Abstract Background: Extramedullary disease (EMD) affects 30% of multiple myeloma (MM) patients, predicting poor overall survival due to aggressive disease kinetics and multi-drug resistance. Understanding 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. 8 patients had peripheral blood plasma (Streck DNA tubes) for circulating tumour DNA (ctDNA). 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, and to explore the presence of mutations in ctDNA. 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. 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. The majority of SV had not been previously identified in these patients. There were increased numbers of CNV/SV with relapsed EMD after therapy, with persistence of the MAPK DM and previously seen CNV/SV. Liquid biopsies show MAPK DM are present in ctDNA at the time of EMD biopsy. The same DM are detectable in the blood prior to development of EMD, suggesting DM clones arise early in disease course. The VAF of DM in ctDNA mirrors PET responses to therapy. Conclusion: MAPK DM were frequent in EMD, and detectable in ctDNA, suggesting roles for both MAPK-targeted therapies and for ctDNA as a biomarker in these patients. A high TMB was identified in patients lacking a MAPK driver mutation; immunotherapy should be considered in this subgroup. Citation Format: Nicholas E. Bingham, Jaynish Shah, Antonia Reale, Daniel Wong, Tiffany Khong, Sridurga Mithraprabhu, Andrew Spencer. Describing the molecular landscape of extramedullary multiple myeloma using whole genome sequencing: Insights into pathology and therapeutic targets [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2225.