landscape. A larger cohort of samples will ascertain patterns of biological processes we present here. These early investigations provides new insights on MM pathogenesis and metastatic dissemination.