Multiple Myeloma Clone Research Articles

Complement C3d enables protective immunity capable of distinguishing spontaneously transformed from non-transformed cells.

We show that intracellular soluble fragment 3d of complement (C3d) induces regression of spontaneous multiple myeloma in mice reducing tumor burden by 10 fold, after 8 weeks. C3d enables cell-mediated immunity to target multiple myeloma clones sparing non-transformed polyclonal B cells and plasma cells with lower mutation loads. We show that C3d increases the expression of ribosomal subunits associated with the translation of defective ribosomal products (DRiPs). C3d also decreases expression of protein arginine methyl transferase (PRMT) 5 which in turn relieves E2f1 repression increasing the expression of Lnc RNAs and derived peptides that evoke anti-tumor cellular immunity. The approach increases MHC-I expression by tumor cells and generates a CMI response that overcomes tumor immune-evasion strategies. Tumors are immunogenic in part because of somatic mutations that originate novel peptides that once presented on MHC engage cell-mediated immunity (CMI). However, in spite of the higher mutation load most tumors evade immunity. We discovered that a component of the complement system (C3d) overcomes tumor immune evasion by augmenting expression of ribosomal proteins and lncRNAs linked to the presentation of novel peptides by tumor cells. C3d induced CMI targets cancer cells sparing non transformed cells uncovering a novel function for complement in immune surveillance.

Patterns of Change in Multiple Myeloma (MM) Clone Size with Autologous Hematopoietic Stem Cell Transplantation (ASCT) Assessed By Next Generation Sequencing (NGS) in Patients (pts) Receiving Modern Therapy

Background Measurable residual disease (MRD) is a quantitative, dynamic prognostic marker of progression free and overall survival in MM. With modern triplet and quadruplet induction strategies deep responses are the norm and the incremental effect of ASCT impact may vary substantially among pts and be influenced by disease and treatment features. However, the accurate characterization of the impact of ASCT is diluted by measuring a disease surrogate (paraprotein) instead of clone size. We examine the quantitative impact of ASCT on MM clone size by serial NGS testing before and after ASCT and evaluate disease and induction treatment features that modulate this impact. Methods We included pts with newly diagnosed MM from five US centers, who received modern triplet and quadruplet induction followed by single melphalan ASCT between 3/2018 and 5/2023 with MRD testing (ClonoSEQ®) pre and post ASCT. ClonoSEQ® platform utilizes NGS to quantify unique immunoglobulin genes rearrangements associated with MM clone, therefore directly capturing clone size. We excluded pts with clone size <10 -5 prior to ASCT. We explored patient-, disease-, and treatment features influencing clone size reduction with AHCT by 1 log 10. We used binary logistic regression to identify factors associated with ≥1 log 10 clone reduction (60-100 days post ASCT). Results One hundred and forty-nine pts with NDMM had pre and post ASCT results and pre ASCT MRD ≥10 -5 and met the inclusion criteria. The median age was 63 years (35-79), 37 pts (25%) ≥70 years, 62% male, 27% Black, 23% ISS3 and 16% RISS3. Seventy-three pts (49%) had high risk chromosomal abnormalities [HRCA, gain/amp(1q), t(4;14), t(14;16), t(14;20) or del(17p)]); 72 pts (48%) had 0 HRCA, 50 (34%) had 1 HRCA, and 23 (15%) had 2+ HRCA. AntiCD38 monoclonal antibody (mAb) was part of induction in 125 (84%) pts [68 pts quadruplet containing carfilzomib; 53 pts quadruplet containing bortezomib], 28 pts received triplets. Eighty-seven pts (58%) had >1log 10 reduction in MRD burden with ASCT. The median log reduction in MRD burden was 1.15 (IQR 0.65-1.75). For pts with 0, 1 and 2+ HRCA, 47% (34/72), 68% (34/50), 74% (17/23) had a >1 log 10 reduction in clone size and the median reduction was 0.96 log 10 (IQR 0.47-1.66), 1.24 log 10 (IQR 0.89-1.91) and 1.5 log 10 (IQR 0.89-2.20), respectively (Figure). Among pts without HRCA, those with hyperdiploid MM had a trend towards less frequent ≥1 log 10 clone size reduction with ASCT (38% vs. 57%, p=0.18). Overall, we saw reduction ≥1 log 10 in clone size in 61% of pts receiving anti-CD38 mAb in induction (N=125) vs. 46% among those who did not (N=24, p= 0.18). Among pts treated with melphalan 200 mg/m 2 (N=113) compared to those who received 140 mg/m 2 (N=36), ≥1 log 10 reduction in clone size was noted in 61% vs. 50% (p=0.25), respectively. In multivariable analysis that included stage, antiCD38 mAb in induction, use of carfilzomib in induction, HRCA and melphalan dose, the presence of HRCA was the only factor associated with greater than 1 log 10 reduction in MRD burden with ASCT [1 HRCA, OR 2.34 (1.07-5.14); ≥2 HRCA, OR 2.71 (0.91-8.03); p=0.049]. Conclusions The impact of high dose melphalan and AHCT on MM clone size is heterogeneous, of greater magnitude in pts with one or more high-risk chromosome abnormalities, and less pronounced in pts with standard risk disease, particularly hyperdiploid MM. The use of antiCD38 mAb in induction regimens or dose of melphalan does not impact the effect of ASCT on MM clone size, likely owing to the distinct mechanism of action of melphalan. This dataset provides context to explore risk- and response-adapted use of ASCT and use of new cellular and non-cellular immunotherapies as post-induction consolidative strategies.

Single Cell Analysis of Bone Marrow Stromal Cells Shows That a Pro-Inflammatory and Stress Response Together with Polarized Differentiation of Stromal Elements Characterize the Bone Marrow Microenvironment during Multiple Myeloma Development

Current knowledge of the biological role of the non-hemopoietic components of the bone marrow (BM) niche to the pathogenesis of multiple myeloma (MM) is poorly understood. We utilized a murine model to interrogate niche forming cells early in the natural history of MM, at a precursor stage (MGUS/smoldering-like) in more detail. Studying interactions between the MM clone and the stromal components of the MM niche are challenging as they represent only approximately 0.2% of the total marrow content and are difficult to isolate. However, by isolating the stromal compartment of the BM by depleting hemopoietic cells via flow sorting combined with the use of single cell RNA sequencing (scRNA-seq) it is possible to perform an in-vivo analysis of the individual cellular components of the stromal microenvironment and to characterize their relative abundance, cellular differentiation state, and transcriptomic profile. To comprehensively assess changes in the stromal cells during the early stage of MM, 5TGM1 cells were intravenously injected into aged (6 month old) KaLwRij mice. KaLwRij mice without MM were used as controls. Stromal cells were isolated following tumor engraftment in the marrow at a precursor stage and enriched as previously described (Baryawno et al, Cell 2019). scRNA-seq was performed on 5 libraries of cells using the 10X Genomics Chromium platform. All data analysis was performed in R. After filtering out low quality cells, 45,030 cells remained for analysis. The 5 libraries were integrated with Seurat and run through a standard clustering workflow involving log-normalization, finding variable features, dimensional reduction, and clustering. Cell type annotation for each cluster was performed manually using established markers for both the stromal and immune compartments. After excluding immune cell populations, 14,219 cells remained. Select subpopulations were re-clustered by isolating the cell types of interest and running them through separate standard clustering workflows. For each population of interest, differentially expressed genes were determined using the Wilcox rank sum test in Seurat, trajectory analysis and pseudotime were computed using Monocle3, and enriched gene sets were computed using GSEAPreranked. We identified 7 distinct populations of stromal cells including mesenchymal stromal cells (MSCs) ( Lepr, Adipoq, and Cxcl12), osteo-lineage cells (OLCs) ( Bglap, Spp1, and Sp7), fibroblasts ( S100a4, Fn1, and Dcn), chondrocytes ( Col2a1, Sox9, and Acan), pericytes ( Acta2, Myh11, and Mcam), and two endothelial cell (ECs) populations, arterial (AEC) and sinusoidal (SEC) ( Cdh5, Cd34, and Pecam1). Compared to normal BM, MM engraftment was associated with numerical differences in stromal cell populations between normal and MM. Gene set enrichment analysis showed an inflammatory and oxidative stress signal associated with the MM microenvironment. Sub-clustering analysis showed MSC differentiation was polarized away from osteocyte formation towards adipocytes with the identification of a novel population only seen in MM. Bone marrow endothelial cell populations were also substantially altered at this early disease stage, with differentiation polarized towards sinusoidal endothelial cells generating a pro-angiogenic /pro-inflammatory phenotype. An increase in cells undergoing endothelial to mesenchymal transition (EndMT) was also present. Taken together, we show, for the first time, existence of remodeling of the stromal populations induced by the MM clone characterized by a pro-inflammatory phenotype together with polarized differentiation. These changes result in the expansion of a number of key populations that increase the MM niche contributing to growth and survival signals and shaping of the content of the immune microenvironment. These changes result in a self-perpetuating signaling loop between cells, which needs to be broken therapeutically in order to stop progression, induce remission and long-term disease outcomes. Conclusion - In early stages of MM pathogenesis, MM cells remodel the stromal microenvironment by altering the amount and function of MSCs and endothelial cells. Through favoring an adipocytic fate of MSCs, endothelial mesenchymal transition and altering the balance between arterial and sinusoidal endothelial cells, MM cells promote an inflammatory environment that contributes to MM development and progression.

The Impact of Soluble BCMA and BCMA Gain on Anti-BCMA Immunotherapies in Multiple Myeloma

B cell maturation antigen (BCMA) targeting chimeric antigen receptor T cells (CAR T) and bispecific T cell engagers (TCE) have remarkable efficacy in multiple myeloma (MM). While BCMA antigen escape, driven by TNFRSF17 deletions or mutations, is a mediator of anti-BCMA CAR T/ TCE resistance, the majority of patients retain BCMA surface expression at progression. Furthermore, patients with high disease burden and extramedullary disease associated with poorer response to anti-BCMA therapies, have high levels of soluble BCMA (sBCMA). The mechanisms mediating this resistance, and in particular the contribution of sBCMA, have not been fully elucidated. In order to assess the effect of sBCMA on anti-BCMA CAR T function, we measured CAR T cells activation with an in-house manufactured anti-BCMA CAR transduced with an NFAT-GFP reporter gene after co-culture with increasing concentrations of sBCMA (0-2500 ng/mL). sBCMA bound to CAR scFv in a dose dependent manner, effectively competing with the fluorophore tagged BCMA peptide binding. Despite their stable engagement, sBCMA failed to induce the expression of GFP, CD69, CD25, or 41BB on CAR T cells. To further examine the functional effect of sBCMA on CAR T cytotoxicity, we generated isogenic OPM2 cell lines with distinct BCMA expression profiles: OPM2 over-expressing BCMA (OPM2_BCMA high) generated by lentiviral transduction of TNFRSF17, and Cas9 BCMA knockout OPM2 (OPM2_BCMA -/-). OPM2_BCMA high cells exhibited one-log-fold increase in surface BCMA expression and 10-fold increase in sBCMA and were more resistant to CAR T mediated lysis. In addition, in co-culture assays, elevated sBCMA impaired OPM2 target cell lysis by anti-BCMA CAR T in a dose dependent-manner. To gain insight into the effect of chronic sBCMA exposure on CAR T, we exposed anti-BCMA CAR T to OPM2 cells secretome in the absence of direct cell to cell contact in a trans-well co-culture system. On day 24, CAR T in co-culture with OPM2_BCMA high (sBCMA levels of 2343 ng/mL) significantly contracted with no viable cells, while CAR T in culture with parental OPM2 (sBCMA level of 267 ng/mL) did persist with up-regulated TIM3 and TIGIT. In contrast CAR T cultured with OPM2_BCMA -/- remained viable with no expression of exhaustion markers. In examining the impact of sBCMA on TCE activity, sBCMA level as low as 50 ng/mL in vitro impaired anti-BCMA TCE binding to K562 cells transduced to stably express BCMA (K562_wtBCMA) . Consistent results were found in cytotoxicity assays wherein the addition of 2 ng/mL of sBCMA was sufficient to induce a reduction in anti-BCMA TCE mediated cytotoxicity of K562_wtBCMA cells by healthy donor peripheral blood mononuclear cells. To identify genomic events that lead to elevated sBCMA expression and subsequent resistance to CAR T/TCE, we conducted bulk whole genome sequencing (100X) on CD138+ bone marrow MM cells obtained from 40 MM patients treated with anti-BCMA CAR T/ TCE. Structural variants (SVs) at or adjacent to TNFRSF17 locus were identified in four patients. These included i) focal copy number gain of TNFRSF17 (3 copies) in a patient who progressed within 3 months post anti-BCMA TCE, ii) subclonal duplication of TNFRSF17 locus in another patient (n=1), as well as iii) focal copy number losses 5‘ or 3‘ to the TNFRSF17 gene locus (n=2). In addition to SVs affecting TNFRSF17 locus, we also identified mutations involving TNFRSF17 transcriptional regulators. Notably, MM clones at progression 4 months post anti-BCMA TCE (n=1) harboured copy number gains (3 copies) of POU2AF1 gene on chr11q23. POU2AF1 encodes a key transcriptional regulator of TNFRSF17 expression. Indeed, sBCMA level from this patient was significantly elevated at relapse at a level (638 ng/mL) correlated with resistance to anti-BCMA TCE. In addition, we identified translocation involving MAP3K14 encoding the NF-kB regulator NIK in a patient with short remission post anti-BCMA CAR T. Of interest, analysis of the CoMMpass dataset revealed significantly higher BCMA transcripts in patients with translocations involving MAP3K14 locus. Our findings highlight the impact of sBCMA levels and its chronicity of exposure on anti-BCMA TCE and CAR T activities in MM. We further identified structural genomic events driving TNFRSF17 over-expression and their correlation to sBCMA levels facilitating tumoral immunotherapeutic escape.

Multiple Myeloma B Cells and Pre-Plasma Cells Are Important Reservoirs for Myeloma Relapse Following Plasma Cell-Directed Therapy and Prevent Cure with Standard Therapies

Multiple Myeloma (MM) is a B cell neoplasm characterized by the accumulation of mature plasma cells (PCs) within the bone marrow (BM). Despite treatment advances, MM remains incurable in the vast majority of patients. Disease relapse typically occurs regardless of treatment with proteasome inhibitors (such as bortezomib and carfilzomib), immunomodulatory drugs (such as lenalidomide and pomalidomide), anti-CD38 monoclonal antibodies (such as daratumumab and isatuximab) or myeloablative melphalan and autologous stem cell transplantation, and also occurs following treatment with immunotherapeutics such as bispecific antibodies and/or CAR-T cells (targeting BCMA, FcRH5 or GPRC5D). This failure to achieve reliable cure in MM, despite the routine attainment of deep treatment responses against PCs, indicates the existence of important intra-tumor heterogeneity and the presence of rare drug-resistant MM cells with full tumorigenic capacity. To identify intra-clonal MM cell subpopulations, we examined BM samples from MM patients using a combination of FACS, single cell resolved immunoflourescence-FISH (IF-FISH), whole exome sequencing (WES), custom-capture targeted deep sequencing (CC-Seq) and single cell RNA sequencing (scRNAseq). We sought to characterize the genomic landscape and to track the dynamic interconnectedness of these intra-clonal subpopulations in patients over time. Using sequential FACS-IF-FISH studies of MM BM samples (n=140) we identified MM clone cellular subpopulations within the BM of MM patients that recapitulate the normal maturation stages between non-malignant post germinal centre B cells and mature PCs. These subpopulations include rare MM cells that resemble CD20 +CD38 -CD138 -Bcma -Irf4 - Xbp1s - B cells, CD20 -CD38 -CD138 - Bcma -Irf4 -Xbp1s - pre-plasmablasts and CD38 +CD138 -Bcma -/lowIrf4 -/+Xbp1s + pre-plasma cells, as well as predominating tumor-bulk CD38 +CD138 +Irf4 +Bcma +Xbp1s + plasma cells. Importantly, MM progenitor cells were typically found to possess all of the chromosomal translocations and copy number variations (CNV) present in MM PCs including whole chromosome ploidy changes, translocations such as t(11;14), t(4;14) and t(14;16), and secondary aberrations such as gain(1q), del(1p) and del(17p). To examine if MM progenitor cells also possess the single nucleotide variations (SNV) present within MM PCs, and are thus fully malignant, and to track MM progenitor subpopulations and their inter-relatedness within patients over time, we performed WES (n=60) plus targeted deep CC-Seq (n=210) on purified cell subpopulations from 17 patients, including 5 patients whom we sampled serially over >3 years. A median of 5 cellular subpopulations were isolated from each BM sample, enriched by factors of up to 1000-fold or more, and each subpopulation was then sequenced to a depth of 4,000-20,000x. Significantly, SNVs that were present in tumor-bulk MM PCs were also regularly detected in MM progenitor cells, although the low frequency of the clonal progenitor cells often prevented capture of complete SNV profiles. At the same time, analyses of serial BM samples from repeat donor patients taken pre- and post-treatment demonstrated that relapsing MM PCs typically did not appear to derive linearly from pre-treatment PCs, as the relapsing PCs commonly lacked a number of SNVs that had been present in pre-treatment PCs. Importantly, we instead observed that relapsed MM plasma cells appeared to consistently derive from MM progenitor cell populations, frequently B cells, as emergent relapse-specific SNVs that were exclusively detected in relapsing post-treatment PCs (but in not pre-treatment PCs) could often also be detected within pre-treatment CD138- CD38- progenitor cells isolated from BM samples that had been collected up to 3 years prior to relapse. We conclude that MM B cells and pre-plasma cell progenitors possess all of the clonal genomic aberrations required for full malignant potential. Tracking of these cells in vivo in patients over time by their SNV profile implicates them as the cellular origin of PC relapse and recurrent MM disease. We propose that cure of MM requires eradication of MM progenitor cells alongside plasma cells. We are currently conducting scRNA-seq and CITE-seq studies of primary MM samples to further characterize MM progenitor cells for gene and protein expression in order to define their optimal therapeutic targets.

Macropinocytosis As a Means of Selectively Targeting RAS-Mutant Multiple Myeloma

Introduction Roughly, 50% of multiple myeloma (MM) is driven by mutant RAS, both at presentation and more at relapse. Currently, in MM there are no clinical approaches to target this disease subset and ongoing strategies to inhibit RAS function or downstream effectors have had limited clinical success in other tumors. This has prompted our team to look into other more indirect modes of RAS-selective targeting. Oncogenic RAS-driven cancers use a unique actin-driven endocytosis pathway, termed macropinocytosis (MP), to increase the nutrient supply and sustain proliferation. To create a strategy for targeting RAS mutant MM, we have investigated whether RAS-mutant MM cells undergo MP and whether this process can be exploited as a therapeutic vulnerability, by utilizing this pro-oncogenic biochemical mechanism to selectively deliver a toxin to the MM clone using a MP selective monobody-drug conjugate (MDC). Methods The MDC relies on a proprietary fibronectin-based scaffold (monobody) identified based on its physiochemical properties that facilitates MP selective internalization, and therefore targets RAS-mutant MM tumor cells, with a very favorable pharmacokinetic profile. MMAE was attached to the monobody with a cathepsin-cleavable linker and a maleimide group for conjugation to the C-terminal free cysteine on the monobody protein (MDC-MMAE). MDC-MMAE was pre-clinically evaluated both in-vitro and in-vivo, in human MM cell lines, patient cells and a doxycycline inducible mutant KRAS cell system. Results In-vitro cell line and patient cell studies demonstrated that RAS-mutant MM cells undergo MP whereas wild type (WT) cells do not. To understand the role of mutant RAS in driving uptake of the MDC, a doxycycline-inducible mutant KRAS V12 KMS11 system was established. The functionality of the inducibility of the cell system was verified using protein expression and membrane ruffling in the presence of doxycycline. Fluorescently-labeled monobody (MDC-Cy5.5) and TMR-Dextran underwent a high level of uptake in the presence of induced mutant RAS. In contrast, in conditions where MP was absent, no uptake of MDC-Cy5.5 and TMR-Dextran was seen. Furthermore, when KMS11 KRASV12 and WT KMS11 were treated with MDC-MMAE, a 2-fold increase in cell death was observed in the mutant RAS cell line compared to wild type RAS. In-vivo studies showed that the pharmacological properties of the drug when conjugated to the monobody enabled faster serum clearance compared to antibody drug conjugates (ADCs) and nanomaterials, while maintaining cellular impermeability allowing enhanced drug uptake via MP. Importantly, in-vivo the MDC-MMAE technology had an improved toxicity profile compared to ADC-MMAE, increasing tolerable drug delivery by upwards of 15-fold. To test the anti-tumor effects in-vivo, the L363 (NRAS Q61), WT KMS11, and KMS11 KRASV12 cell lines were subcutaneously injected into MM murine models. After tumors were established, 0.5mg/kg MDC-MMAE was administered every 7 days. Mutant RAS L363 and KMS11 KRASV12 tumors treated with MDC-MMAE exhibited roughly a 6-fold decrease in tumor size after 21 days while WT KMS11 tumors showed no significant difference in tumor volume. MDC-MMAE at 5mg/kg showed complete tumor regression as a single agent in L363 tumors. Improved outcomes were seen with MDC-MMAE in combination with standard of care options at lower doses. Additionally, MDC-MMAE has displayed enhanced immunogenic cell death that has not been seen with other chemotherapeutic options, making it an ideal partner for immunotherapy combination. Conclusion Limited clinical options exist for the treatment of mutant RAS MM tumors. We show that by taking advantage of MP, a process upregulated in RAS-mutant cancers, MDC-MMAE specifically targets and inhibits MM growth and improves survival in murine models. It can also be combined with other MM standard of care drugs and with immunotherapy drugs to have a potentially important clinical effect. In collaboration with Tezcat Biosciences, the MDC-MMAE technology has achieved a phase II SBIR for advancement to clinical application.

Mass Spectrometry-Based Assessment of M-Protein in Peripheral Blood during Maintenance Therapy in Multiple Myeloma (MM) in the Phase III ATLAS Trial

Introduction Minimal residual disease (MRD) assessment in bone marrow (BM) has emerged as one of the strongest prognostic factors in MM. Mass spectrometry (MS), capable of detecting MM-derived monoclonal proteins in peripheral blood with high sensitivity, has the potential to complement standard methods. While its role in evaluating residual disease and its prognostic performance compared to routine MRD assessments is not yet established, other unanswered questions remain: (1) What is the role of MS in response assessment for patients without a high disease burden baseline sample? (2) What is the optimal timing of MS assessment, considering the long half-life of immunoglobulins? Methods Stored serum samples collected at different timepoints [screening, cycle 6 (C6), C12, C18, C24, C36] from patients treated in the ATLAS trial, were analyzed using the EXENT® Immunoglobulin isotypes (GAM) assay (EXENT® Solution, in development by The Binding Site, part of Thermo Fisher Scientific), which utilizes MALDI-TOF MS. ATLAS is an ongoing phase 3 trial that randomized 180 patients with newly diagnosed MM after autologous stem cell transplantation (ASCT) to maintenance therapy with carfilzomib, lenalidomide, and dexamethasone (KRd) or lenalidomide alone (R). In the KRd arm, patients with standard-risk cytogenetics who achieved MRD-negativity after cycle 6 de-escalated therapy to receive R, from C9. No baseline diagnostic samples were available, and samples were not available for every patient at every timepoint. A sample was called positive by MS when the EXENT® solution confirmed the presence of an M protein, matching the known (diagnostic) isotype. BM MRD was assessed at the same timepoints using multicolor flow cytometry (MFC) with a limit of detection (LoD) of 10 -5 and next-generation sequencing (NGS, clonoSEQ, Adaptive, LoD 10 -6). Progression-free survival (PFS) was analyzed using the Kaplan-Meier method, with log-rank test for comparisons between groups. The data cutoff (31-Dec-2021) was the same as was used in the ATLAS publication by Dytfeld et al. Lancet Oncology 2023. Results 585 samples from 138 patients were analyzed by MS. There were 349 paired BM MRD results by NGS; patients with MRD 10 -5 were considered MRD(+). The overall agreement between the two methods equaled 70%, with a comparable number of patients being MS+/NGS- (56/349, 16%) and MS-/NGS+ (50/349, 14%). The agreement at screening was 61%, but it improved at further timepoints and reached the highest level at C18 (74%). For BM MFC, there were 499 paired samples. There was 67% agreement, and the discrepancy was driven mostly by MS+/MFC- patients (125/499, 25%), whereas MS-/MFC+ was less frequent (40/499, 8%). The agreement was lowest at screening (60%), similarly to the NGS-based assessment, and reached the highest level at C18 (73%). Based on these findings, we concluded that C18 may be the optimal timepoint to assess prognostic role of MS results. Using the landmark method, MS(-) following C18 was associated with superior PFS compared to MS(+) [HR=0.39 (95% CI: 0.17-0.91), p=0.025; Figure 1]. MFC [HR=0.13 (0.04-0.35), p=0.0002], and NGS [HR=0.15 (0.03-0.63), p=0.003] statuses were also associated with PFS using this landmark method. Although small numbers of patients preclude definitive conclusions, we observed a similar effect size in outcomes among NGS(-)/MS(-) vs NGS(-)/MS(+) patients [HR=0.14 (0.01-1.58), p=0.06] and MFC(-)/MS(-) vs MFC(-)/MS(+) [HR=0.40 (0.13-1.27), p=0.11], both favoring BM MRD(-) with concurrent MS(-). Out of the 45 patients who were initially MS(+) and subsequently converted to MS(-), 23 (51%) occurred following C12 or later. Patients who converted from MS(+) to MS(-) had similar PFS to those with sustained MS(-) throughout the analyzed period [HR=0.74 (0.32-1.68), p=0.47; Figure 2], with the majority of PFS events in the converted group occurring during first 18 months of observation. Conclusions MS-based disease assessment in the post-ASCT setting is feasible in many cases, if information on the original MM clone is available by traditional methods. Our results suggest that the optimal timepoint for such evaluation can be as long as 18 months after ASCT. The EXENT® assay may be complementary to BM-based techniques for MRD assessment, allowing for more accurate prognostication when used in combination. Further studies are needed to confirm its exact timing and role in the disease evaluation algorithms.

Characterization of JNJ-79635322, a Novel BCMAxGPRC5DxCD3 T-Cell Redirecting Trispecific Antibody, for the Treatment of Multiple Myeloma

Background: Multiple myeloma (MM) is a genetically complex and heterogenous malignancy with a poor survival rate and accounts for approximately 10% to 15% of all hematologic cancers. Despite advances in myeloma therapy, MM remains incurable. Deep and durable clinical responses have been observed when targeting the tumor associated antigens BCMA and GPRC5D with immunotherapies. Additionally, data presented at ASCO this year demonstrated a further improvement in ORR when BCMA and GPRC5D bispecifics were given in combination to relapsed or refractory MM patients. These data suggest that broader antigen coverage by redirecting T cells to two tumor surface antigens is an effective means to harness the immune system to deplete cancer cells. Targeting two tumor antigens may also reduce the likelihood of antigen escape as a primary reason for relapse. Here, we describe JNJ-79635322, a trispecific antibody targeting B-cell maturation antigen (BCMA) and G-Protein-coupled receptor class 5 member D (GPRC5D), which are both highly expressed on plasmablasts and plasma cells in myeloma patient samples. Dual antigen recognition on plasma cells with a trispecific T-cell engaging antibody engineered with a CD3 arm has the potential to enhance tumor binding through avidity that could result in efficient depletion of malignant clonal populations and prevent tumor antigen loss mediated resistance. Methods: JNJ-79635322 is a trispecific antibody composed of an anti-CD3 binding domain an anti-BCMA binding domain, and an anti-GPRC5D binding domain. It was selected based on biophysical assessment and showed activity in multiple myeloma models. Results: JNJ-79635322 induced potent cytotoxicity in various myeloma cell lines in vitro with concomitant T-cell activation. Importantly, JNJ-79635322 was able to deplete both dual (BCMA and GPRC5D) and single (BCMA or GPRC5D) target expressing cells efficiently. In addition, dose dependent depletion of H929 myeloma cells was observed in a more physiological healthy fresh whole blood co-culture assay. In vivo, JNJ-79635322 exhibited potent antitumor activity compared to vehicle and antibody controls in a murine MM xenograft prevention model (single target expressing clonal H929 cells) and two tumor regression models (RPMI 8226 and MM.1S cells). Conclusion: JNJ-79635322 is a potential first-in-class trispecific antibody targeting BCMA and GPRC5D with the ability to deplete dual and single target expressing MM clones in preclinical studies vitro and in vivo. A Phase 1 dose-escalating study of JNJ-79635322 in myeloma patients is ongoing (NCT05652335).

Proteomic Alteration in the Progression of Multiple Myeloma: A Comprehensive Review.

Multiple myeloma (MM) is an incurable hematologic malignancy. Most MM patients are diagnosed at a late stage because the early symptoms of the disease can be uncertain and nonspecific, often resembling other, more common conditions. Additionally, MM patients are commonly associated with rapid relapse and an inevitable refractory phase. MM is characterized by the abnormal proliferation of monoclonal plasma cells in the bone marrow. During the progression of MM, massive genomic alterations occur that target multiple signaling pathways and are accompanied by a multistep process involving differentiation, proliferation, and invasion. Moreover, the transformation of healthy plasma cell biology into genetically heterogeneous MM clones is driven by a variety of post-translational protein modifications (PTMs), which has complicated the discovery of effective treatments. PTMs have been identified as the most promising candidates for biomarker detection, and further research has been recommended to develop promising surrogate markers. Proteomics research has begun in MM, and a comprehensive literature review is available. However, proteomics applications in MM have yet to make significant progress. Exploration of proteomic alterations in MM is worthwhile to improve understanding of the pathophysiology of MM and to search for new treatment targets. Proteomics studies using mass spectrometry (MS) in conjunction with robust bioinformatics tools are an excellent way to learn more about protein changes and modifications during disease progression MM. This article addresses in depth the proteomic changes associated with MM disease transformation.

Newly Diagnosed, Untreated, Multiple Myeloma (MM) Patient Samples Already Harbor Cereblon (CRBN) Exon 10 Deletions Associated with Drug Resistance

Background: Acquisition of drug resistance, particularly immunomodulatory drugs (IMiDs) in MM is one of the most important challenges in patient treatment today. Early on in the disease, the plasma cells in the MM clone are often sensitive to chemotherapy. However, over time, after exposure to chemotherapy, the cells acquire increasing genomic complexity and mutations leading to resistance to therapy and ultimately patient death. Cereblon (CRBN) is a binding protein, which is required for the action of the widely used IMiDs such as lenalidomide and pomolidomide. CRBN is also essential to the newer CRBN E3 ligase modulator drugs and proteolysis targeting chimeras. We recently described an algorithm based on digital gene expression profiling of purified CD138(+) MM cells from patients at different clinical stages that we term the "MM-IP-7", which correlated with clinical drug resistance, poor survival, and disease progression following combination treatment containing IMIDs and/or PIs (XY Zhu et al, eHaem 2022). Given the recent description of genetic changes in the CRBN gene that are associated with IMiD resistance particularly the exon 10-spliced CRBN transcripts that increase in incidence in parallel with IMiD-refractory states (S. Gooding et al, Blood 2021), we sought to build an integrated, single-platform test to measure both gene expression levels and genetic deletions. Methods: We designed a novel CRBN splicing isoform probe with homology to both exon 9 and exon 11. This probe will thus capture CRBN mRNA sequences in which exon 10 is deleted and portions of exons 9 and 11 and juxtaposed. We also included probes to CRBN overall (Exons 5 and 6). These probes were combined with the MM-IP-7 probes to create a dual gene expression and genetic assay that was used to analyze 30 cases of newly diagnosed MM, 30 cases of end stage MM, and 3 MM cell lines (MM1.S, OPM2, XG1). To define increased exon 10 deleted allele frequency, we calculated the ratio of exon 10 deletion/total exon 10. Cases with apparent exon 10 deletion by expression profiling, were further evaluated with RT-PCR. Results: 40% MM samples in the end stage group versus 23% of samples in the newly diagnosed group were identified with more than 20% exon 10 deleted mRNA as compared to total CRBN mRNA levels. Three cases were shown to have more than 45% exon 10 deleted mRNA, including two newly diagnosed cases and one end stage case. RT-PCR confirmed exon 10 deletion in a newly diagnosed patient sample with sufficient remaining RNA that was tested (cloning and sequencing for confirmation are underway). Cell line results showed very low exon 10 splicing variant, which correlated with our mRNAseq data for these lines. Conclusions: Our findings demonstrate that inclusion of probes bridging CRBN exon 10 deletion with homology to exons 9 and 11, is feasible approach to use on the gene expression platform, which allows for combined evaluation of expression and genetic changes. CRBN exon 10 deletion is a known treatment resistance feature in MM, which may already be present at diagnosis and selected by subsequent therapy with possible therapeutic planning implications. Additional studies to confirm the results and expand the study set are underway.

Unraveling unique features of plasma cell clones in POEMS syndrome with single-cell analysis.

POEMS syndrome is a rare monoclonal plasma cell disorder, with unique symptoms distinct from those of other plasma cell neoplasms, including high serum VEGF levels. Because the prospective isolation of POEMS clones has not yet been successful, their real nature remains unclear. Herein, we performed single-cell RNA-Seq of BM plasma cells from patients with POEMS syndrome and identified POEMS clones that had Ig λ light chain (IGL) sequences (IGLV1-36, -40, -44, and -47) with amino acid changes specific to POEMS syndrome. The proportions of POEMS clones in plasma cells were markedly smaller than in patients with multiple myeloma (MM) and patients with monoclonal gammopathy of undetermined significance (MGUS). Single-cell transcriptomes revealed that POEMS clones were CD19+, CD138+, and MHC class IIlo, which allowed for their prospective isolation. POEMS clones expressed significantly lower levels of c-MYC and CCND1 than MM clones, accounting for their small size. VEGF mRNA was not upregulated in POEMS clones, directly indicating that VEGF is not produced by POEMS clones. These results reveal unique features of POEMS clones and enhance our understanding of the pathogenesis of POEMS syndrome.

Immunophenotypic Characteristics of Bone Marrow Microenvironment Cellular Composition at the Biochemical Progression of Multiple Myeloma.

Multiple myeloma (MM) relapses are inevitable in the majority of patients, and in addition to genetic changes in the MM clone, the immune profile of the bone marrow (BM) plays a key role in this process. Biochemical progression or relapse (BR) precedes clinical relapse in a significant proportion of patients with MM. In the present study, we used flow cytometry to assess the cellular composition of the BM microenvironment in MM patients with confirmed BR. Fifteen distinct cells subsets in the BM were evaluated with the panel of antibodies used routinely for MRD monitoring in MM in 52 patients with MM (MRD-negative n = 20, BR n = 20, and clinically relapsed MM, RMM n = 12). The median percentage of MM cells detected in BR patients was 0.90% versus not detectable in MRD-negative patients and of 3.0% in RMM cohort. Compared to the MRD-negative group, BR status was associated with an increase in the percentage of lymphoid subpopulations, including memory B cells (p = 0.003), CD27+T cells (p = 0.002), and NK/NKT cells (p < 0.001). Moreover, a decrease in B-cell precursors (p < 0.001) and neutrophils (p = 0.006) was observed. There were no significant differences in the composition of the BM cell subpopulations between the BR and RMM groups. Our results indicate the involvement of B-, T-, and NK cells in the process of losing immune surveillance over the MM clone that leads to relapse. It can be speculated that similar studies of a larger cohort of BR patients can potentially identify a group of patients for which an early treatment intervention would be beneficial.

PB1954: COMPARING STR PROFILES OF DNA ISOLATED FROM DIVERSE TUMOR SITES IN MULTIPLE MYELOMA

Background: Multiple myeloma (MM) course is often complicated by extramedullary lesions - plasmacytomas, which pathogenesis is still not completely understood. Genome instability underlies dissemination in various neoplasias, including MM. An important manifestation of genetic instability is the loss of heterozygosity, i.e., the loss of the second allele in the regions of the genome featuring inherited polymorphisms. STR profiling is a method that allows simultaneously assessing the degree of DNA degradation and giving an integral assessment of the stability of the tumor genome. To assess the heterogeneity of MM, not only the tumor substrate in the bone marrow and plasmacytoma but also in circulating cell-free DNA (cfDNA) present in the peripheral blood plasma could be effectively analyzed. Aims: To compare STR profiles of DNA isolated from various anatomical locations (plasma, bone marrow, plasmacytoma) in MM patients. Methods: The prospective study included 21 patients with newly diagnosed MM (11 women, 10 men) aged 39 to 82 years (median 53 years). MM was diagnosed according to the IMWG-2014 criteria. In 15 patients, MM was complicated by plasmacytoma, in 6 of them a tumor biopsy was performed and tumor DNA was isolated. In all patients, plasma circulating cfDNA and control DNA were isolated from buccal epithelium using a commercial kit (Qiagen, Germany) according to the manufacturer’s protocol. In 11 patients, positive immunomagnetic selection of CD138+ bone marrow cells was performed using a monoclonal antibody to CD138 (Miltenyi Biotec, Germany). STR-profiling using COrDIS Plus panel (Gordiz LLC, Russia) of isolated DNA samples (21 plasma circulating cfDNA samples, 11 CD138+ DNA samples of bone marrow cells, 6 plasmacytoma DNA samples) was performed. STR profiles were analyzed using GeneMapper v.4 software (Applied Biosystems, USA). Results: In 10 out of 11 patients, analysis of STR profiles in CD138+ bone marrow cells revealed loss of heterozygosity (LOH) variants with different allelic load, suggesting either a deletion/copy number neutral LOH, or a duplication of one of the two alleles affecting from one to six STR loci for every patient. In all patients for whom plasmacytoma DNA was isolated (n=6), various variants of LOH were also identified (from 1 to 6 loci) (Table 1). Of the 21 plasma circulating cfDNA samples, LOH variants were detected in ten cases (48%) (from 1 to 4 loci), in the other 10 cases, no tumor DNA was detected, the samples matched the control DNA from the buccal epithelium; only in one case, the result turned out to be non-informative due to a significant degree of degradation of the cfDNA. STR profiles of DNA isolated from different tumor sites of the same patient in most cases were identical. In one case aberrant STR loci in plasma circulating cfDNA and bone marrow CD138+ cells were different (patient No. 20). In this patient, MM was complicated by plasmacytomas of various anatomical locations (Th7, 8th rib). One can speculate that the discrepancy between plasma circulating cfDNA and bone marrow CD138+ STR profiles indicates the presence of various MM clones in different tumor loci. Image:Summary/Conclusion: Evaluation of plasma circulating cfDNA might be an effective approach for molecular diagnosis in MM. In 48% of MM patients, it is possible to detect tumor cfDNA in the plasma at the onset of the disease. Comparison of DNA STR profiles from different lesions might reveal the diversity of tumor clones in MM.

Monitoring multiple myeloma in the peripheral blood based on cell-free DNA and circulating plasma cells

With the advent of novel, highly effective therapies for multiple myeloma (MM), classical serologic monitoring appears insufficient for response assessment and prediction of relapse. Moreover, serologic studies in MM are hampered by interference of therapeutic antibodies. The detection of malignant plasma cell clones by next generation sequencing (NGS) or multiparameter flow cytometry (MFC) circumvents these difficulties and can be performed in the peripheral blood (pB) by targeting circulating cell-free DNA (cfDNA) or circulating plasma cells (CPCs), thus also avoiding an invasive sampling procedure. Here, we applied NGS of VJ light chain (LC) rearrangements in cfDNA and MFC of magnetically-enriched CD138-positive CPCs (me-MFC) to investigate disease burden in unselected MM patients. Sequencing was successful for 114/130 (87.7%) cfDNA samples and me-MFC results were analyzable for 196/205 (95.6%) samples. MM clones were detectable in 38.9% of samples taken at initial diagnosis or relapse (ID/RD), but only in 11.8% of samples taken during complete remission (CR). Circulating MM plasma cells were present in 83.3% of ID/RD samples and 9.9% of CR samples. Residual disease assessment by NGS or me-MFC in samples taken during very good partial remission or CR was 80% concordant. Notably, 4/4 (NGS) and 5/8 (me-MFC) positive CR samples were from patients with oligo- or non-secretory myeloma. The time to progression was shorter if there was evidence of residual myeloma in the pB. Together, our findings indicate that our two novel analytical approaches accurately indicate the course of MM and may be particularly valuable for monitoring patients with serologically non-trackable disease.

Dual Targeting of Multiple Myeloma Stem Cells and Myeloid-Derived Suppressor Cells for Treatment of Chemotherapy-Resistant Multiple Myeloma.

Here we review the insights and lessons learned from early clinical trials of T-cell engaging bispecific antibodies (BsABs) as a new class of biotherapeutic drug candidates with clinical impact potential for the treatment of multiple myeloma (MM). BsABs are capable of redirecting host T-cell cytotoxicity in an MHC-independent manner to malignant MM clones as well as immunosuppressive myeloid-derived suppressor cells (MDSC). T-cell engaging BsAB targeting the BCMA antigen may help delay disease progression in MM by destroying the MM cells. T-cell engaging BsAB targeting the CD38 antigen may help delay disease progression in MM by depleting both the malignant MM clones and the MDSC in the bone marrow microenvironment (BMME). BsABs may facilitate the development of a new therapeutic paradigm for achieving improved survival in MM by altering the immunosuppressive BMME. T-cell engaging BsiABs targeting the CD123 antigen may help delay disease progression in MM by depleting the MDSC in the BMME and destroying the MM stem cells that also carry the CD123 antigen on their surface.

Targeting DNA2 Overcomes Myeloma Cells' Metabolic Reprogramming in Response to DNA Damage

DNA damage resistance is a major barrier to effective DNA-damaging anticancer therapy in multiple myeloma (MM). To discover novel mechanisms through which MM cells overcome DNA damage, we investigated how MM cells become resistant to antisense therapy targeting ILF2, an important DNA damage regulator in MM (Marchesini et. al., Cancer Cell 2017). We continuously treated JJN3 and KMS11 cells with an ILF2-targeting antisense oligonucleotide (ILF2 ASOs) or control non-targeting antisense oligonucleotide (NT ASOs). Whereas KMS11 cells maintained a high level of DNA damage activation and a significantly increased rate of apoptosis after 3 weeks of ILF2 ASOs treatment, JJN3 cells overcame ILF2 ASO-induced DNA damage activation and became resistant to ILF2 ASOs treatment.To evaluate whether continuous ILF2 ASOs exposure could lead to the selection of MM clones intrinsically resistant to ILF2 ASO-induced DNA damage, we performed single-cell RNA seq (scRNA-seq) analysis of JJN3 cells treated with NT or ILF2 ASOs for 3 weeks. Our analysis divided JJN3 cells into 2 main clusters that were independent of treatment (Fig. 1A), suggesting that persistent exposure to ILF2 ASOs did not induce clonal selection. Differential gene expression analysis of NT ASO- and ILF2 ASO-treated cells in each of these clusters revealed that DNA damage resistant ILF2 ASO-treated cells had significantly upregulated oxidative phosphorylation (OXPHOS), DNA repair signaling, and reactive oxidative species (ROS). Consistent with these results, metabolomic analysis of JJN3 cells after long-term exposure to ILF2-ASOs showed a significant enrichment of tricarboxylic acid cycle (TCA) intermediates (Fig. 1B). ILF2-ASO-resistant MM cells were significantly more sensitive to the OXPHOS inhibitor IACS-010759 than ILF2-ASO-sensitive cells were. These data suggest that MM cells can undergo an adaptive metabolic rewiring to restore energy balance and promote survival in response to DNA damage.We then hypothesized that ILF2-ASO-resistant cells' metabolic reprogramming relies on the repair of DNA damage induced by ILF2 depletion or by the generation of ROS from activated mitochondrial metabolism and that targeting DNA repair proteins involved in these processes overcomes DNA damage resistance. We used a CRISPR/Cas9 library screening strategy to identify DNA repair genes whose loss of function suppresses MM cells' ability to overcome ILF2-ASO-induced DNA damage. Compared with those in NT-ASO-treated cells, DNA2-targeting sgRNAs were significantly depleted after 3 weeks of treatment in ILF2-ASO-treated JJN3 cells but not in ILF2-ASO-treated KMS11 cells. These data suggest that DNA2 is needed to promote resistance to ILF2 depletion. Accordingly, the DNA2 inhibitor NSC105808 (NSC) significantly enhanced ILF2-ASO-induced apoptosis in JJN3 cells. These data gain added significance in light of previous findings that DNA2 is a nuclear and mitochondrial DNA nuclease/helicase that enables cancer cells to counteract the DNA replication stress and mitochondrial oxidative DNA damage induced by DNA-damaging agents. Accordingly, we observed that DNA2 was mainly localized into the mitochondria of MM cells.To dissect the mechanisms of DNA2 inhibition-induced synthetic lethality, we evaluated whether DNA2 activity is essential to maintain activated OXPHOS, which ILF2-ASO-resistant cells require to survive. The quantification of mitochondrial respiratory activity in NT-ASO-and ILF2-ASO-treated MM cells exposed to NSC for 72 hours showed that DNA2 activity inhibition significantly decreased the oxygen consumption rate while increasing ROS production in only ILF2-depleted cells. Transmission electron microscopy analysis showed that NSC-treated ILF2-depleted cells had fragmented mitochondrial cristae structures, whose perturbations affect the OXPHOS system structure and impair cell metabolism. These data suggest that DNA2 is essential to counteract oxidative DNA damage and maintain mitochondrial respiration after MM cells' metabolic reprogramming.In conclusion, our study has revealed a novel mechanism through which MM cells can overcome DNA damage activation. Further studies will clarify whether targeting DNA2 is synthetically lethal in tumors with increased demand of mitochondrial metabolism. [Display omitted] DisclosuresKonopleva: Genentech: Consultancy, Honoraria, Other: grant support, Research Funding; AbbVie: Consultancy, Honoraria, Other: Grant Support, Research Funding; Sanofi: Other: grant support, Research Funding; Ablynx: Other: grant support, Research Funding; Reata Pharmaceuticals: Current holder of stock options in a privately-held company, Patents & Royalties: intellectual property rights; Agios: Other: grant support, Research Funding; Cellectis: Other: grant support; Rafael Pharmaceuticals: Other: grant support, Research Funding; Calithera: Other: grant support, Research Funding; Forty Seven: Other: grant support, Research Funding; Ascentage: Other: grant support, Research Funding; AstraZeneca: Other: grant support, Research Funding; F. Hoffmann-La Roche: Consultancy, Honoraria, Other: grant support; Stemline Therapeutics: Research Funding; Novartis: Other: research funding pending, Patents & Royalties: intellectual property rights; Eli Lilly: Patents & Royalties: intellectual property rights, Research Funding; KisoJi: Research Funding.

P-106: Targeting DNA2 overcomes myeloma cells’ metabolic reprogramming in response to DNA damage

<h3>Background</h3> DNA damage resistance is a major barrier to effective DNA-damaging anticancer therapy in multiple myeloma (MM). To discover novel mechanisms through which MM cells overcome DNA damage, we investigated how MM cells become resistant to antisense therapy targeting ILF2, an important DNA damage regulator in 1q21 MM. <h3>Method</h3> We continuously treated MM cells with an ILF2-targeting antisense (ILF2-ASO) or control non-targeting antisense (NT-ASO) for 3 weeks to see if ILF2-ASO exposure leads to the selection of MM clones intrinsically resistant to DNA damage or activates compensatory mechanisms to overcome ILF2 depletion-induced DNA damage. <h3>Results</h3> Single-cell RNA sequencing (scRNA-seq) analysis revealed that DNA damage-resistant ILF2-ASO-treated cells had significantly upregulated oxidative phosphorylation (OXPHOS), DNA repair signaling, and reactive oxidative species (ROS). Metabolomic analysis of MM cells after long-term exposure to ILF2-ASO showed a significant enrichment of tricarboxylic acid cycle (TCA) intermediates. Consistent with these results, ILF2-ASO-resistant MM cells were significantly more sensitive to the OXPHOS inhibitor IACS-010759 than ILF2-ASO-sensitive cells were. These data suggest that MM cells can undergo an adaptive metabolic rewiring to restore energy balance and promote survival in response to DNA damage. We then hypothesized that ILF2-ASO-resistant cells' metabolic reprogramming relies on the repair of DNA damage induced by ILF2 depletion or by the generation of ROS from activated mitochondrial metabolism and that targeting DNA repair proteins involved in these processes overcomes DNA damage resistance. We used a CRISPR/Cas9 screening strategy to identify DNA repair genes whose loss of function suppresses MM cells' ability to overcome ILF2-ASO-induced DNA damage. Compared with those in NT-ASO-treated cells, DNA2-targeting sgRNAs in ILF2-ASO-treated JJN3 cells were significantly depleted after 3 weeks of treatment, suggesting that DNA2 is needed to promote resistance to ILF2 depletion. Accordingly, the DNA2 inhibitor NSC105808 (NSC) significantly enhanced ILF2-ASO-induced apoptosis. To dissect the mechanisms of DNA2 inhibition-induced synthetic lethality, we evaluated whether DNA2 activity is essential to maintain activated OXPHOS, which ILF2-ASO-resistant cells require to survive. The quantification of mitochondrial respiratory activity in NT-ASO- and ILF2-ASO-treated MM cells exposed to NSC for 3 days showed that DNA2 activity inhibition significantly decreased the oxygen consumption rate while increasing ROS production in only ILF2-depleted cells. Transmission electron microscopy analysis showed that NSC-treated ILF2-depleted cells had fragmented mitochondrial cristae structures, whose perturbations affect the OXPHOS system structure and impair cell metabolism. <h3>Conclusion</h3> In conclusion, our study has revealed a novel mechanism through which MM cells counteract oxidative DNA damage and maintain mitochondrial respiration after metabolic reprogramming.

The Prognostic Role of MYC Structural Variants Identified by NGS and FISH in Multiple Myeloma.

Structural variants (SV) of the MYC gene region are common in multiple myeloma and influence disease progression. However, the prognostic significance of different MYC SVs in multiple myeloma has not been clearly established. We conducted a retrospective study of multiple myeloma comparing MYC SV subtypes identified by next-generation sequencing (NGS) and FISH to MYC expression and disease survival using 140 cases from Mayo Clinic and 658 cases from the MMRF CoMMpass study. MYC SVs were found in 41% of cases and were classified into nine subtypes. A correlation between the presence of a MYC SV and increased MYC expression was identified. Among the nine MYC subtypes, the non-immunoglobulin (non-Ig) insertion subtype was independently associated with improved outcomes, while the Ig insertion subtype, specifically involving the IgL gene partner, was independently associated with poorer outcomes compared with other MYC SV subtypes. Although the FISH methodology failed to detect approximately 70% of all MYC SVs, those detected by FISH were associated with elevated MYC gene expression and poor outcomes suggesting a different pathogenic role for FISH-detected MYC subtypes compared with other MYC subtypes. Understanding the impact of different MYC SVs on disease outcome is necessary for the reliable interpretation of MYC SVs in multiple myeloma. NGS approaches should be considered as a replacement technique for a more comprehensive evaluation of the multiple myeloma clone.

The Pliancy of Plasma Cell Differentiation Status Conceals a Gradient of Chromosomal Instability in Newly Diagnosed Multiple Myeloma Patients

IntroductionChromosomal instability (CIN) is a driving force of myelomagenesis: the continuous modification of plasma cell (PC) genomes favours the acquisition of progressively more DNA alterations, clonal evolution and heterogeneity, thereby promoting tumour development. Similarly, the pliancy of Multiple Myeloma (MM) plasma cells differentiation status acts as adaptive strategy to exogenous stress (e.g. in response to therapy). However, the genomic background that supports any diverse plasma cell differentiation phenotype has not yet been inferred.AimTo correlate the genomic background with the phenotypic plasticity of MM clone(s) at diagnosis, in order to stratify patients (pts) according to both the level of CIN and their PC differentiation stages, and ultimately to evaluate the impact of this stratification on the disease outcome.Patients and MethodsA total of 64 newly diagnosed pts were included in the present study. Nearly all pts (54/64) received a PI-based treatment as a front-line therapy. In each patient, both the BM CD138+/CD38+ PC and CD19+ B cell compartments were characterized by 6-color multiparametric flow citometry analysis, combining CD138-PE, CD38-PE-Cy7, CD20-APC, CD19-APC-Cy7, CD27-FITC, CD45-FITC, CD28-APC, CD44-FITC, CD54-APC, CD81-PerCP-Cy5.5, CD56-APC and SHH-PE, as a functional marker of Hedgehog pathway activation (Miltenyi Biotech). Whole copy number abnormalities (CNAs) characterization of CD138+ purified BM PCs was carried out by SNP array hybridization with Cytoscan HD array (Affymetrix).ResultsAccording to the detected CIN, as described both by total CNAs and portion of genome changed (GC), three major subgroups were identified: the first one, including 21 pts with high CIN (medium tot. CNAs = 550, % GC ≥ 25%); the second one including 25 pts with a intermediate CIN (medium tot. CNAs = 315, % GC = 10-25%) and the third one including 18 pts with low CIN (medium tot. CNAs = 105, % GC ≤ 10%). As expected, in pts with high CIN, more than a quarter of unstable genome was due to the hyperdiploidy; however, they were also characterized by a higher prevalence of high-risk features. Indeed, 1p deletion (FAF1), 16q deletion (WWOX, FANCA) and 17p deletion (TP53) were the most recurrent abnormalities, and almost exclusively associated to high CIN pts (p<.05). A concordance analysis between patients' subgroup reveals that the presence of these three deletions positively correlates with an overall deregulation of other tumour suppressor genes, distributed along the genome, such as RB1, LATS2, TNFAIP3, ST13 and NF1 (p<.05). This particular genomic make-up was also associated to a marked oncogenes amplification on MYC, MYCN, IRF4, EGFR, CCND1 and AKT2 genes (p<.05), which probably reflects their activation.A detailed immunophenotypic analysis of the three subgroups of pts showed that high CIN background mainly characterized mature PCs (17/21 = 81%) (Fig. 1), as described by: a) a significant deregulation of both CD19 and CD81 markers; b) a higher expression of CD28 and CD44, which usually characterized advanced disease stages; c) a reduced expression of CD20, CD27 and CD45, commonly associated to preceding PC differentiation stages (p<.05). Accordingly, considering the MM clone(s) as a mixture of different B-cell lineage populations, the high CIN pts advanced differentiation status was also confirmed by a reduced Hedgehog pathway activation in the CD19+ B cells compartment (median SHH expression CD19-/CD81- vs. CD19+/CD81+: 11,3% vs. 98,1%; p<.05), as well as by a declining of CD138-/low/CD19+/CD20-/CD38high/CD27high plasmablast population, which probably highlighted a more quiescent immature reservoir pool.Finally, the presence of more mature PCs with high CIN characterizes pts carrying baseline clinical features associated to bad prognosis (e.g. PET lesions, k/l ratio, ISS III, β2-microglobulin; p<.05). In addition, these pts tend to obtain high quality response rates (≥CR) to PI induction therapy.ConclusionA high level of genomic complexity correlates with an advanced PCs differentiation stages in newly diagnosed MM patients, and this is lastly associated with a prevalence of poor prognosis features. Chromosomal instability, together with cellular phenotypic pliancy, represents an important, yet poorly defined, mechanism by which MM clone(s) accelerate their own evolution and survival.Acknowledgments: AIRC, AIL, Fondazione Berlucchi. [Display omitted] DisclosuresMartinelli:Ariad/Incyte: Consultancy; Johnson&Johnson: Consultancy; Amgen: Consultancy; Celgene: Consultancy; Pfizer: Consultancy; Roche: Consultancy.

Definition of a Prognostic Risk-Classifier Based on SNPs Array Analysis of Copy Number Alterations in Newly Diagnosed Transplant-Eligible Multiple Myeloma Patients

BACKGROUND. The genomic heterogeneity that characterizes all Multiple Myeloma (MM) patients (pts) is the consequence of an evolutionary process operating during the pre-neoplastic phase of the disease. Whatever genomic event occurs, it accounts for the de-regulated expression of either one of the cyclin D or other key regulators of cell cycle progression. Therefore, a deficient regulation of cell cycle control is considered as a hallmark of MM. Moreover, among the MM genomic heterogeneity, several lesions are recurrent and their presence significantly impacts the prognosis.AIM. To deeply characterize the MM clone of newly diagnosed MM pts by means of a genomic approach, aimed to identify Copy Number Alterations (CNAs) affecting clinical outcomes.PATIENTS AND METHODS. We analyzed 247 pts who were enrolled in the EMN02/HO95 phase III study and were eligible for randomization to receive standard dose intensification therapy with bortezomib-melphalan-prednisone (VMP) (n=93) or high dose intensification therapy with autologous stem cell transplantation (ASCT) (n=154) after a V-based induction. Genomic data were obtained from all the pts, whose highly purified CD138+ BM plasma cells were profiled by SNPs array. Any CNA ≥50kb was evaluated. At a median follow-up of 34 months (m), the estimated progression-free (PFS) and overall survival (OS) was 63% and 85%, respectively.RESULTS. In order to identify genetically homogeneous subgroups of pts, we first dissected the genomic complexity observed in the overall population. As expected, most pts had a complex genomic background, while occasionally few of them (23/247, 9%) carried only one of the following alterations: CN losses (L) on chr. 13q, CN gains (G) on chr.1q, hyperdiploidy and any IgH translocations. Remarkably, these 4 alterations were also the most frequently observed across the overall pt population, and their combination identified 16 subgroups harbouring a progressively more complex genomic profile. This suggests that these 4 alterations - either combined or alone - are both necessary and sufficient to describe the genomic background of the whole population. Indeed, all the other observed CNAs have a lower frequency and were randomly scattered among the 16 subgroups.When the clinical outcomes of these subgroups were analyzed, it was found that those with inferior outcomes carried at the same time both CN-L on chr.13q and CN-G on chr.1q, regardless of others genomic lesions. Based on this, we re-stratified pts according to the absence or presence of 1 or both of these lesions, to define the following 3 groups: group 1 (64 pts), carrying both CN-L on chr.13q and CN-G on chr.1q; group 2 (94 pts), carrying either CN-L on chr.13q or CN-G on chr.1q; group 3 (89 pts), lacking both these lesions.Providing that pts included in these groups were correctly randomized in a 1:1 ratio to receive either VMP or ASCT, we observed that the 34-m estimates of PFS was significantly shorter in group 1 (48%, 61% and 72% respectively; p=0.0021). OS rates were 72% in group 1 vs 88% in group 2 and 91% in group 3 (p=0.0001).PFS hazard ratio (HR) of pts in group 1 (HR=2,51, 95%CI: 1.48-4.25, p=0.0006) was comparable to that of pts carrying either del(17p) (HR=2.09, 95%CI: 1.20-3.65) or t(4;14) (HR=1.84, 95%CI: 1.14-2.99), resulting as an independent factor in predicting PFS in a Cox multivariate analysis (both from del(17p) and t(4;14), p=0.01). PFS benefit correlated with ASCT in comparison with VMP was statistically significant in groups 2 and 3, while only a trend was observed in group 1 (table 1).The genomic background of pts belonging to group 1 was characterized by the presence of several genomic aberrations, which overall severely compromise genes involved in the control of cell cycle progression (Rb1, CKS1B, MDM4, MCL1, genes coding for the DREAM-complex components, YAP1, MYC, FANCA).CONCLUSIONS. Based on an evolutionary rationale, we proposed a simply, CNAs-based risk-classifier, which was able to segregate our cohort of pts with newly diagnosed MM into 3 well-populated groups, thus recapitulating most of the genomic aberrations commonly described in MM. The 3 groups have different clinical outcomes and PFS benefit imparted from ASCT as compared to VMP. According to the observed genomic background, a strong de-regulation of cell cycle control might be responsible of the dismal outcome of pts carrying both CN-L on chr.13 and CN-G on chr. 1q.Supported by AIRC, Fond. del Monte Bo e Ra [Display omitted] DisclosuresLarocca:Janssen: Honoraria; Celgene: Honoraria; Bristol-Myers Squibb: Honoraria; Amgen: Honoraria. Martinelli:Ariad/Incyte: Consultancy; Pfizer: Consultancy; Celgene: Consultancy; Amgen: Consultancy; Johnson&Johnson: Consultancy; Roche: Consultancy. Sonneveld:Celgene, Amgen, Janssen, Karyopharm, Takeda: Consultancy, Honoraria, Research Funding; Celgene Corporation, Amgen, Janssen, Karyopharm, PharmaMar, SkylineDx: Honoraria; Celgene Corporation, Amgen, Janssen, Karyopharm, SkylineDx, PharmaMar: Consultancy.