Abstract: Risk of developing myelodysplastic syndrome (MDS) is significantly increased in both multiple myeloma (MM) and monoclonal gammopathy of undetermined significance, suggesting that it is therapy independent. However, the incidence and sequelae of dysplastic hematopoiesis at diagnosis are unknown. Here, we used multidimensional flow cytometry (MFC) to prospectively screen for the presence of MDS-associated phenotypic alterations (MDS-PA) in the bone marrow of 285 patients with MM enrolled in the PETHEMA/GEM2012MENOS65 trial (#NCT01916252). We investigated the clinical significance of monocytic MDS-PA in a larger series of 1252 patients enrolled in 4 PETHEMA/GEM protocols. At diagnosis, 33 (11.6%) of 285 cases displayed MDS-PA. Bulk and single-cell–targeted sequencing of MDS recurrently mutated genes in CD34+ progenitors (and dysplastic lineages) from 67 patients revealed clonal hematopoiesis in 13 (50%) of 26 cases with MDS-PA vs 9 (22%) of 41 without MDS-PA; TET2 and NRAS were the most frequently mutated genes. Dynamics of MDS-PA at diagnosis and after autologous transplant were evaluated in 86 of 285 patients and showed that in most cases (69 of 86 [80%]), MDS-PA either persisted or remained absent in patients with or without MDS-PA at diagnosis, respectively. Noteworthy, MDS-associated mutations infrequently emerged after high-dose therapy. Based on MFC profiling, patients with MDS-PA have altered hematopoiesis and T regulatory cell distribution in the tumor microenvironment. Importantly, the presence of monocytic MDS-PA at diagnosis anticipated greater risk of hematologic toxicity and was independently associated with inferior progression-free survival (hazard ratio, 1.5; P = .02) and overall survival (hazard ratio, 1.7; P = .01). This study reveals the biological and clinical significance of dysplastic hematopoiesis in newly diagnosed MM, which can be screened with moderate sensitivity using cost-effective MFC.

Abstract: Background Bone disease (BD) is a hallmark of multiple myeloma (MM) and is characterized by severe skeleton damage, reduced quality of life and overall survival (1-2). Several findings indicated that IL-17 producing CD4+ T cells (Th17) play a central role in triggering MMBD and support MM cell growth mainly by IL-17 production. There is compelling evidence that miR-21 is a central player in Th17 effector functions. Our preliminary data have shown that miR-21 is highly upregulated in MM-Th17 isolated from patients with active BD as compared to MM with no active BD and controls. We found that inhibition of miR-21 in naive T cells (miR-21i-T cells) impaired differentiation towards Th17 in vitro, by reducing interleukin (IL)-17, IL-22, RANKL and RORC, leading to abrogation of osteoclast (OCL) bone resorption. Aims Based on these premises, we sought to explore miR-21 related underlying molecular networks that support pathogenic Th17 differentiation and function. As miRNAs may exert direct and indirect effects on gene expression and at post-transcriptional level, we performed a global head-to-head comparison by RNA-seq and proteomic -phosphoproteomic analysis on miR-21i-Th17. Then, we recapitulated and validated our findings in NOD/SCID gNULL mice, injected intratibially with miR-21i-T cells and MM cells. Methods RNAseq and proteomic/phosphoproteomic assays have been performed on in vitro differentiated Th17 cells originated from scramble control (SC) or miR-21i transfected naïve T cells (SC-Th17 and miR-21i-Th17 respectively) from 3 healthy donors through MARS-seq protocol adapted for bulk RNA and proteome/phosphoproteome analysis . Data have been analyzed through R by using different packages including limma, DESEQ2 and pheatmap. To perfom global proteome/phosphoproteome analysis, we conducted a mass spectrometry study of phosphopeptides protein extract from SC-Th17 and miR-21i-Th17, enriched using SCX-IMAC/TiO2. High-resolution LC-Ms/MS data were processed using Proteome Discoverer software Results In the presence of miR-21i, we found 109 upregulated and 22 downregulated proteins in the global proteome analysis of Th17 cells, while 90 and 18 phosphoproteins were up and down modulated, respectively. Paired analysis showed that 46 proteins are modulated in expression but not in phosphorylation, 23 proteins are modulated in phosphorylation but not in expression, while 85 proteins are modulated in both conditions. These data suggest that selective miRNA modulation interferes with a specific and limited group of proteins/phosphoproteins according to cell type and despite predicted pleiotropic miRNA activity. To understand whether miR-21i-Th17 undergo a "molecular reprogramming", we evaluated gene expression by RNA seq Analysis of miR-21-related molecular pathways in Th17 cells and found upregulation of STAT-1/-5a-5b, downregulation of STAT-3 and redirection of Th17 to Th1/activated like cells as shown by a pair-to-pair RNAseq and proteome/phosphoproteome analysis. These data indicate that miR-21 plays a central role in driving Th17 differentiation and function in a proinflammatory milieu such as MM-Bone marrow microenvironment (BMM). However, when miR-21 activity is strongly counteracted, pathogenic Th17 can switch to a Th1 like phenotype (STAT 1 dependent gene/protein upregulation). This switch may partly explain the attenuation of MMBD observed in vitro. To confirm our observation in vivo, we injected intratibially miR-21i exposed- or scramble miR (SC) exposed-naïve CD4+ T cells together with MM cells into gamma null SCID mice. We observed that mice injected with SC CD4+ naïve T cells presented severe local skeleton damage, while bone structure was preserved in miR-21i naïve CD4+ T cells injected mice. Conclusions Our data highlight the relevance of miR-21 in supporting Th17 mediated MMBD onset and progression. The possibility to "reprogram" MM Th17 by miR-21 modulation opens a new avenue to develop miR-21 targeting therapeutic strategies to counteract BMM-dependent MM development and related-BD. Figure Disclosures Paiva: Amgen, Bristol-Myers Squibb, Celgene, Janssen, Merck, Novartis, Roche, and Sanofi; unrestricted grants from Celgene, EngMab, Sanofi, and Takeda; and consultancy for Celgene, Janssen, and Sanofi: Consultancy, Honoraria, Research Funding, Speakers Bureau.

Abstract: In MM patients relapsing after MRD-negativity, the disease could reemerge from immature cells or from undetectable MRD. However, it remains unknown if immature cells have the same genetic background as MM plasma cells (PCs), as well as the amount of MRD that persists below the limit of detection (LOD) of next-generation techniques. To obtain further insight, we compared the biological landscape of MM PCs at diagnosis to that of CD34 progenitors, B cells and normal PCs isolated from patients with negative MRD by next-generation flow (NGF) after treatment. We performed whole-exome sequencing (WES, mean depth: 90x) with the 10XGenomics Exome Solution for low DNA-input as well as deep NGS of B-cell receptor immunoglobulin (BcR IG) gene rearrangements (mean, 69,975 sequences), in a total of 68 cell-samples isolated from the bone marrow (BM) of 7 MM patients with MRD-negativity by EuroFlow NGF after induction with VRD and auto-transplant (GEM2012MENOS65 trial). Patients with negative MRD were intentionally selected to avoid contamination with MM PCs during sorting of CD34 progenitors, B-cell precursors, mature B cells and normal PCs after induction and transplant. We investigated in these populations the presence of somatic mutations and clonotypic BcR Ig rearrangements detectable in MM PCs sorted at diagnosis, using peripheral blood T cells as germline control. We also performed WES in matched diagnostic MM PCs and MRD cells persisting after VRD induction in 14 cases as control. In another 6 patients with untreated MM, we performed single-cell RNA and BcR IG sequencing (scRNA/BcRIGseq) of total BM B cells and PCs (n=16,380) to investigate before treatment, if the clonotypic BcR IG sequence of MM PCs was detectable in other B cell stages defined by their molecular phenotype. We used multidimensional flow cytometry (MFC) to investigate the frequency of B cell clonality in BM samples from a larger series of 195 newly-diagnosed MM patients, prospectively enrolled in the GEM-CLARIDEX trial. Somatic mutations present in diagnostic MM PCs were detectable in the lymphopoiesis of 5/7 patients achieving MRD-negativity after treatment. In one case, out of 55 mutations present in diagnostic MM PCs, a single mutation in PCSK1N (VAF: 0.30) was detectable in normal PCs. In the other four patients, a total of 85 mutations were present in MM PCs and up to 10 (median VAF, 0.16) were found all the way from CD34 progenitors into B-cell precursors, mature B cells and normal PCs, but not in T cells. Of note, most mutations were reproducibly detected in each cell type after induction and after transplant. All somatic mutations shared by MM PCs and normal cells were non-recurrent, and genes recurrently mutated in MM (eg. ACTG1, ATM, DIS3, FAM46C, KRAS, LTB, MAX, TRAF3) were found in MM PCs but never in normal cells. Copy number alterations (CNA) were found only in MM PCs. By contrast, up to 513/827 (62%) mutations and 48/67 (72%) CNA were detectable in matched diagnostic MM PCs and persistent MRD cells, indicating that the few somatic variants present in normal cells were unlikely related to contaminating MRD below NGF's LOD. Accordingly, MM clonotypic BcR IG rearrangements were detectable in normal PCs (4/7patients) and in immature B cells (5/7 patients) but at much lower frequencies (mean of 0.02% in both). Of note, 9 additional clonotypes (mean 8.4%) were found in MM PCs of 5/7 patients (range, 1-3). scRNR/BcRIGseq unveiled that clonotypic cells were confined mostly but not entirely within PC clusters, and that in 1 patient another clonotype was detectable in mature B cells. Accordingly, using MFC we found in a larger series that 25/195 (13%) of newly-diagnosed MM patients display B-cell clonality (median of 0.7% BM clonal B cells, range 0.02%-6.3%). In conclusion, we show for the first time that MM patients bear somatic mutations in CD34 progenitors that specifically differentiate into the B cell lineage, likely before the disease onset. Because diagnostic, MRD (and relapse) MM PCs display great genetic similarity, these results suggest that undetectable MRD & lt;10-6 rather than normal cells with a few non-recurrent mutations are responsible for relapses after MRD-negativity. This study also challenges our understanding of myelomagenesis and clonal heterogeneity, and proposes that mutated lymphopoiesis may increase risk of developing B cell and PC oligoclonality, which precedes secondary driver mutations or CNA leading to the expansion of MM PCs. Disclosures Puig: Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Takeda, Amgen: Consultancy, Honoraria; The Binding Site: Honoraria; Janssen: Consultancy, Honoraria, Research Funding. Martinez-Lopez:BMS: Honoraria, Other: Advisory boards; Janssen: Honoraria, Other: Advisory boards and Non-Financial Support ; Amgen: Honoraria, Other: Non-Financial Support ; Celgene: Honoraria, Other: Advisory boards and Non-Financial Support ; Incyte: Honoraria, Other: Advisory boards; Novartis: Honoraria, Other: Advisory boards; VIVIA Biotech: Honoraria; F. Hoffmann-La Roche Ltd: Honoraria. Lahuerta:Takeda, Amgen, Celgene and Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees. Rosinol Dachs:Janssen, Celgene, Amgen and Takeda: Honoraria. Bladé:Jansen, Celgene, Takeda, Amgen and Oncopeptides: Honoraria. Mateos:EDO: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; GSK: Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Adaptive: Honoraria; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pharmamar: Membership on an entity's Board of Directors or advisory committees. San-Miguel:Amgen, Bristol-Myers Squibb, Celgene, Janssen, MSD, Novartis, Roche, Sanofi, and Takeda: Consultancy, Honoraria. Paiva:Amgen, Bristol-Myers Squibb, Celgene, Janssen, Merck, Novartis, Roche, and Sanofi; unrestricted grants from Celgene, EngMab, Sanofi, and Takeda; and consultancy for Celgene, Janssen, and Sanofi: Consultancy, Honoraria, Research Funding, Speakers Bureau.

Abstract: Granulocytic myeloid-derived suppressor cells (G-MDSCs) promote tumor growth and immunosuppression in multiple myeloma (MM). However, their phenotype is not well established for accurate monitoring or clinical translation. We aimed to provide the phenotypic profile of G-MDSCs based on their prognostic significance in MM, immunosuppressive potential, and molecular program. The preestablished phenotype of G-MDSCs was evaluated in bone marrow samples from controls and MM patients using multidimensional flow cytometry; surprisingly, we found that CD11b+CD14−CD15+CD33+HLADR− cells overlapped with common eosinophils and neutrophils, which were not expanded in MM patients. Therefore, we relied on automated clustering to unbiasedly identify all granulocytic subsets in the tumor microenvironment: basophils, eosinophils, and immature, intermediate, and mature neutrophils. In a series of 267 newly diagnosed MM patients (GEM2012MENOS65 trial), only the frequency of mature neutrophils at diagnosis was significantly associated with patient outcome, and a high mature neutrophil/T-cell ratio resulted in inferior progression-free survival (P & lt; .001). Upon fluorescence-activated cell sorting of each neutrophil subset, T-cell proliferation decreased in the presence of mature neutrophils (0.5-fold; P = .016), and the cytotoxic potential of T cells engaged by a BCMA×CD3-bispecific antibody increased notably with the depletion of mature neutrophils (fourfold; P = .0007). Most interestingly, RNA sequencing of the 3 subsets revealed that G-MDSC–related genes were specifically upregulated in mature neutrophils from MM patients vs controls because of differential chromatin accessibility. Taken together, our results establish a correlation between the clinical significance, immunosuppressive potential, and transcriptional network of well-defined neutrophil subsets, providing for the first time a set of optimal markers (CD11b/CD13/CD16) for accurate monitoring of G-MDSCs in MM.

Abstract: Large-scale immune monitoring is becoming routinely used in clinical trials to identify determinants of treatment responsiveness, particularly to immunotherapies. Flow cytometry remains one of the most versatile and high throughput approaches for single-cell analysis; however, manual interpretation of multidimensional data poses a challenge when attempting to capture full cellular diversity and provide reproducible results. We present FlowCT, a semi-automated workspace empowered to analyze large data sets. It includes pre-processing, normalization, multiple dimensionality reduction techniques, automated clustering, and predictive modeling tools. As a proof of concept, we used FlowCT to compare the T-cell compartment in bone marrow (BM) with peripheral blood (PB) from patients with smoldering multiple myeloma (SMM), identify minimally invasive immune biomarkers of progression from smoldering to active MM, define prognostic T-cell subsets in the BM of patients with active MM after treatment intensification, and assess the longitudinal effect of maintenance therapy in BM T cells. A total of 354 samples were analyzed and immune signatures predictive of malignant transformation were identified in 150 patients with SMM (hazard ratio [HR], 1.7; P & lt; .001). We also determined progression-free survival (HR, 4.09; P & lt; .0001) and overall survival (HR, 3.12; P = .047) in 100 patients with active MM. New data also emerged about stem cell memory T cells, the concordance between immune profiles in BM and PB, and the immunomodulatory effect of maintenance therapy. FlowCT is a new open-source computational approach that can be readily implemented by research laboratories to perform quality control, analyze high-dimensional data, unveil cellular diversity, and objectively identify biomarkers in large immune monitoring studies. These trials were registered at www.clinicaltrials.gov as #NCT01916252 and #NCT02406144.

Abstract: Tumor recognition by T cells is essential for antitumor immunity. A comprehensive characterization of T cell diversity may be key to understanding the success of immunomodulatory drugs and failure of PD-1 blockade in tumors such as multiple myeloma (MM). Here, we use single-cell RNA and T cell receptor sequencing to characterize bone marrow T cells from healthy adults ( n  = 4) and patients with precursor ( n  = 8) and full-blown MM ( n  = 10). Large T cell clones from patients with MM expressed multiple immune checkpoints, suggesting a potentially dysfunctional phenotype. Dual targeting of PD-1 + LAG3 or PD-1 + TIGIT partially restored their function in mice with MM. We identify phenotypic hallmarks of large intratumoral T cell clones, and demonstrate that the CD27 − and CD27 + T cell ratio, measured by flow cytometry, may serve as a surrogate of clonal T cell expansions and an independent prognostic factor in 543 patients with MM treated with lenalidomide-based treatment combinations.