P-335: Small nucleotide, copy number and structural variants cooperate to hijack driver genes in extramedullary progression of myeloma

Introduction: Extramedullary disease (EMD) affects up to 30% of multiple myeloma (MM) patients, predicting poor overall survival due to aggressive disease kinetics and multi-drug resistance. Elucidating EMD genomics may inform targeted treatment approaches.

Methods: We obtained EMD samples from 15 patients (n=8 fresh and n=7 formalin-fixed, paraffin embedded). Second EMD biopsies were taken in n=3, with n=1 having 3 sequential EMD biopsies. Germline variants were excluded using buccal swab DNA. DNA was extracted using QIAGEN DNeasy kit prior to whole genome sequencing (WGS) (30x; xGen PRISM library preparation; Illumina Novaseq).
Bioinformatics identified short nucleotide variants (SNVs), copy number variations (CNVs) and structural variants (SV) using the Broad Institute GATK best practice pipeline, CNVpytor and Manta, respectively. Droplet digital PCR was used to confirm SNV and CNV findings.

Results: The median age at diagnosis of MM was 52 years (n=4 primary EMD, n=11 secondary EMD). 46% were hyperdiploid.

Driver mutations (DM) in the MAPK pathway were seen in 80%, primarily at codon 61 of NRAS and KRAS (n=5, n=3); only two non-p.Q61 mutations were seen (NRAS p.G13R and KRAS p.A146V). Three patients had activating BRAF mutations (two p.V600E, one p.G469A). Variant allele frequencies (VAF) of DM suggested clonal rather than subclonal level. A quarter of patients (26.6%) had loss of function TP53 mutations.
In those with no MAPK DM identified, the median whole genome SNV was 77,142 with a tumour mutational burden (TMB) of 15 mutations/Mb, compared to 20,543 and 3.2 in DM patients. The median SNV and TMB increased with relapse or progression of EMD.

CNV/SV analysis identified gain(3q), gain(1q), del(1p) and monosomy 13/ del(13q) in 93%, 86%, 46% and 73% respectively. Gains of BRAF (66%) and MYC (53%) and loss of TP53 (40%) were frequent; with SNV, 20% had biallelic loss. Secondary translocations were seen in 40% of patients, involving MYC, FGFR3, CCND2 and CCND3 in 20%, 13%, 6% and 6%, respectively, and partnered with IGH, IGL and TXNDC5. There was a median of 44 SV per patient. The majority of SV had not been previously identified in these patients and frequently involved published MM driver genes or known super-enhancers such as H1FX.

Sequential biopsies of EMD demonstrated temporospatial persistence of DM, with the same DM detected at different anatomical sites and at separate timepoints. There were increased numbers of CNV/SV with relapsed EMD after therapy, consistent with a role in disease progression and drug resistance.

Conclusions: The MAPK DM, high TMB and persistent genomic instability suggest roles for MAPK-targeted therapies, immunotherapies and DNA damage repair pathway inhibitors, respectively, in EMD. Recurrent codon 61 mutations in RAS suggest a specific role in EMD progression.