Novak, Joseph

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.

Abstract IgM MGUS and Waldenstrom Macroglobulinemia (WM) represent a disease spectrum with highly dissimilar therapeutic management ranging from observation to chemoimmunotherapy. The current classification relies solely on clinical features and does not explain the heterogeneity that exists within each of these conditions. To shed light on the biology that may account for the clinical differences, we used bone marrow (BM) CD19+/CD138+ sorted cells and matched BM plasma from 32 patients (pts) (7 IgM MGUS, 25 WM) and 5 healthy controls to perform the first comprehensive multiomics approach including whole exome sequencing, RNA seq, proteomics and metabolomics. Applying principal component analysis to gene expression profiling, most of WM pts clustered together, while a small subset of them grouped separately with MGUS pts, suggesting a biologic dichotomy within WM. The healthy controls formed a distinct group from most WM and MGUS. We then applied a non-negative matrix factorization consensus clustering to the gene expression data and identified three robust clusters. Cluster 1 (C1) included only WM pts, cluster 2 (C2) included both WM and MGUS pts, and cluster 3 (C3) included all controls as well as a small number of WM and MGUS pts. When mutations commonly identified in WM were analyzed, there was no difference among the three groups (excluding controls) in mutation burden of MYD88 L265P and CXCR4. Interestingly, aberrant expression of TNFAIP3 seemed a distinct feature of C1 as deletion of 6q (which encodes for TNFAIP3) and TNFAIP3 mutations were each significantly enriched in C1 (47%) compared to C2 (0%) and C3 (20%; p=0.04). Individual clusters associated with specific transcriptional signature and clinical features. While C1 displayed enrichment of cell growth, downregulation of inflammatory pathways (eg IL6 and IL8 signaling) and aggressive behavior, C2 showed increased inflammatory signaling and cell survival with indolent behavior. C3 had an intermediate feature with combined proliferative and inflammatory signatures. In accordance with the transcriptomic data, the hallmark of C1 was upregulation of proliferation-associated proteins (eg AKT, MAPK) and downregulation of inflammatory proteins (eg IL4, IL10) while the opposite was observed in C2. Once more, C3 confirmed intermediate features with combined upregulation of proliferation and inflammatory proteins. The metabolism was rewired towards fatty acid catabolism in C1, glycolysis in C2 and anabolism in C3. Accordingly, C1 showed undetectable concentration of 3-hydroxybutyric acid as opposed to C2 which had increased levels of malic and lactic acids, as end products of fatty acid oxidation and glycolysis respectively. Those metabolites had intermediate levels in C3. In conclusion, we identified three molecular clusters with distinct clinical, proteomic and metabolomic features, suggesting a potential biologic classification that may have therapeutic implications. Citation Format: Patrizia Mondello, Jonas Paludo, Joseph Novak, Kerstin Wenzl, Shahrzad Jalali, Jordan Krull, Esteban Braggio, Surendra Dasari, Michelle Manske, Jithma Abeykoon, Asher Chanan-Khan, Robert Kyle, Morie Gertz, Zhi Zhang Yang, Anne Novak, Stephen Ansell. Molecular clusters and functional drivers of IgM monoclonal gammopathies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3487.