The Mutational Landscape of Extramedullary Multiple Myeloma Reveals Novel Biologic Insights and Potential Therapeutic Targets for Exploration

Background: Patients with relapsed/refractory multiple myeloma demonstrate an increasing incidence of extramedullary disease (EMD), noted in up to 30% of patients. Presence of EMD portends a markedly inferior prognosis with limited response to salvage therapies, including immune effector therapy. Understanding the molecular alterations in EMD is key to exploring novel therapeutic approaches but remains to be well-characterized. Here we describe the mutational landscape and biologic pathways with alterations in a large cohort of patients with EMD. Methods: We included 18 paraffin-embedded tumor tissues from true EMD biopsies (non-bone contiguous disease) for whole exome sequencing (WES); biopsies from paraskeletal/bone-contiguous soft tissue were not considered. Germline sequencing was performed utilizing cryopreserved peripheral blood mononuclear cells (PBMCs) at initial diagnosis from patients without evidence of circulating tumor cells. Additionally, we studied 12 CD138-sorted cryopreserved bone marrow (BM) aspirates collected at the time of development of EMD. Following standard DNA extraction and library construction practices, we performed WES on the EMD, BM and PBMC samples. Variant calling was performed using consensus calls observed in at least 2 out of the 3 callers (Mutect2, VarScan, Strelka2) and these were included for downstream analysis. Synonymous and low-impact variants were filtered. Pathway analysis was performed using the PanCancer driver gene sets and compared using Fisher's exact test. Results: The median age of patients with EMD was 65.6 years (range 46.2-86 years). Seven (39%) patients had de novo (previously untreated) EMD and 11 (61%) had secondary EMD (developed at relapse). The MAPK pathway was frequently mutated, most commonly involving NRAS (33%), KRAS (22%) and BRAF (22%) genes. These mutations were mutually exclusive. Alterations in TP53 were noted in 11% of EMD samples. Single nucleotide variants (SNVs) in epigenetic modulators were frequently noted in EMD, with the most prevalent alterations seen in ARID1A (33%), KMT2C (28%), EP300 (22%) and KMT2B (22%). Both ARID1A and EP300 are postulated to play a role in IRF4 regulation and plasma cell differentiation. Additionally, inhibition of EP300 can be a viable therapeutic strategy in MM through disruption of IRF4 signaling. We noted frequent mutations in the roundabout (ROBO) genes ROBO1 and ROBO2, observed in 22% and 28% of EMD samples, respectively. ROBO1 has been demonstrated to be necessary for myeloma cell adhesion to BM stromal and endothelial cells, with ROBO1 knockout MM cells infused in mice demonstrating a tendency for extramedullary dissemination. Similarly, variants in LRP1B, a frequently altered large putative tumor suppressor gene in the LDL receptor family, were noted in 33 % of EMD samples. Depletion of LRP1B has been demonstrated to promote anchorage-independent growth, cell migration and invasion in vitro. Notably, in the paired BM samples, no SNVs were noted in ARID1A, LRP1B, ROBO1 and ROBO2 genes (q-value not significant). In paired samples, among the shared mutations of interest, we noted expansion of the BRAF clone in 2 EMD samples [variant allele frequency (VAF) of 3% and 13% in BM and 58% and 29% in the paired samples, respectively]. An expansion of a shared KRAS mutation was noted in another EMD sample (VAF of 15% in BM and 29% in EMD). The frequently altered cellular pathways in EMD samples (n=18) included the MAPK pathway (89%), chromatin histone modifier pathway (83%), transcriptional regulation (83%), immune signaling (72%) and RNA abundance (72%). In the cohort of paired EMD and BM samples (n=24), notable enrichment was evident in EMD for MAPK pathway alterations [91% in EMD vs. 25% in BM q=0.02] and chromatin histone modifier pathway (83% in EMD vs. 42% in BM, q <0.01). Conclusions: Patients withEMD demonstrate a near universal presence of alterations in the MAPK pathway. Variants in chromatin modifiers regulating plasma cell differentiation and genes involved in BM stromal adhesion and homing of plasma cells were frequently noted, potentially facilitating the extramedullary spread of myeloma cells. Functional studies are needed to evaluate these hypotheses. Targeting EP300, ARID1A and downstream MEK/ERK inhibition as part of combination therapies may represent promising therapeutic approaches for further exploration in EMD.