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 <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.
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.
Background: The broad use of immunomodulatory drugs (IMiDs) and the breakthrough of novel immunotherapies in MM, urge the optimization of immune monitoring to help tailoring treatment based on better ...prediction of patients' response according to their immune status. For example, current T cells immune monitoring is of limited value because the phenotype of tumor-reactive T cells is uncertain.
Aims: To characterize the MM immune microenvironment at the single-cell level and to identify clinically relevant subsets for effective immune monitoring.
Methods: We used a semi-automated pipeline to unveil full cellular diversity based on unbiased clustering, in a large flow cytometry dataset of 86 newly-diagnosed MM patients enrolled in the PETHEMA/GEM2012MENOS65 clinical trial, including immune monitoring at diagnosis, after induction with bortezomib, lenalidomide, dexamethasone (VRD), autologous transplant and VRD consolidation. Immunophenotyping was performed using the first 8-color combination (CD19, CD27, CD38, CD45, CD56, CD81, CD117, CD138) of the next-generation flow (NGF) panel for MRD assessment. Results were then validated in additional 145 patients enrolled in the same trial. Deep characterization of T cells was performed using 17-color multidimensional flow cytometry (TIM3, CD160, TIGIT, CD57, CD8, PD1, CD45RA, CD56, BTLA, CD4, CD3, CD39, CD137, CTLA4, CCR7, CD16, CD27) and combined single-cell (sc) RNA/TCR sequencing (10xGenomics).
Results: Simultaneous analysis of the entire dataset (n=333 files) unbiasedly identified 25 cell clusters (including 9 myeloid and 13 lymphocytes subsets) in the MM immune microenvironment. Afterwards, we correlated a total of 120 immune parameters derived from the cellular abundance of each cluster and specific cell ratios determined at all time points, with a total of 20 clinical parameters including the International Staging System (ISS) and FISH cytogenetics. Twelve variables had significant impact in progression-free survival (PFS) and the ratio between CD27- vs CD27+ T cells emerged as an independent prognostic factor (HR:0.09, p=0.04) together with the ISS in a Cox regression model. The 3-year PFS rates of patients with high vs low CD27-/CD27+ ratios were 94% vs 71% (p=0.02), respectively; these findings being confirmed in the validation dataset. Thus, we observed in the entire cohort (n=231) that a prognostic score including the CD27-/CD27+ T cell ratio (HR:0.21, p=0.013) and ISS (HR:1.41, p=0.015) outperformed each parameter alone (HR:0.06, p=0.007). To gain further insight into the biological significance of the CD27-/CD27+ T cell ratio, we performed scRNA/TCRseq in 44,969 lymphocytes from 9 MM patients. Downstream analysis unveiled that CD27- T cells were mostly CD8 and included senescent, effector and exhausted clusters. By contrast, CD27+ T cells were mainly CD4 and the remaining CD8 T cells had a predominant immune suppressive phenotype (ie. high GZMK, TIGIT, LAG3 and PD1 expression levels). Such T cell clustering was validated by 17-color multidimensional flow cytometry that confirmed the cellular distribution identified by scRNAseq, as well as higher reactivity for PD1, TIGIT, BTLA and TIM3 in CD27+ vs CD27- T cells. Simultaneous scTCRseq revealed a total of 90 different clonotypes (median of 12 per patient). Interestingly, most clonotypes where found in CD27- (74/90) as opposed to CD27+ T cells and, using the VDJB database, the CDR3 sequences of clonotypic effector/exhausted CD27- T cells were predicted to recognize MM-related epitopes such as MLANA, HM1.24 (CD319), TKT, or IMP2. In selected patients, we performed exome- and RNA-sequencing of tumor cells and analyzed their HLA profile. Using the T Cell Epitopes - MHC Binding Prediction tool from the IEDB Analysis Resource, we found expression of mutated genes (e.g. UBXN1, UPF2, GNB1L) predicted to bind MHC class I molecules on tumor cells and potentially recognized by autologous clonotypic CD27- T cells.
Conclusion: We show for the first time that potential MM-reactive T cells are CD27-negative and that their abundance in the immune microenvironment of newly-diagnosed MM patients is prognostic, possibly due to their reactivation after treatment with IMiDs and autologous transplant. Because NGF is broadly used, these results are readily applicable for effective T cell immune monitoring.
Puig:Janssen: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Amgen: Consultancy, Honoraria; The Binding Site: Honoraria; Takeda: Consultancy, Honoraria. Rosinol Dachs:Janssen, Celgene, Amgen and Takeda: Honoraria. Oriol:Janssen: Consultancy; Takeda: Consultancy, Speakers Bureau; Amgen: Consultancy, Speakers Bureau; Celgene Corporation: Consultancy, Speakers Bureau. Rios:Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees. Sureda:Takeda: Consultancy, Honoraria, Speakers Bureau; Novartis: Honoraria; Gilead: Honoraria; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Honoraria; Roche: Honoraria; Sanofi: Honoraria; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel Support; Amgen: Membership on an entity's Board of Directors or advisory committees. De La Rubia:Takeda: Consultancy; Janssen: Consultancy; Celgene Corporation: Consultancy; AMGEN: Consultancy; AbbVie: Consultancy. Mateos:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Adaptive: Honoraria; EDO: 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; GSK: Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees. Lahuerta:Takeda, Amgen, Celgene and Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees. Bladé:Irctures: Honoraria; Janssen, Celgene, Amgen, Takeda: 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.
Background: Deep understanding of the complexity and diversity of the tumor immune microenvironment (TIME) and its influence on response to therapy is needed to improve the ability to predict, ...monitor and guide immunotherapeutic responsiveness. Among different cell types in the MM-TIME, granulocytic MDSCs (G-MDSCs) have a prominent role in promoting tumor growth and inducing immune suppression; however, their identification and monitoring is imprecise because the phenotypic profile of MDSCs in the MM-TIME is not well-established.
Aim: To provide the detailed phenotypic profile of G-MDSCs based on the immune suppressive potential, gene regulatory network and clinical significance of distinct granulocytic subsets in the MM-TIME.
Methods: First, we used multidimensional flow cytometry (MFC) to evaluate the preestablished phenotype of G-MDSCs in bone marrow (BM) samples from controls (n=4) and MM patients (n=5). We then used principal component analysis (PCA) to unbiasedly identify different granulocytic subsets in the MM-TIME, and FACS for in vitro experiments to determine their immune suppressive potential (n=9) and for RNAseq to analyze the molecular profile of G-MDSCs in MM (n=5) vs controls (n=5). Subsequently, the clinical significance of the different granulocytic subsets was investigated by comparing their numbers at diagnosis, in MM patients (n=124) achieving MRD-negativity vs MRD-positivity after treatment with VRD induction (x6) followed by autologous transplant and VRD consolidation (x2) (GEM2012MENOS65 clinical trial).
Results: In humans, G-MDSCs have been defined as a unique cluster displaying a CD11b-, CD14-, CD15+, CD33+ and HLADR- phenotype, comprising 1% of total BM nucleated cells in healthy individuals and approximately 25% in MM patients. However, we found that the percentage of cells with a CD11b-CD14-CD15+CD33+HLADR- phenotype was similar in the BM of controls and MM patients (median of 8% in both, P>.99). Since these cells were not expanded in MM and represented only 24% of total neutrophils, we next used MFC and PCA to unbiasedly identify other cell clusters within neutrophils. Accordingly, 3 major subsets were identified in neutrophils from controls and MM patients, based on homogeneous CD14-CD15+CD33+HLADR- expression but differential reactivity against CD11b, CD13 and CD16: CD11b-CD13lo/-CD16- (19% and 24%), CD11b+CD13lo/-CD16- (46% and 47%) and CD11b+CD13+CD16+ (35% and 29%). Afterwards, we used FACSorting to deplete or isolate individually, each of the 3 neutrophil subsets from the BM MM-TIME and determine its immune suppressive potential in 2 functional assays: 1) the proliferation rate of autologous T cells in presence of CD3/CD28 stimulatory beads and, 2) the cytotoxic potential of autologous T-cells against MM cells using a BCMAxCD3 bispecific antibody. Interestingly, we noted a significant decrease in T cell proliferation when these were stimulated in the presence of CD11b+CD13+CD16+ neutrophils (0.5-fold, p =.03) but not the CD11b-CD13lo/-CD16- and CD11b+CD13lo/-CD16- subsets. In addition, we noted that the cytotoxic potential of T cells engaged by the BCMAxCD3 bispecific antibody significantly increased with the depletion of CD11b+CD13lo/-CD16- and CD11b+CD13+CD16+ subsets (3-fold and 4-fold, respectively; p ≤.04) but not CD11b-CD13lo/-CD16- neutrophils. Furthermore, RNAseq of the 3 subsets in controls and MM patients revealed that genes related with the IL-4, IL-10 and IL-13 immunosuppressive pathways were specifically upregulated in the CD11b+CD13+CD16+ subset. Finally, based on the surrogacy between the achievement of MRD-negativity and prolonged survival, we compared the distribution of the 3 granulocytic subsets in the BM-TIME at diagnosis and observed that patients reaching MRD-negativity (n=56) displayed significantly lower percentages of total neutrophils (46% vs 52%, p =.002), particularly of the CD11b+CD13lo/-CD16- (11% vs 15%, p =.003) and CD11b+CD13+CD16+ (31% vs 35%, p =.07) subsets vs MRD-positive cases (n=68).
Conclusions: We have determined the correlation between the phenotypic, molecular and immunosuppressive potential of unique granulocytic subsets. Thus, we have identified optimal markers for monitoring G-MDCSs in patients with MM (ie. CD11b, CD13, CD16) and unveiled that, in contrast to previous findings, the more mature granulocytes are the only stages with immunosuppressive potential.
Puig:Celgene: Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria. Martinez Lopez:Celgene: Research Funding, Speakers Bureau; Bristol Myers Squibb: Research Funding, Speakers Bureau; Novartis: Research Funding, Speakers Bureau; Janssen: Research Funding, Speakers Bureau. Oriol:Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Rios:Amgen, Celgene, Janssen, and Takeda: Consultancy. Rosinol:Janssen, Celgene, Amgen, Takeda: Honoraria. Mateos:Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees; GSK: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; GSK: Consultancy, Membership on an entity's Board of Directors or advisory committees. Lahuerta:Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: 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; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees. Bladé:Celgene: Honoraria; Janssen: Honoraria; Amgen: Honoraria. San-Miguel:Janssen: Honoraria; Celgene: Honoraria; Amgen: Honoraria; BMS: Honoraria; Novartis: Honoraria; Sanofi: Honoraria; Roche: Honoraria.
Background: Although MYD88 L265P is highly frequent in WM, by itself is insufficient to explain disease progression since most cases with IgM MGUS also have mutated MYD88. In fact, the percentage of ...MYD88 L265P in CD19+ cells isolated from WM patients is typically <100%, which questions if this mutation initiates the formation of B-cell clones. Furthermore, a few WM patients have detectable MYD88 L265P in total bone marrow (BM) cells and not in CD19+ selected B cells, raising the possibility that other hematopoietic cells carry the MYD88 mutation. However, no one has investigated if the pathogenesis of WM is related to somatic mutations occurring at the hematopoietic stem cell level, similarly to what has been shown in CLL or hairy cell leukemia.
Aim: Define the cellular origin of WM by comparing the genetic landscape of WM cells to that of CD34 progenitors, B cell precursors and residual normal B cells.
Methods: We used multidimensional FACSorting to isolate a total of 43 cell subsets from BM aspirates of 8 WM patients: CD34+ progenitors, B cell precursors, residual normal B cells (if detectable), WM B cells, plasma cells (PCs) and T cells (germline control). Whole-exome sequencing (WES, mean depth 74x) was performed with the 10XGenomics Exome Solution for low DNA-input due to very low numbers of some cell types. We also performed single-cell RNA and B-cell receptor sequencing (scRNA/BCRseq) in total BM B cells and PCs (n=32,720) from 3 IgM MGUS and 2 WM patients. Accordingly, the clonotypic BCR detected in WM cells was unbiasedly investigated in all B cell maturation stages defined according to their molecular phenotype. In parallel, MYD88p.L252P (orthologous position of the human L265P mutation) transgenic mice were crossed with conditional Sca1Cre, Mb1Cre, and Cγ1Cre mice to selectively induce in vivo expression of MYD88 mutation in CD34 progenitors, B cell precursors and germinal center B cells, respectively. Upon immunization, mice from each cohort were necropsied at 5, 10 and 15 months of age and screened for the presence of hematological disease.
Results: All 8 WM patients showed MYD88 L265P and 3 had mutated CXCR4. Notably, we found MYD88 L265P in B cell precursors from 1/8 cases and in residual normal B cells from 3/8 patients, which were confirmed by ASO-PCR. In addition, CXCR4 was simultaneously mutated in B cell precursors and WM B cells from one patient. Overall, CD34+ progenitors, B-cell precursors and residual normal B cells shared a median of 1 (range, 0-4; mean VAF, 0.16), 2 (range, 1-5; mean VAF, 0.14), and 4 (range, 1-13; mean VAF, 0.26) non-synonymous mutations with WM B cells. Some mutations were found all the way from CD34+ progenitors to WM B cells and PCs. Interestingly, concordance between the mutational landscape of WM B cells and PCs was <100% (median of 85%, range: 25%-100%), suggesting that not all WB B cells differentiate into PCs.
A median of 7 (range, 2-19; mean VAF, 0.39) mutations were unique to WM B cells. Accordingly, many clonal mutations in WM B cells were undetectable in normal cells. Thus, the few somatic mutations observed in patients' lymphopoiesis could not result from contamination during FACSorting since in such cases, all clonal mutations would be detectable in normal cells. Of note, while somatic mutations were systematically detected in normal cells from all patients, no copy number alterations (CNA) present in WM cells were detectable in normal cells. scRNA/BCRseq unveiled that clonotypic cells were confined mostly within mature B cell and PC clusters in IgM MGUS, whereas a fraction of clonotypic cells from WM patients showed a transcriptional profile overlapping with that of B cell precursors.
In mice, induced expression of mutated MYD88 led to a moderate increase in the number of B220+CD138+ plasmablasts and B220-CD138+ PCs in lymphoid tissues and BM, but no signs of clonality or hematological disease. Interestingly, such increment was more evident in mice with activation of mutated MYD88 in CD34+ progenitors and B-cell precursors vs mice with MYD88 L252P induced in germinal center B cells.
Conclusions: We show for the first time that WM patients have somatic mutations, including MYD88 L265P and in CXCR4, at the B cell progenitor level. Taken together, this study suggests that in some patients, WM could develop from B cell clones carrying MYD88 L265P rather than it being the initiating event, and that other mutations or CNA are required for the expansion of B cells and PCs with the WM phenotype.
Roccaro:Janssen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Transcan2-ERANET: Research Funding; AstraZeneca: Research Funding; European Hematology Association: Research Funding; Transcan2-ERANET: Research Funding; Associazione Italiana per al Ricerca sul Cancro (AIRC): Research Funding; Associazione Italiana per al Ricerca sul Cancro (AIRC): Research Funding; European Hematology Association: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Research Funding; Amgen: 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.
Mitogenic stimulation of lymphocytes involves alkalinization of intracellular pH (pHi). Subsequent pHi regulation may involve HCO3− extrusion through Cl−/HCO3− exchangers and/or Na+‐HCO3− ...co‐transporters with acid‐loading capability. Abnormalities in these mechanisms could result in immune dysfunctions, as suggested by the CD8+ T‐cell expansion encountered in mice lacking Ae2 (a widely expressed acid loader with electroneutral and Na+‐independent Cl−/HCO3− anion‐exchange activity). Here we report that CD8+ T cells but not CD4+ T cells or other lymphocyte populations, are crucially dependent on Ae2 for pHi regulation. While total lymphocytes (including isolated CD4+ T cells) exhibit Ae1 expression and Na+‐HCO3− co‐transport with acidifying potential, CD8+ T cells lack these acid‐loading mechanisms. In Ae2‐KO mice, CD4+ but not CD8+ T cells upregulate these potential Ae2 surrogates. As a consequence, Ae2‐KO CD8+ T cells exhibit alkalinized pHi, and dramatically increase their pHi upon CD3 stimulation. Moreover, stimulated Ae2‐deficient CD8+ T cells show enhanced intracellular production of IL‐2 and membrane expression of its receptor IL‐2Rα, together with increased cell proliferation and activation. These findings demonstrate that CD8+ T cells are critically dependent on Ae2 for pHi homeostasis and tuning of cell proliferation and activation. Ae2 thus constitutes a novel target to modulate CD8+ T‐cell responses.
Background: MM and AL are the two most common malignant monoclonal gammopathies. Both diseases result from the accumulation of clonal PCs, but their clinical behavior is significantly different ...suggesting fundamental differences in disease biology. Previous attempts to identify genetic hallmarks that could explain such differences have been unsuccessful. Furthermore, it is unknown if MM and AL arise from the same or different normal PC counterparts.
Aim: To define a transcriptional atlas of the normal PC development in peripheral blood (PB) and bone marrow (BM) for comparison with the transcriptional programs of clonal PCs in MM and AL.
Methods: A total of 93 subjects were studied. In 7 healthy adults (HA), PB PCs were phenotypically sorted according to heavy-chain isotypes (IgG, IgA and IgM). In addition, 5 different BM PCs subsets were isolated based on the differential expression of CD19, CD39, CD81 and CD56, due to their ascribed role in dissecting unique BM PC differentiation states. Clonal PCs from patients with MM (n=38) and AL (n=41) were isolated by FACS according to patient-specific aberrant phenotypes. Due to small numbers of PCs sorted from each subset in HA and clonal PCs in AL patients, we used an RNAseq method optimized for limited cell numbers. Differential expression across all pairwise comparisons between groups was analyzed with Deseq2 R package followed by k-means clustering of genes in R. Single-cell RNAseq (scRNAseq, 10xGenomics) was performed in a total of 35,910 PCs from 3 HA, 2 MM and 2 AL. We used Seurat R package to remove batch effect followed by canonical correlation to perform an integrated analysis of all single PCs from HA, MM and AL subjects.
Results: Principal component analysis of RNAseq data unveiled two major clusters of normal PCs: those in PB and those in BM (with some transcriptional diversity between CD19+ and CD19- PCs), whereas the CD19+CD39+CD81+CD56- BM subset co-localized with PB and CD39- BM PCs (Panel A). Clonal PCs from MM and AL patients clustered together, and both displayed some transcriptional variance related to the spatial location of normal PCs (i.e. PB or BM). In total, 2174 genes were found significantly deregulated after cross-comparing the 10 PC groups (adj.p-value<0.01, logFC>1) and semi-supervised k-means clustering unveiled 8 transcriptional modules (Panel B). Namely, the transition from PB into BM PCs was characterized by genes related to proliferation (clusters 1 & 2), whereas CD39+ and CD39- BM PC subsets differed on the expression of genes associated with proliferation, homing, and metabolism (1, 2, 4 & 6). Thus, CD19+CD39+CD81+CD56- BM PCs emerged as a novel subset that bridges new-born PB with long-lived (CD39-) BM PCs. Interestingly, clonal PCs from MM and AL shared transcriptional programs related to quiescence (5 & 6) with long-lived BM PCs; however, skewing of polyclonal immunoglobulin gene expression (3) and active gene transcription (8) emerged as hallmarks of the neoplastic transformation from normal, long-lived PCs into clonal PCs. That notwithstanding, the later displayed expression levels of the proliferation and homing transcriptional modules (1 & 4) similar to new-born PB and CD39+ BM PCs. Of note, a small transcriptional cluster of genes related to ribosome biogenesis (7) was significantly more expressed in MM than AL. These findings led us to integrate scRNAseq profiles of normal and clonal BM PCs from MM and AL patients, to define PC clusters based on their transcriptional program rather than their normal vs malignant status (Panel C). This strategy unveiled 11 different PC clusters with unequal distribution between groups. Thus, more than half of clonal PCs in MM and AL were assigned to a cluster that is also predominant in normal PCs (1). By contrast, other clusters with a transcriptional program similar to that of new-born PCs (2 & 5) became rarer in MM and AL. Furthermore, a cluster of PCs with an immature-like phenotype (6) was detectable in MM but almost absent in AL.
Conclusions: This is the first integrated analysis of the transcriptional programs of normal PC subsets and clonal PCs in MM and AL, both at the bulk and single-cell levels. Our results unveil shared and exclusive transcriptional states in normal and clonal PCs, together with unique differences between clonal PCs in MM and AL. Thus, we provide here a fundamental resource to understand normal PC development and the cellular origin of both malignant monoclonal gammopathies.
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Puig:Takeda: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Research Funding; Celgene: Honoraria, Research Funding. Ocio:Pharmamar: Consultancy; AbbVie: Consultancy; Janssen: Consultancy, Honoraria; Seattle Genetics: Consultancy; BMS: Consultancy; Takeda: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Sanofi: Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Mundipharma: Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Array Pharmaceuticals: Research Funding. Oriol:Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Martinez Lopez:Bristol Myers Squibb: Research Funding, Speakers Bureau; Janssen: Research Funding, Speakers Bureau; Novartis: Research Funding, Speakers Bureau; Celgene: Research Funding, Speakers Bureau. Mateos:Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; GSK: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; GSK: Consultancy, Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees. Lahuerta: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; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees. San-Miguel:Sanofi: Consultancy; Takeda: Consultancy; Novartis: Consultancy; MSD: Consultancy; Janssen: Consultancy; Celgene: Consultancy; Brystol-Myers Squibb: Consultancy; Amgen: Consultancy; Roche: Membership on an entity's Board of Directors or advisory committees.
Normal cell counterparts of solid and myeloid tumors accumulate mutations years before disease onset; whether this occurs in B lymphocytes before lymphoma remains uncertain. We sequenced multiple ...stages of the B lineage in elderly individuals and patients with lymphoplasmacytic lymphoma, a singular disease for studying lymphomagenesis because of the high prevalence of mutated
MYD88
. We observed similar accumulation of random mutations in B lineages from both cohorts and unexpectedly found
MYD88
L265P
in normal precursor and mature B lymphocytes from patients with lymphoma. We uncovered genetic and transcriptional pathways driving malignant transformation and leveraged these to model lymphoplasmacytic lymphoma in mice, based on mutated
MYD88
in B cell precursors and
BCL2
overexpression. Thus,
MYD88
L265P
is a preneoplastic event, which challenges the current understanding of lymphomagenesis and may have implications for early detection of B cell lymphomas.
MYD88
L265P
occurs in between a normal mutated lymphopoiesis and additional genetic alterations during lymphomagenesis.
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 < .001). We also determined progression-free survival (HR, 4.09; P < .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.
•FlowCT is a new computational workspace for unveiling cellular diversity and objectively identifying biomarkers in large immune monitoring studies.•FlowCT identified T-cell biomarkers predictive of malignant transformation and survival in SMM and active MM data sets.
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Primary biliary cirrhosis (PBC) is a chronic cholestatic disease of unknown etiopathogenesis showing progressive autoimmune-mediated cholangitis. In PBC patients, the liver and lymphocytes exhibit ...diminished expression of AE2/SLC4A2, a Cl-/HCO3- anion exchanger involved in biliary bicarbonate secretion and intracellular pH regulation. Decreased AE2 expression may be pathogenic as Ae2a,b(-/-) mice reproduce hepatobiliary and immunological features resembling PBC. To understand the role of AE2 deficiency for autoimmunity predisposition we focused on the phenotypic changes of T cells that occur over the life-span of Ae2a,b(-/-) mice. At early ages (1-9 months), knockout mice had reduced numbers of intrahepatic T cells, which exhibited increased activation, programmed-cell-death (PD)-1 expression, and apoptosis. Moreover, young knockouts had upregulated PD-1 ligand (PD-L1) on bile-duct cells, and administration of neutralizing anti-PD-L1 antibodies prevented their intrahepatic T-cell deletion. Older (≥ 10 months) knockouts, however, showed intrahepatic accumulation of cytotoxic CD8(+) T cells with downregulated PD-1 and diminished apoptosis. In-vitro DNA demethylation with 5-aza-2'-deoxycytidine partially reverted PD-1 downregulation of intrahepatic CD8(+) T cells from aged knockouts.
Early in life, AE2 deficiency results in intrahepatic T-cell activation and PD-1/PD-L1 mediated deletion. With aging, intrahepatic CD8+ T cells epigenetically suppress PD-1, and their consequential expansion and further activation favor autoimmune cholangitis.
Background: Despite significant improvements in the treatment of MM, the outcome of patients with HR cytogenetics remains poor despite similar complete remission (CR) rates as compared to SR cases. ...Relapses among patients in CR are attributed to the persistence of MRD, but knowledge about the impact of MRD in patients with SR and HR cytogenetics, treated with modern therapies and monitored with next-generation techniques, is limited. Similarly, there is virtually no data about in vivo mechanisms of resistance in SR and HR MM; however, since MRD represents those very few cells that are resistant to treatment, it could be hypothesized that profiling MRD cells may shed light into the mechanisms of resistance in both SR and HR patients.
Aim: To determine the clinical impact of MRD in MM patients with SR vs HR cytogenetics, and to identify transcriptional mechanisms determining MRD resistance by investigating the transcriptome of MRD cells in both patient subgroups.
Methods: This study was conducted in a series of 390 patients enrolled in the PETHEMA/GEM2012 trial (6 induction cycles with VRD followed by ASCT and 2 courses of consolidation with VRD). FISH was analyzed on CD138 purified PCs at diagnosis. MRD was predefined to be prospectively assessed following induction, transplant and consolidation, using next-generation flow (NGF) according to EuroFlow. In 40 patients 28 with SR and 12 with HR cytogenetics: i.e., t(4;14), t(14;16) and/or del(17p), diagnostic and MRD tumor cells persisting after VRD-induction were isolated by FACS according to patient-specific aberrant phenotypes. Due to the small number of sorted MRD cells (median of 25,600) we used a 3' end RNAseq method optimized for generating libraries from low-input starting material (MARSeq). Differential expression analyses were performed with DESeq2 R package.
Results: At the latest time-point in which MRD was assessed, MRD-positive rates progressively increased (p =.006) from SR patients (148/300, 49%) to cases with t(4;14) (24/42, 57%) and del(17p) (29/38, 76%). Furthermore, MRD levels were significantly superior in patients with del(17p) compared to SR FISH (0.02% vs 0.006%, p =.009), while MRD levels in patients with t(4;14) (0.004%) were similar to those in SR MM. Only 10 patients had a t(14;16) and 4 were MRD-positive.
Among patients achieving MRD-negativity (<2x10-6), 3-year progression-free survival (PFS) rates were similar for those with SR FISH, t(4;14) and del(17p) (90%, 100% and 89%; p >.05). Conversely, 3-year PFS rates for MRD-positive patients decreased from those having SR FISH to those with t(4;14) and del(17p) (59%, 46% and 24%, respectively), with statistically significant differences between the first and the latest subgroups (p <.001).
Since clearance of MRD notably lowered the risk of relapse and persistence of MRD significantly shortened the PFS in each cytogenetic group (p ≤.001), we investigated the unique features of MRD cells persisting after VRD-induction by comparing their transcriptome to that of patient-matched tumor cells at diagnosis (n=40). Accordingly, MRD cells showed 763 genes significantly deregulated (Padj <.05), including a cluster of proteasome subunits and proteasome related genes (i.e. PSMB5, PSMC3IP, BTRC, HUWE1, FBXL20 and TRIM69). Gene set enrichment analysis unveiled biologic determinants of MRD resistance such as the IL6-JAK-STAT signaling pathway in SR patients and the ROS pathway in HR patients (FDR <0.1). Interestingly, the number of genes deregulated in MRD cells of SR patients was 9-fold higher than HR cases suggesting that, whereas in SR MM, a few tumor cells with specific gene regulatory networks may have higher probability to persist VRD induction, the presence of HR cytogenetic alterations is associated per se, with a transcriptional program that allows a few MRD cells to persist treatment.
Conclusions: This is one of the largest studies integrating patients' cytogenetics and MRD status. Our results, based on intensive treatment and MRD monitoring using NGF, unveil that achieving MRD-negativity may overcome the poor prognosis of HR cytogenetics. By contrast, persistent MRD significantly reduces PFS rates, particularly in patients with del(17p). Interestingly, MRD cells from SR and HR patients may have different transcriptional mechanisms leading to VRD resistance, and further understanding of these could provide knowledge on how to eradicate MRD in both patient subgroups.
Puig:Takeda: Consultancy, Honoraria; Celgene: Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding. Garcia-Sanz:Affimed: Research Funding. Martinez-Lopez:BMS: Research Funding; Pfizer: Research Funding; Vivia: Honoraria; Celgene: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Novartis: Research Funding. Oriol:Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Rios:Amgen, Celgene, Janssen, and Takeda: Consultancy. De La Rubia:Ablynx: Consultancy, Other: Member of Advisory Board. Mateos:GSK: Consultancy, Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees; GSK: Consultancy, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Lahuerta:Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: 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; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees. Bladé:Janssen: Honoraria. San-Miguel:Amgen: Honoraria; BMS: Honoraria; Novartis: Honoraria; Sanofi: Honoraria; Celgene: Honoraria; Roche: Honoraria; Janssen: Honoraria.
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