Normal Plasma Cells Research Articles

Pharmacodynamic Effects of Nirogacestat, a Gamma Secretase Inhibitor, on B-Cell Maturation Antigen in Healthy Participants

Purpose: B-cell maturation antigen (BCMA) is expressed on the cell membrane of normal plasma cells (PCs) and multiple myeloma (MM) cells and is the target of several investigational agents and approved products for the treatment of MM. Low BCMA receptor density may be associated with lower response rates, less durable responses, or resistance to BCMA therapies. Mechanistically, BCMA is cleaved from the cell surface by the enzyme gamma secretase (GS), which results in reduced levels of membrane bound BCMA (mbBCMA) and generation of soluble BCMA (sBCMA). GS inhibitors (GSIs) have been shown to increase mbBCMA, and potentiation of the activity of several BCMA-targeted therapies has been demonstrated in vitro and in clinical studies in combination with GSIs. While the effect of GS inhibition on mbBCMA has been reproducibly characterized in vitro, the effect on BCMA dynamics has yet to be adequately characterized in humans. Therefore, this study sought to evaluate the pharmacodynamics of the GSI nirogacestat on mbBCMA on PCs in healthy participants. Methods: An adaptive design clinical study in healthy participants used quantitative flow cytometry to evaluate mbBCMA receptor density on PCs collected from bone marrow (BM) and whole blood (WB) following administration of nirogacestat. In the first cohort, 9 participants received a single dose of nirogacestat 150 mg, and mbBCMA density on PCs isolated from BM and WB was measured at baseline and 4, 8, 24, or 48 hours post-dose. Changes in the percentage of mbBCMA+ PCs, serum concentrations of sBCMA, and serum concentrations of nirogacestat were also determined. Based on the results of the first cohort, participants were enrolled in a second cohort in which different dose levels, treatment schedules, and post-dose collection time points were included to refine the exposure-response relationship and to better understand the dynamics of mbBCMA. Results: Nirogacestat dosing in healthy participants resulted in a rapid increase in mbBCMA density on PCs isolated from BM and WB. Maximal increases in mbBCMA (8- to 12-fold) were observed from 4 to 8 hours post-dose and returned to baseline levels by 24 to 48 hours. These changes in mbBCMA density correlated with serum concentrations of nirogacestat. The responses in BM and WB were generally similar, but there were some differences in mbBCMA dynamics. The serum nirogacestat concentrations and mbBCMA density values were used to develop a pharmacokinetic/pharmacodynamic (PK/PD) model that describes mbBCMA dynamics relative to GSI concentrations, including receptor density on PCs in both BM and WB. Results of the final PK/PD model will be presented. Conclusions: Administration of a single dose of nirogacestat in healthy participants resulted in a robust but temporary increase in mbBCMA receptor density on PCs in both BM and WB that correlated with nirogacestat serum concentrations, indicating that the turnover of mbBCMA on PCs is rapid and its abundance on the cell surface is directly regulated by GS activity. PK/PD modeling showed that sustained serum concentrations of nirogacestat are necessary for maintaining elevated levels of mbBCMA receptor density, thereby suggesting that the schedule of nirogacestat dosing may have direct implications on the potentiation of BCMA-targeting therapies. This PK/PD study supports the use of nirogacestat to increase mbBCMA levels on myeloma cells in patients receiving BCMA-directed therapy. Ongoing and planned studies are evaluating mbBCMA dynamics with nirogacestat combined with BCMA-targeted treatments in patients with MM.

Delineating CDK9- Regulated Molecular Events for the Development of Rationally Derived Multiple Myeloma Treatment Strategies

Abstract Background: Despite major advances in multiple myeloma (MM) therapy over the last 2 decades, most patients relapse. The identification of novel targets and the development of derived treatment approaches are therefore urgently needed. Aberrant expression of various cyclin-dependent kinases (CDKs) in solid and hematologic malignancies including MM, results in the loss of proliferative control and enhanced survival. The serine-threonine kinase CDK9, a subunit of pTEFb, in particular, is a major transcriptional regulator of numerous oncogenes. Past studies have suggested CDK9 as a potential therapeutic target in MM. However, CDK9-regulated molecular events in MM are only partly understood. By delineating CDK9-dependent pathophysiologic effects, the present study proposes rationally derived anti-CDK9-containing novel MM treatment strategies to improve patient outcome. Methods: Following expression profiling, CRISPR loss-of-function screens and correlation analyses in MM cell line and patient cells, the regulatory impact of CDK9 on downstream target genes was outlined using genomic as well as pharmacological approaches in 2D/3D MM models of the tumor microenvironment. Functionally, CDK9-regulated molecular effects as well as anti-MM activity of anti-CDK9-containing rationally derived treatment combinations were determined by gene arrays, qPCR, flow cytometry, and western blot, proliferation and survival analyses. Results: Strongly suggested by a significant induction of CDK9 mRNA expression levels progressing from normal plasma cells to cells from patients with MGUS, SMM and MM; siRNA and CRISP loss-of-function screens across various MM cell lines verified their dependency on CDK9. Correlative expression levels indicated a functional role of CDK9 (but not for CDK2 and CDK7) on Mcl-1, cMyc, Mdm2, and RNA Pol II, but not other genes (e.g., Bcl-2) in the CCLE as well as CoMMpass and GSE5900/GSE2658 MM patient datasets. Indeed, siRNA-mediated CDK9 silencing decreased protein levels of Mcl-1, cMyc, Mdm2, and RNA Pol II, and consequently tumor cell survival. Similarly, the novel, selective CDK9-directed proteolysis-targeting chimera Thal-sns-032 reduced mRNA/ protein levels of CDK9 as well as of Mcl-1, cMyc, RNA Pol II, and Mdm2, and increased p53 levels in a dose- and time-dependent manner, both in MM cell line and primary cells. In contrast, Thal-sns-032 did not inhibit survival of peripheral blood mononuclear cells (PBMCs) from healthy donors. Furthermore, Thal-sns-032 induced downregulation of CDK9, Mcl-1 and cMyc in MM.1S co-cultured with BMSCs cells. Of note, Thal-sns-032- mediated anti-MM effects were abrogated upon its wash-out. Moreover, knockdown CDK9 by shRNA inhibited proliferation and survival, both in MM tumor cell- and tumor cell/BMSC co-cultures. Rationally derived combination strategies of Thal-sns-032 with venetoclax, navitoclax, Selinexor or Carfilzomib as well as other investigational and established MM therapies induced synergistic anti-MM effects in MM cells or BMSC co-cultures. Conclusion: In summary, by delineating CDK9-regulated molecular events in MM, our studies strongly support the therapeutic role of targeted CDK9-therapy and rationally derived MM combination treatment strategies.

Increased Levels of Circulating Plasma Cells in Patients with Newly Diagnosed Multiple Myeloma Are Independently Associated with Poor Prognosis

The presence of circulating tumor cells (CTCs) has long been suggested as a valuable prognostic biomarker in Multiple Myeloma (MM). Recently, increased baseline CTC levels in patients with newly-diagnosed MM (NDMM) have been associated with adverse outcomes in the GEM2012MENOS65/GEM2014MAIN and FORTE trials. Since January 2017, we have been evaluating the presence of CTCs utilizing Next-generation Flow cytometry (NGF) with a median limit of detection (LOD) reaching 2x10-6. NGF assessment has been performed according to the EuroFlow guidelines for sample preparation, antibodies, cytometer settings and analysis of data. Currently, we have analyzed 525 matched BM and PB samples from NDMM patients and have found clonal plasma cells in 100% of BM and 89.1% (468/525) of PB samples respectively. Patients presented with ISS III/R-ISS III, high-risk cytogenetics, serum β2 microglobulin above median and hemoglobin levels ≤ 8.5 g/dL had increased CTC levels. Inversely, the absence of CTCs or their low prevalence (<10-5) was associated with an increased normal plasma cell compartment within the BM (R2=0.82, P < .0001). No association was detected between the CTC number and the therapeutic response to induction treatment in accordance with previous reports. Nevertheless, over a median follow-up monitoring of 42 months (range: 3-66 months), a running log-rank test with a step increase of 0.1% CTCs, highlighted an optimal cut-off point for PFS risk assessment at the level of 2x10-4. In the multivariable analysis, NDMM patients with CTCs above this cut-off showed a 2.2-fold higher risk of progression which was independent from ISS, induction scheme and baseline cytogenetics (HR: 2.2, 95% CI: 1.24 - 3.28, P< .001). Our large number of matched samples allowed for a phenotypic comparison between BM clonal cells and CTCs. The majority of patients with detectable CTCs (86%) showed a matched phenotypic profile of aberrant plasma cell in the two sites. However, 66 patients showed phenotypic discrepancies based on one of the following patterns: i) all phenotypic subsets were present in both BM and PB but with significantly altered ratios (≥20% of relative prevalence) for at least two concomitant subsets; ii) presence of ≥ 1 phenotypic subsets (with a minimum relative prevalence of 20% of all clonal cells) only in the BM; and iii) presence of ≥ 1 phenotypic subsets only in the PB. Remarkably, patients with phenotypic discrepancies had significantly higher CTC levels than those with a phenotypic agreement on the two sites (P < .001), together with signs of a more diffuse disease pattern, as evidenced by the imaging results. It is likely that these patients may share a patchy BM infiltration (and/or even extramedullary disease) and accumulate circulating clonal cells from various sites (thus leading to increased tumor burden) with a spatial clonal heterogeneity, which, as a total, may differ from a specific phenotype derived from a particular BM site. The biological differences among patients with differential baseline CTCs expand beyond the tumor cells. Performing a deep phenotypic approach for the identification of 32 distinct immune subsets, we analyzed the immune profile of both ΒΜ and PB in NDMM patients differing to their CTC levels by two log decimals. Patients with CTCs ≥ 5x10-3 had substantially increased levels of total T cells (especially CD8+), NK cells, tumor associated macrophages (TAMs) and an elevated ratio of memory to naïve B cells (P < .0001 for all subsets) in their BM microenvironment than those with less than 5x10-5 CTCs. The same differences, though with weaker statistical significance, applied also in PB, where, we could also identify a clear immune signature with characteristics of tumor suppression and T cell exhaustion for NDMM with the high CTC burden. Altogether, our results confirm the negative and independent prognostic impact of increased CTC levels in the NDMM and further imply its relevance in the real-world setting to establish improved risk-stratification models. Moreover, since the liquid biopsy is a better representative of the entire tumor load than a tissue biopsy sample, the analysis of CTCs may serve as the new hallmark for the real-time evaluation of a patient's disease and/or immune status.

Novel Antigens LMA and Kma Are Expressed on Malignant Bone Marrow Plasma Cells from Patients with Relapsed Refractory Multiple Myeloma and Plasma Cell Dyscrasias

Introduction Previous studies have identified the lambda myeloma antigen (LMA) and kappa myeloma antigen (KMA) on the surface of malignant plasma cells in myeloma patient bone marrow samples, human myeloma cell lines and other plasma cell dyscrasias1-3. LMA and KMA are not present on normal B cells and are found on occasional mononuclear cells in tonsillar tissue and secondary mucosal lymphoid tissue. Targeting KMA and LMA through cellular and other immunological therapies has the potential advantage of leaving a population of normal plasma cells untouched and minimising the risk of serious post-treatment infection. Characteristics of the antibodies called KappaMab and LambdaMabs (10B3 and 7F11) that bind to specific conformational epitopes in the constant regions of these surface-bound light chains have been described in clinical trials and in vitro experiments1, 2, 4-6. Here, we present data on a larger series of patients with plasma cell dyscrasias, demonstrating the expression of LMA on plasma cells from patients in whom myeloma evolves from monoclonal gammopathy of unknown significance (MGUS). We also describe the difference in phenotype between LMA and KMA expressing malignant plasma cells and the frequent expression of KMA in relapsed refractory myeloma patient (RRMM) samples. Methods Patient BM samples (κ=49 and λ=31) were analysed using multiparametric FCM immunophenotyping with APC labelled LambdaMab (10B3) and KappaMab Fab'2 fragments, CD38, CD138, CD269 (BCMA), CD319 (SLAM F7), CD56 and CD45 monoclonal antibodies. Plasma cells were identified as previously described by initial gating using CD38 and CD138. LMA and KMA expression was determined and compared with the other cell markers. Results Nine of 12 (75%) newly diagnosed λ-type patients with MGUS or SMM expressed LMA on the surface of BM plasma cells. Longitudinal data from two patients was available. One patient showed LMA expression and antigen density increasing on the surface of bone marrow plasma cells during progression from MGUS (2020) to smoldering myeloma (SMM) (2022) (% of plasma cells positive for LMA rose from 61% to 92%, antigen density increased from 532 to 5164 molecules of PE/cell). In a second individual, LMA expression and antigen density increased on bone marrow plasma cells during progression from SMM (2020) to myeloma (2022) (% of plasma cells positive for LMA rose from 94% to 100%, antigen density increased from 3981 to 8941 molecules of PE/cell). KMA expression continued to be observed on plasma cells in 4 of 6 patients after autologous stem cell transplant. KMA was expressed in 86% of patients with ĸRRMM (n=14). LMA expression was observed in 2 of 4 patients with λRRMM. Both KMA and LMA antigen densities were higher than BCMA in the RRMM population (KMA mean 1802, range 468-7943 vs BCMA mean 1306, range 350-2630 molecules of PE/cell), (LMA mean 2439, range 263-6664 vs BCMA mean 817, range 537-1065 molecules of PE/cell). Conclusions In an enlarged cohort of patients with myeloma, we have confirmed that KMA and LMA are expressed at all stages of disease. Our data examining longitudinal expression in individual patients suggests that expression of LMA on BM plasma cells increases from MGUS to MM. The majority of patients with relapsed refractory kappa myeloma expressed KMA. Further data is required to confirm LMA expression in this population. The antigen density of both LMA and KMA is higher than BCMA on RRMM BM plasma cells. KMA and LMA expression is independent of serum Ig and FLC levels and % marrow plasma cells. These data show that KappaMab and LambdaMab have therapeutic potential in the treatment of myeloma patients, especially in the setting of relapsed refractory disease where the combination of high antigen density and restriction of the target antigen to the malignant subpopulation of plasma cells will confer valuable clinical benefit.

Ultra-Sensitive Assessment of Measurable Residual Disease (MRD) in Peripheral Blood (PB) of Multiple Myeloma (MM) Patients Using Bloodflow

Background: Assessing MRD in bone marrow (BM) using next-generation flow (NGF) or sequencing precludes periodic evaluations because of its invasiveness. MRD assessment in PB could overcome this limitation, but its clinical value is not established and the negative predictive value (NPV) when compared to BM is <70%. This is because tumor burden in PB is ~3log lower than in BM, and methods capable of detecting MRD below 10-6 are thus needed for improved concordance. We first aimed at investigating the prognostic value of MRD assessment in PB using NGF. Our second aim was to develop a new method with increased sensitivity. Methods: MRD was evaluated using NGF in PB of 138 MM patients (pts) enrolled in the GEM2014MAIN trial. PB samples were collected after the second year of maintenance, when pts stopped treatment if MRD negative (in BM), or continued on therapy for three additional years if MRD positive at that time. Reaching a minimum sensitivity of 10-7 requires analyzing ≥ 2x108 cells (~50mL of PB). To avoid high staining costs and impractically long acquisition periods, a new method integrating immunomagnetic enrichment using MACS® MicroBeads prior NGF was developed and coined as BloodFlow. Large (~50mL) PB volumes were magnetically labeled and processed via MACS® columns, and ~100µL aliquots enriched with circulating plasma cells (PC) were analyzed using EuroFlow NGF. The concordance between MRD assessment using BloodFlow (in PB) vs NGF (in PB and/or BM) was analyzed in 389 samples from 351 MM pts. Results: Of the 138 pts enrolled in the GEM2014MAIN trial having MRD assessed in PB, 15 (12%) showed positive MRD. Their median progression-free survival (PFS) since MRD testing was 22 months, which was significantly inferior vs those with undetectable MRD in PB (median not reached, Fig 1A). The respective rates of PFS at two years were 50% and 98% (HR: 11.7; p<.0001). Among the 123 pts with undetectable MRD in PB, 33 (27%) showed persistent MRD in BM and inferior PFS vs those with undetectable MRD in PB and BM. The respective rates of PFS at two years were 62% and 100% (p<.0001). The results from this part of the study confirmed the prognostic value of MRD assessment using NGF in PB, and emphasized the importance of increased sensitivity to reduce the number of false-negative results in PB vs BM. In the second part of the study, an optimized BloodFlow protocol was developed after comparing various lysing methods and MicroBeads combinations for optimal enrichment of circulating PC. Initial testing in PB samples from healthy individuals showed on average an 82-fold increment in the number of circulating normal PC with BloodFlow vsNGF. Dilution experiments with MM cell lines showed detection of up to 1x10-7 tumor cells. The performance of BloodFlow vs NGF in PB was compared in 353 samples collected in different treatment scenarios. BloodFlow detected MRD in 33/353 (9%) samples. The lowest MRD level was 6x10-8. Of 33 positive samples using BloodFlow, 19 (58%) were negative by NGF (Fig 1B). All negative cases according to BloodFlow were also negative by NGF. Subsequently, we compared the performance of BloodFlow vs NGF in 199-paired PB and BM samples. Concordance was observed in 137 (69%) double-negative and 19 (9.5%) double-positive samples. MRD was detected in BM and not in 41 (20.5%) PB paired-samples, while 2 (1%) were negative in BM but positive in PB (both showing MRD reappearance in BM soon after). Thus, BloodFlow showed a NPV of 77% when compared to NGF in BM. MRD assessment during induction and intensification was the feature more frequently associated with a false-negative result using BloodFlow (26/41 [63%]), followed by reduced PB cellularity (15/41 [37%]) and MRD levels < 10-5 in BM (12/41 [29%]). In the GEM2014MAIN trial, 2 of 4 pts with positive MRD in PB using BloodFlow progressed, whereas none of the 29 pts having undetectable MRD relapsed thus far. Conclusion: MRD assessment in PB using NGF was prognostic in pts under maintenance or observation. Notwithstanding, a new method (BloodFlow) was developed to increase the NPV and showed an unprecedented sensitivity to detect MRD down to 10-8 in PB. BloodFlow detected MRD in PB more frequently than NGF, with a consequent decrease in the number of cases with persistent MRD in BM while undetectable in PB, which were more frequent during early and intensive treatment stages. These results suggest the possibility of periodic and ultra-sensitive MRD assessment in PB during maintenance/observation. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Characterization of BCMA Expression in Circulating Rare Single Cells of Patients with Plasma Cell Neoplasms.

B-cell maturation antigen (BCMA), a key regulator of B-cell proliferation and survival, is highly expressed in almost all cases of plasma cell neoplasms and B-lymphoproliferative malignancies. BCMA is a robust biomarker of plasma cells and a therapeutic target with substantial clinical significance. However, the expression of BCMA in circulating tumor cells of patients with hematological malignancies has not been validated for the detection of circulating plasma and B cells. The application of BCMA as a biomarker in single-cell detection and profiling of circulating tumor cells in patients' blood could enable early disease profiling and therapy response monitoring. Here, we report the development and validation of a slide-based immunofluorescence assay (i.e., CD138, BCMA, CD45, DAPI) for enrichment-free detection, quantification, and morphogenomic characterization of BCMA-expressing cells in patients (N = 9) with plasma cell neoplasms. Varying morphological subtypes of circulating BCMA-expressing cells were detected across the CD138(+/-) and CD45(+/-) compartments, representing candidate clonotypic post-germinal center B cells, plasmablasts, and both normal and malignant plasma cells. Genomic analysis by single-cell sequencing and correlation to clinical FISH cytogenetics provides validation, with data showing that patients across the different neoplastic states carry both normal and altered BCMA-expressing cells. Furthermore, altered cells harbor cytogenetic events detected by clinical FISH. The reported enrichment-free liquid biopsy approach has potential applications as a single-cell methodology for the early detection of BCMA+ B-lymphoid malignancies and in monitoring therapy response for patients undergoing anti-BCMA treatments.

ERBB1/EGFR and JAK3 Tyrosine Kinases as Potential Therapeutic Targets in High-Risk Multiple Myeloma.

Our main objective was to identify abundantly expressed tyrosine kinases in multiple myeloma (MM) as potential therapeutic targets. We first compared the transcriptomes of malignant plasma cells from newly diagnosed MM patients who were risk-categorized based on the patient-specific EMC-92/SKY-92 gene expression signature values vs. normal plasma cells from healthy volunteers using archived datasets from the HOVON65/GMMG-HD4 randomized Phase 3 study evaluating the clinical efficacy of bortezomib induction/maintenance versus classic cytotoxic drugs and thalidomide maintenance. In particular, ERBB1/EGFR was significantly overexpressed in MM cells in comparison to normal control plasma cells, and it was differentially overexpressed in MM cells from high-risk patients. Amplified expression of EGFR/ERBB1 mRNA in MM cells was positively correlated with increased expression levels of mRNAs for several DNA binding proteins and transcription factors with known upregulating activity on EGFR/ERBB1 gene expression. MM patients with the highest ERBB1/EGFR expression level had significantly shorter PFS and OS times than patients with the lowest ERBB1/EGFR expression level. High expression levels of EGFR/ERBB1 were associated with significantly increased hazard ratios for unfavorable PFS and OS outcomes in both univariate and multivariate Cox proportional hazards models. The impact of high EGFR/ERBB1 expression on the PFS and OS outcomes remained significant even after accounting for the prognostic effects of other covariates. These results regarding the prognostic effect of EGFR/ERBB1 expression were validated using the MMRF-CoMMpass RNAseq dataset generated in patients treated with more recently applied drug combinations included in contemporary induction regimens. Our findings provide new insights regarding the molecular mechanism and potential clinical significance of upregulated EGFR/ERBB1 expression in MM.

BCMA-targeting chimeric antigen receptor T-cell therapy for multiple myeloma

Multiple myeloma (MM) remains an incurable hematologic malignancy, despite the development of numerous innovative therapies during the past two decades. Immunotherapies are changing the treatment paradigm of MM and have improved the overall response and survival of patients with relapsed/refractory (RR) MM. B cell maturation antigen (BCMA), selectively expressed in normal and malignant plasma cells, has been targeted by several immunotherapeutic modalities. Chimeric antigen receptor (CAR) T cells, the breakthrough in cancer immunotherapy, have revolutionized the treatment of B cell malignancies and remarkably improved the prognosis of RRMM. BCMA-targeting CAR T cell therapy is the most developed CAR T cell therapy for MM, and the US Food and Drug Administration has already approved idecabtagene vicleucel (Ide-cel) and ciltacabtagene autoleucel (Cilta-cel) for MM. However, the development of novel BCMA-targeting CAR T cell therapies remains in progress. This review focuses on BCMA-targeting CAR T cell therapy, covering all stages of investigational progress, including the innovative preclinical studies, the initial phase I clinical trials, and the more developed phase II clinical trials. It also discusses possible measures to improve the efficacy and safety of this therapy.

Abstract 641: Single-cell RNA sequencing of rare circulating tumor cells in precursor myeloma patients reveals molecular underpinnings of tumor cell circulation

Abstract Background: Multiple Myeloma (MM) is a hematological malignancy characterized by abnormal proliferation of terminally differentiated plasma cells (PCs) in the bone marrow (BM). MM is almost always preceded by the precursor stage smoldering multiple myeloma (SMM). BM biopsies are useful to monitor disease progression, but they are invasive and not routinely collected from patients for disease monitoring during precursor stages. Profiling circulating tumor cells (CTCs) from peripheral blood (PB) could aid early detection, disease monitoring, and biomarker identification to predict patients at high risk of progression that may benefit from early therapeutic intervention. Methods: Paired PB and BM aspirates were collected from 40 SMM patients enrolled in the PCROWD study (IRB #14-174) at Dana-Farber Cancer Institute. Malignant PCs were enriched by magnetic bead-based methods and underwent 5’ single-cell RNA sequencing (scRNA-seq) and single-cell B-cell receptor sequencing (scBCR-seq) (10x Genomics). Results: We analyzed 105,246 BM PCs and 33,234 PB PCs from 15 patients. To differentiate malignant from normal PCs, we used clonal V(D)J rearrangements, assessed by concurrent scBCR-seq. A total of 86,986 BM tumor cells and 8,718 CTCs were captured. A median of 5, 26, and 47 CTCs were present per mL of blood from low, intermediate, and high-risk SMM patients as defined by the International Myeloma Working Group (IMWG) “20/2/20” criteria, suggesting sequencing-based CTC enumeration corresponds to prognosis. High levels of driver genes commonly upregulated in patients with specific translocations, including CCND1 and MAF, were detected in both BM tumor and CTC clusters in 3 patients with t(11;14) and t(14;16) confirmed by fluorescence in situ hybridization (FISH) clinical testing, and 2 additional patients with inconclusive FISH results (Wilcoxon, q &amp;lt;10-3), supporting the idea of CTC-based prognostication. Differential expression (DE) analysis revealed 8 genes that were significantly upregulated and 3 genes that were significantly downregulated in CTCs compared to BM tumor cells robustly across 15 paired samples. Gene set enrichment analysis (GSEA) revealed genes DE in CTCs are associated with TNF-α and NF-κB signaling, which are commonly induced by extrinsic factors in the bone marrow milieu, providing insight into the biology of tumor cell circulation. Conclusions: This study highlights the utility of scRNA-seq for molecular profiling of CTCs, even in asymptomatic low tumor burden disease. Additional analyses are ongoing in the expanded cohort of 40 patients with paired samples to help gain further insight into CTC heterogeneity. Overall, this study will help enable the design of new molecular liquid biopsy-based approaches to diagnosis, disease monitoring, and biological insights to improve treatment strategies for precursor myeloma patients. Citation Format: Elizabeth D. Lightbody, Danielle T. Firer, Romanos Sklavenitis-Pistofidis, Michael Agius, Ankit K. Dutta, Michelle Aranha, Jean-Baptiste Alberge, Laura Hevenor, Nang Kham Su, Cody Boehner, Erica Horowitz, Jacqueline Perry, Anna Cowan, Hadley Barr, Anna Justis, Daniel Auclair, Catherine R. Marinac, Gad Getz, Irene Ghobrial. Single-cell RNA sequencing of rare circulating tumor cells in precursor myeloma patients reveals molecular underpinnings of tumor cell circulation [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 641.

Abstract 2291: microRNA/mRNA integrated analysis of multiple myeloma transcriptomics

Abstract Myeloma is plasma cell disorder, mostly effects adults over 60 years. Non-coding RNAs are emerging field and play vital role in development of disease. Major non-coding RNAs are miRNAs, lncRNAs, circRNAs and sn/snoRNAs. We analyzed restricted and publically available RNA-seq and small RNA-seq data sets for biomarkers identification and their involvement in myeloma. We obtained restricted data from “BluePrint”, which contains 11 myeloma plasma cells and 4 normal tonsil plasma cells (EGAS00001001110). We identified 1534 genes are differentially regulated (2-fold cut-off, &amp;gt;10-FC 218 genes). Top 10 upregulated genes were: EDNRB (1246-FC), SCUBE1 (737-FC), MC4R (691-FC), NDNF (601-FC), PTGS2 (567-FC), GPRC5D (531-FC), MFAP3L (467-FC), CCND1 (381-FC), CXCL12 (344-FC) and BTBD3 (326-FC) ; top 10 downregulated were: EBF1 (-111-FC), HLA-DRB1(-133-FC), CPXM1 (-171-FC), LOC642131 (-171-FC), IGHV1OR15-3 (-171-FC), HLA-DRB5 (-204-FC), PRAMENP (-210-FC), DTX1 (-220-FC), CD22 (-337-FC) and RFTN1 (-356-FC). Publically available small RNA-seq data downloaded and analyzed for miRNAs, lncRNAs, circRNAs and sn/snoRNAs which contains 3 healthy donor’s plasma cells and 3 newly diagnosed myeloma patient plasma cells (PRJNA377345). We used mirDIP portal to analyze miRNA and mRNA differentially expressed data, predicated from more than 13 databases showed major role of miR-152-3p (targets 28 mRNAs), miR-93-5p (targets 19 mRNAs), miR-301a-3p (targets 13 mRNAs), miR-29c-3p (targets 12 mRNAs), and miR-144-3p (targets 9 mRNAs). Integrated analysis can provide valuable information from the transcriptomics data and effect of miRNAs on mRNAs. Citation Format: Srinivas V. Koduru. microRNA/mRNA integrated analysis of multiple myeloma transcriptomics [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 2291.

Abstract 2298: OTUD6B is a dependency in multiple myeloma that drives S-phase entry via MYC activation

Abstract Multiple myeloma (MM) is the second most common hematological malignancy and remains incurable, thus demanding for new therapeutic targets. While the pathophysiology of MM is poorly understood, the substantial responsiveness of MM patients to proteasomal inhibitors (PIs) like bortezomib or carfilzomib hints towards a central role of the ubiquitin proteasome system (UPS). Deubiquitylases (DUBs) are therapeutically targetable components of the UPS, whose inhibition can destabilize oncoproteins. However, the identities of oncoprotein-regulating DUBs remain largely elusive. To identify new vulnerabilities in MM, a CRISPR/Cas9 screen targeting all human DUBs was performed. For validated candidates, phenotypical analysis regarding proliferation and cell cycle progression was performed, as well affinity and non-affinity mass spectrometry-based screens to identify substrates. We thereby identified OTUD6B as a novel oncogene that drives G1/S-transition. LIN28B, a suppressor of microRNA biogenesis, was delineated as both a cell cycle-specific deubiquitylation substrate and activator of OTUD6B. RNA-Seq and qPCR analyses of OTUD6B and LIN28B depleted MM cells revealed that the stabilization of LIN28B drives MYC expression and activity at the G1/S transition, which in turn allows for rapid S-phase entry. Thus, silencing of OTUD6B as well as LIN28B inhibited MM outgrowth in xenograft experiments. Analyses of large MM patient cohorts revealed a progressive increase of OTUD6B expression along the transition from normal plasma cells to MGUS to MM and that high expression of OTUD6B was associated with a significantly adverse overall survival. Furthermore, OTUD6B expression was found to strongly correlate with MYC expression and significantly reduced progression-free survival in patients treated with the PI bortezomib. Knockout of OTUD6B in MM cells significantly enhance the anti-myeloma activity of the drug when using sub-lethal doses. Together, these results validate OTUD6B as a new therapeutically targetable oncogene, dependency, and prognostic factor in MM, that eventually serves as a master regulator of MYC activity to drive cell cycle progression. Citation Format: Ria Spallek, Carmen Paulmann, Oleksandra Karpiuk, Jana Zecha, Susan Klaeger, Isabell Schaeffer, Rupert Öllinger, Thomas Engleitner, Jan Krönke, Matthias Wirth, Ullrich Keller, Roland Rad, Bernahrd Kuster, Florian Bassermann. OTUD6B is a dependency in multiple myeloma that drives S-phase entry via MYC activation [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 2298.

69-Year-Old Man With Dysuria and Right Lower Abdominal Pain

69-Year-Old Man With Dysuria and Right Lower Abdominal Pain

MO173: Clinical Profile of Plasma Cell Dyscrasias and Their Renal Outcomes

Abstract BACKGROUND AND AIMS The involvement of kidney in plasma cell dyscrasias is widespread, often referred to as myeloma kidney. At the time of presentation, nearly 50% of patients have renal involvement which is associated with higher mortality [1, 2]. Multiple myeloma is more common in African Americans, with male predilection and median age about 65–70 years [3]. Two major causes of renal insufficiency are light chain cast nephropathy and hyperkalcaemia [4]. Flow cytometry plays an important role in diagnosis of plasma cell dyscrasias. Myeloma cells infrequently express CD19 and variable CD45 in contrast to normal plasma cells. Approximately 70% of myeloma cells will express CD56, which is typically negative in normal plasma cells and in plasma cell leukaemia [5]. Studies addressing the relation of flow cytometry parameters with respect to prognosis and outcome in plasma cell dyscrasias are sparse in Asian population. Hence, this research was conducted to study the clinical profile and correlation of flow cytometry parameters with renal outcomes in patients with plasma cell dyscrasias. METHOD All consecutive patients, aged above 18 years and diagnosed with plasma cell dyscrasias from January 2016 to June 2021 were included. RESULTS Of 165 patients, 115 were males (69.7%), with mean age 58.2 ± 6.4 years. Pallor was seen in 126 (76.4%), bone pains in 90 (54.5%), lytic lesions in 63 (38.2%) and fractures in 37 (22.4%) patients. Severe anaemia (&amp;lt;7 g/ dL) was seen in 35 (21.2%) patients. At presentation, deranged renal parameters with eGFR &amp;lt; 60 mL/min/1.73 m2 were seen in 89 (53.9%) patients, of which 55 (61.8%) patients progressed to chronic kidney disease and 8 (8.9%) patients were continued on maintenance hemodialysis. Nephrotic syndrome presentation was seen in 26 (15.8%) patients. Infections were seen in 65 (39.4%) patients, of which most common infections were respiratory (25, 38.5%), blood stream (18, 27.7%) and urinary tract (12, 18.5%) infections. Serum free light chains were more than 800 mg/L in 71 patients, of them high kappa and lambda levels are seen in 41 (24.8%), and 30 (18.2%) patients, respectively. Most common immunoglobulin seen was IgG (63.6%), followed by IgA (15.8%) and IgE (2.4%). Flow cytometry parameters are shown in Table 1. Remission was attained with chemotherapy in 139 patients (64.3%), and bone marrow transplantation was done in 27 patients (16.4%). Relapse of disease was seen in 31 patients (18.8%) and worsening of renal parameters at relapse was seen in 15 (48.4%) patients. A total of 35 (21.2%) patients expired. Correlation of flow cytometry parameters with renal outcomes showed presence of CD56 with worse renal outcomes (P = .032), and presence of CD117 with favourable renal outcomes (P = .003) with statistical significance. Presence of CD56 and CD138 had increased risk of mortality with significant P value (P = .04, P = .01, respectively). CONCLUSION Flow cytometry parameters may play a role in predicting the prognosis and renal outcomes in patients of plasma cell dyscrasias. Our study showed presence of CD56 with worse renal outcome and mortality, while CD117 presence with favourable renal outcome. Table 1. Flow cytometry parameters in patients with plasma cell dyscrasias CD markers (n = 165) Present Absent CD19 21/165 (12.7%) 144/165 (87.3%) CD38 53/165 (32.1%) 112/165 (67.9%) CD45 46/165 (27.9%) 119/165 (73.9%) CD56 134/165 (81.2%) 31/165 (18.8%) CD117 86/165 (52.1%) 79/165 (48.9%) CD138 46/165 (27.9%) 119/165 (73.9%) KAPPA 93/165 (56.4%) 72/165 (44.6%) LAMBDA 71/165 (43%) 94/165 (57%)

Long-term CR Multiple Myeloma Patients Show Cured or MGUS-like Minimal Residual Disease Pattern by Next Generation Flow.

In the era of novel agents, many multiple myeloma patients can achieve a complete remission, but most of them relapse, and minimal residual disease detection can play a crucial role. Next-generation flow (NGF) can detect monoclonal plasma cells with a sensitivity of 10-6. Little is known about long-term remission patients (> 2 years) and in particular, if more sensitive techniques such as NGF can still detect minimal disease in those patients. Aim of the study was to analyze patients with MM in response to NGF at > 2 years of sustained remission after several treatments. MRD was studied by NGF in bone marrow aspirates according to Euroflow Consortium indications. 62 patients with sustained CR at >2 years were studied, MRD+ status was detected at a threshold cut-off of 10-6 in 32/62 (52%); 4/15 (27%) patients were MRD positive at >5 years of remission and they displayed a prevalence of normal vs abnormal monoclonal plasma cell immune-phenotype (MGUS-like). NGF is a powerful technique to detect MRD. Myeloma patients in prolonged sustained complete remission can show in high percentage an MRD negative status or MGUS like.

Enrichment-Free Single-Cell Detection and Morphogenomic Profiling of Myeloma Patient Samples to Delineate Circulating Rare Plasma Cell Clones.

Multiple myeloma is an incurable malignancy that initiates from a bone marrow resident clonal plasma cell and acquires successive mutational changes and genomic alterations, eventually resulting in tumor burden accumulation and end-organ damage. It has been recently recognized that myeloma secondary genomic events result in extensive sub-clonal heterogeneity both in localized bone marrow areas and circulating peripheral blood plasma cells. Rare genomic subclones, including myeloma initiating cells, could be the drivers of disease progression and recurrence. Additionally, evaluation of rare myeloma cells in blood for disease monitoring has numerous advantages over invasive bone marrow biopsies. To this end, an unbiased method for detecting rare cells and delineating their genomic makeup enables disease detection and monitoring in conditions with low abundant cancer cells. In this study, we applied an enrichment-free four-plex (CD138, CD56, CD45, DAPI) immunofluorescence assay and single-cell DNA sequencing for morphogenomic characterization of plasma cells to detect and delineate common and rare plasma cells and discriminate between normal and malignant plasma cells in paired blood and bone marrow aspirates from five patients with newly diagnosed myeloma (N = 4) and monoclonal gammopathy of undetermined significance (n = 1). Morphological analysis confirms CD138+CD56+ cells in the peripheral blood carry genomic alterations that are clonally identical to those in the bone marrow. A subset of altered CD138+CD56- cells are also found in the peripheral blood consistent with the known variability in CD56 expression as a marker of plasma cell malignancy. Bone marrow tumor clinical cytogenetics is highly correlated with the single-cell copy number alterations of the liquid biopsy rare cells. A subset of rare cells harbors genetic alterations not detected by standard clinical diagnostic methods of random localized bone marrow biopsies. This enrichment-free morphogenomic approach detects and characterizes rare cell populations derived from the liquid biopsies that are consistent with clinical diagnosis and have the potential to extend our understanding of subclonality at the single-cell level in this disease. Assay validation in larger patient cohorts has the potential to offer liquid biopsy for disease monitoring with similar or improved disease detection as traditional blind bone marrow biopsies.

&lt;i&gt;MAGE-C1&lt;/i&gt; gene and mage-c1 protein expression comparison in primary multiple myeloma patients

Background . Nowadays, hematology is a dynamically developing science due to the in-depth study of the molecular mechanisms of a particular disease. A better understanding of oncohematological diseases biology makes it possible to synthesize new targeted drugs, which have a favorable therapeutic effect. In particular, in multiple myeloma, after the introduction of proteasome inhibitors and immunomodulatory drugs into clinical practice, an improvement in overall survival was observed. However, characteristics of the mechanisms of transformation normal plasma cells into malignant ones are still difficult; therefore, the study of the pathobiological basis of multiple myeloma is currently an urgent task. The objective : to evaluate the possible influence of MAGE-C1 gene expression and the presence of mage-c1 protein in patients with newly diagnosed multiple myeloma on the anti-tumor response after bortezomib-containing therapy. Materials and methods . A prospective study included 33 multiple myeloma patients. The diagnosis was established according to International Myeloma Working Group criteria (IMWG, 2014). In 32 patients the induction therapy included bortezomib-containing courses, in one patient lenalidomide was included in the first-line regimens. The MAGE-C1 gene expression by real-time polymerase chain reaction and mage-c1 protein by immunohistochemistry in plasma cells bone marrow, were determined for all patients at the debut of multiple myeloma. As a control group was examined the bone marrow material of healthy donors. Results . When assessment the statistical relationship between the expression of MAGE-C1 gene and mage-c1 protein, it was found that there was no high expression of mage-c1 protein at low values of MAGE-C1 gene expression. At the same time, high expression of the gene was always associated with protein expression above normal values. The analysis aimed at finding the relationship between MAGE-C1 gene and mage-c1 protein detection and the degree of antitumor response after 6 courses of induction therapy showed that high expression of the studied parameters was associated with a worse response to bortezomib-containing treatment. Conclusion . We confirmed that the results of the two methods were comparable. Single factor analysis showed that patients with decreased MAGE-C1 gene and mage-c1 protein expression levels achieved a significantly higher antitumor response to bortezomib-containing regimens, while high expression was accompanied by refractoriness to bortezomib.

Relevance of polyclonal plasma cells and post-therapy immunomodulation in measurable residual disease assessment in multiple myeloma.

Immunophenotypic profile and post-therapy alteration in antigenic expression were evaluated in normal, reactive, and aberrant plasma cells (NPC, RPC, and APC) for impact on measurable residual disease (MRD) assessment in multiple myeloma (MM). Samples from non-MM staging marrow (n =30), Hodgkin's lymphoma (n =30) and MM (n =724) were prospectively evaluated for expression profiles of NPC, RPC, and APC using antigens recommended in consensus guidelines. Polyclonal NPC-RPC demonstrated aberrations for all antigens evaluated with a higher frequency of aberrancies in post-therapy samples compared to treatment naïve samples (p <0.001%). Immunomodulation in APC was observed in 79% of post-therapy samples with a change in expression of 1, 2, and ≥3 antigens in 19.9%, 15.6%, and 43.5% samples, respectively. In 13.4% of samples, APC showed no aberrancy and aberrant status was assigned based on cytoplasmic light chain restriction (cyLCR) alone. 9% samples with an admixture of NPC and APC displayed normal cytoplasmic kappa to lambda ratio (cyKLR) when the percentage of APC of total PC (neoplastic plasma cell index, NPCI), was below 25% and 50% for kappa and lambda restricted cases, respectively. The panorama and high frequency of antigenic aberrations on polyclonal PC signify the importance of MRD assay validation on a large cohort under normal and reactive conditions. Frequent Immunophenotypic shifts in APC re-confirm the redundancy of baseline immunophenotype for MRD evaluation. Small clones of APC may be missed by assessment of cyKLR alone and therefore, surface marker aberrancy supported by cyLCR is required for definitive assignment of residual APC.

Redefining CD56 as a Biomarker and Therapeutic Target in Multiple Myeloma.

Multiple myeloma is an incurable, genetically heterogeneous disease, without available tailored therapeutic approaches. CD56 signaling promotes multiple myeloma growth and adhesion, by activating CREB1 target genes, MCL1 and BCL2. Inhibition of CREB1 alone or in combination with lenalidomide is an unexplored synthetic lethal approach in CD56-expressing patients with multiple myeloma.

Understanding the Bioactivity and Prognostic Implication of Commonly Used Surface Antigens in Multiple Myeloma.

Multiple myeloma (MM) progression is dependent on its interaction with the bone marrow microenvironment and the immune system and is mediated by key surface antigens. Some antigens promote adhesion to the bone marrow matrix and stromal cells, while others are involved in intercellular interactions that result in differentiation of B-cells to plasma cells (PC). These interactions are also involved in malignant transformation of the normal PC to MM PC as well as disease progression. Here, we review selected surface antigens that are commonly used in the flow cytometry analysis of MM for identification of plasma cells (PC) and the discrimination between normal and malignant PC as well as prognostication. These include the markers: CD38, CD138, CD45, CD19, CD117, CD56, CD81, CD27, and CD28. Furthermore, we will discuss the novel marker CD24 and its involvement in MM. The bioactivity of each antigen is reviewed, as well as its expression on normal vs. malignant PC, prognostic implications, and therapeutic utility. Understanding the role of these specific surface antigens, as well as complex co-expressions of combinations of antigens, may allow for a more personalized prognostic monitoring and treatment of MM patients.

A novel protein encoded by circHNRNPU promotes multiple myeloma progression by regulating the bone marrow microenvironment and alternative splicing

BackgroudMultiple myeloma (MM) is an incurable plasma cell malignancy in the bone marrow (BM), while immunoglobulin D type of MM (IgD MM) is a very rare but most severe subtype in all MM cases. Therefore, systemic study on IgD MM is purposeful to disclose the recurrent and refractory features in both IgD and other types of MM, and beneficial to the development of potent therapeutic strategy on MM.MethodsAgilent SBC-ceRNA microarray chips were employed to examine 3 normal plasma cell samples (NPCs), 5 lgD MM samples and 5 lgG MM samples, respectively. Sanger sequencing, RNase R digestion and qPCR assays were used to detect the existence and expression of circHNRNPU. BaseScope™ RNA ISH assay was performed to test circHNRNPU levels in paraffin-embedded MM tissues. The protein encoded by circHNRNPU was identified by LC-MS/MS, which was named as circHNRNPU_603aa. The function of circHNRNPU_603aa on cellular proliferation and cell cycle was assessed by MTT test, colony formation assay, flow cytometry and MM xenograft mouse model in vivo. RIP-seq, RIP-PCR and WB analysis for ubiquitination were performed to explore the potential mechanism of circHNRNPU_603aa in MM. Exosomes were isolated from the culture supernatant of MM cells by ultracentrifugation and characterized by Transmission Electron Microscope and WB confirmation of exosomes markers Alix and CD9.ResultsCircHNRNPU was one of the top most abundant and differentially expressed circRNA in IgD MM relative to lgG and NPCs samples. Increased circHNRNPU was associated with poor outcomes in four independent MM patient cohorts. Intriguingly, MM cells secreted circHNRNPU, which encoded a protein named as circHNRNPU_603aa. Overexpressed circHNRNPU_603aa promoted MM cell proliferation in vitro and in vivo, in contrast knockdown of circHNRNPU_603aa by siRNA abrogated these effects. Due to circHNRNPU_603aa including RNA-binding RGG-box region, it regulated SKP2 exon skipping, thereby competitively inhibited c-Myc ubiquitin so as to stabilize c-Myc in MM. MM cells secreted circHNRNPU through exosomes to interfere with various cells in the BM microenvironment.ConclusionOur findings demonstrate that circHNRNPU_603aa is a promising diagnostic and therapeutic marker in both MM cells and BM niche.