Although light-chain amyloidosis (AL) and multiple myeloma (MM) are characterized by tumor plasma cell (PC) expansion in bone marrow (BM), their clinical presentation differs. Previous attempts to ...identify unique pathogenic mechanisms behind such differences were unsuccessful, and no studies have investigated the differentiation stage of tumor PCs in patients with AL and MM. We sought to define a transcriptional atlas of normal PC development in secondary lymphoid organs (SLOs), peripheral blood (PB), and BM for comparison with the transcriptional programs (TPs) of tumor PCs in AL, MM, and monoclonal gammopathy of undetermined significance (MGUS). Based on bulk and single-cell RNA sequencing, we observed 13 TPs during transition of normal PCs throughout SLOs, PB, and BM. We further noted the following: CD39 outperforms CD19 to discriminate newborn from long-lived BM-PCs; tumor PCs expressed the most advantageous TPs of normal PC differentiation; AL shares greater similarity to SLO-PCs whereas MM is transcriptionally closer to PB-PCs and newborn BM-PCs; patients with AL and MM enriched in immature TPs had inferior survival; and protein N-linked glycosylation–related TPs are upregulated in AL. Collectively, we provide a novel resource to understand normal PC development and the transcriptional reorganization of AL and other monoclonal gammopathies.
•Tumor cells express transcriptional programs of both immature and mature stages of normal PC development.•Survival of patients with AL and MM is inferior when tumor cells express transcriptional programs of more immature normal PC stages.
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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.
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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 immune system reacts to viral infection with cellular and humoral responses. Thus, myelo- and lympho-suppression caused by cancer itself as well as cytotoxic treatment may pose a ...challenge to COVID-19 patients with solid and hematological tumors, but severe events from initial onset of COVID-19 appear to be more frequent in blood malignancies vs other cancer types. Preliminary data showed lower neutrophil and lymphocyte counts in COVID-19 patients bearing hematological cancer, but there are conflicting results supporting that both worsening of lymphopenia during COVID-19 and its depth prior to infection had a beneficial impact on survival. Thus, greater knowledge on the immune status of hematological patients may be useful to optimize prevention, risk stratification and treatment strategies.
Aim: Analyze the immune status of COVID-19 patients with or without solid and hematological cancer.
Methods: We use multidimensional flow cytometry (MFC) to analyze immune profiles in peripheral blood samples of 515 COVID-19 patients at presentation. Data was analyzed with a semi-automated pipeline that performs batch-analyses of MFC data to avoid variability intrinsic to manual analysis, and unveils full cellular diversity based on unbiased clustering. In 14 cases, deep immunophenotyping of B- and T-cells was performed and six myeloid- and dendritic-cell subsets were FACSorted for transcriptome analysis using RNAseq.
Results: Of the 515 COVID-19 patients, 15 and 10 had solid and hematological tumors, respectively. Those with hematological cancer showed similar frequency of hospitalization than those with solid tumors (90% and 93%, respectively), which was modestly higher to that observed in persons without an active tumor (76%). By contrast, the frequency of hematological cases requiring intensive care (50%) and dying from COVID-19 (30%) was significantly higher to that observed in patients with no active tumor (5.5% and 4%, respectively), or with solid cancer (both 0%).
Based on semi-automated analysis of MFC data, we systematically quantified a total of 19 cell types in PB that included 6 myeloid and 13 lymphoid subsets. Patients with hematological malignancies displayed altered immune profiles with significantly decreased absolute numbers of classical and intermediate monocytes, immunoregulatory and cytotoxic NK cells, double-negative, double-positive, CD4 and CD56- γδ T cells, as well as of mature B cells when compared to those with no tumor.
Unsupervised hierarchical analysis of RNAseq data from basophils, myeloid and plasmacytoid dendritic cells, classical and non-classical monocytes and neutrophils showed considerable clustering of samples from hematological cases. Furthermore, a variable number of differentially expressed genes was found in all six cell types between COVID-19 patients with or without blood cancer. Genes related to NF-κB and STAT transcription factors as well as genes encoding toll-like receptors and proinflammatory interleukin receptors, all of which described to be implicated in the response and evasion of innate sensing by coronaviruses, were differentially expressed in many of these cell types. Deep phenotypic characterization of T- and B-cell compartments in PB of COVID-19 patients with (N = 4) or without (N = 10) hematological cancer showed that the relative distribution of antigen-dependent maturation stages within the T-cell compartment was generally similar between both groups. However, some hematological cases displayed profound alterations in virtually all of the 16 B-cell subsets analyzed, with a notorious reduction in memory B cells expressing IgG and IgA subclasses.
We next compared immune responses from presentation to last follow-up in COVID-19 patients with hematological cancer and favorable (N = 3) vs fatal (N = 3) outcome. Interestingly, we found opposite kinetics in myeloid cell types such as eosinophils and neutrophils, decreasing numbers of various T cell subsets, as well as lower mature B cells and circulating PCs at presentation together with a decrease in B cell counts in deceased cases.
Conclusions: Our study exposes for the first time that hematological patients show a constellation of immune alterations that could compromise the response to the infection caused by SARS-CoV-2, suggesting an association between impaired immune responses and poorer outcomes in COVID-19 patients with hematological malignancies.
Paiva:SkylineDx: Consultancy; Takeda: Consultancy, Honoraria, Research Funding; Roche: Research Funding; Adaptive: Honoraria; Amgen: Honoraria; Janssen: Consultancy, Honoraria; Karyopharm: Consultancy, Honoraria; Kite: Consultancy; Sanofi: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau.
Myelodysplastic syndromes (MDS) are hematopoietic stem cell (HSC) malignancies characterized by ineffective hematopoiesis, with increased incidence in older individuals. Here we analyze the ...transcriptome of human HSCs purified from young and older healthy adults, as well as MDS patients, identifying transcriptional alterations following different patterns of expression. While aging-associated lesions seem to predispose HSCs to myeloid transformation, disease-specific alterations may trigger MDS development. Among MDS-specific lesions, we detect the upregulation of the transcription factor DNA Damage Inducible Transcript 3 (DDIT3). Overexpression of DDIT3 in human healthy HSCs induces an MDS-like transcriptional state, and dyserythropoiesis, an effect associated with a failure in the activation of transcriptional programs required for normal erythroid differentiation. Moreover, DDIT3 knockdown in CD34
cells from MDS patients with anemia is able to restore erythropoiesis. These results identify DDIT3 as a driver of dyserythropoiesis, and a potential therapeutic target to restore the inefficient erythroid differentiation characterizing MDS patients.
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Background: CTCs may be responsible for MM spreading and accordingly, their numbers in peripheral blood (PB) could be a potential surrogate marker for the rate of dissemination and overall tumor ...burden in bone marrow (BM). In such case, CTCs may be a powerful biomarker of malignant transformation and disease aggressiveness.
Aim: To investigate the clinical significance of CTCs in patients with smoldering (SMM), newly diagnosed (NDMM) and relapsed/refractory MM (RRMM), and to compare the transcriptional profile of CTCs across the disease spectrum.
Methods: Next-generation flow (NGF) cytometry was used to assess the percentage of CTCs in PB of 1,157 patients: 316 with SMM, 650 with NDMM and 191 with RRMM. In each disease setting, patients were sub-classified into three groups with undetectable, low and high percentage of CTCs. Cutoffs were defined using maximally selected rank statistics adjusted for time to progression (TTP) in SMM and progression free survival (PFS) in NDMM/RRMM. A subset of SMM patients (n=86) was enrolled in GEM-CESAR. Transplant eligible (n=374) and ineligible (n=276) NDMM, as well as RRMM patients, were homogenously treated according to the GEM2012MENOS65, GEM-CLARIDEX and GEM-KYCYDEX clinical trials, respectively. In 40 patients (2 SMM, 33 NDMM and 5 RRMM) paired CTCs and BM tumor cells were FACSorted and their transcriptional profile was analyzed using RNAseq. Differentially expressed genes were investigated using DESeq2.
Results: CTCs were detected in 248/316 (78%), 597/650 (92%) and 170/192 (89%) of SMM, NDMM and RRMM patients. Median CTC frequencies were: 0.001% (0.05 CTCs/µL), 0.01% (0.64 CTCs/µL) and 0.005% (0.22 CTCs/µL), respectively. There were 79 genes differentially expressed between patient-matched CTCs and BM tumor cells (e.g., FLNA, EMP3, LGALS9, MUC1). These were functionally related with TNFα signaling and inflammatory response (enriched in CTCs), as well as to cell cycle and MYC targets (enriched in BM tumor cells). Interestingly, the enrichment of these signatures in CTCs and BM tumor cells was progressively more pronounced from SMM to NDMM and RRMM. Altogether, these data suggest that the CTC-based dissemination potential peaks at the stage of NDMM, which could be related to greater inflammation in BM and cell cycle arrest driving tumor cell egression into PB.
There were significant associations between the percentage of CTCs and the 2/20/20 IMWG risk model in SMM, the ISS in NDMM, and high-risk cytogenetics in all three-disease settings. Untreated SMM patients (n=230) with high CTC levels (≥0.02%) showed ultra-high risk of transformation vs those with low and undetectable CTCs (median TTP of 11 months vs not reached NR in both; P < .0001). Notably, SMM patients with ≥0.02% CTCs enrolled in GEM-CESAR have not reached a median TTP; thus, early intervention abrogated the poor prognosis of high CTC levels. Transplant-eligible NDMM patients stratified by undetectable, low and high (≥0.2%) CTC levels showed median PFS of NR, 78 and 47 months, respectively (P < .0001). Significant risk stratification was further observed in transplant ineligible NDMM (median PFS: NR, 31 and 14 months, respectively, P = .002) and RRMM (median PFS: NR, 24 and 7 months, respectively, P = .004).
In untreated SMM, multivariate analysis of TTP including CTCs, serum M-component (>2 g/dL), sFLC ratio (>20) and BM plasma cells (>20%) selected CTCs as an independent prognostic factor (hazard ratio HR: 1.61, P = .015) together with the M-component and sFLC ratio. In NDMM, multivariate analysis of PFS including CTCs, BM plasma cells counts by morphology and flow cytometry, ISS, LDH, cytogenetics and transplant eligibility showed that high CTC levels had independent prognostic value (HR: 1.43, P = .003). Only the achievement of undetectable measurable residual disease (MRD) abrogated the poor prognosis of high CTC levels.
Conclusions: This is the largest study investigating the role of CTCs in smoldering and active MM. Our results show that tumor cells are continuously trafficking in PB, possibly through a dynamic mechanism of egression that peaks in NDMM. Evaluation of CTCs in PB outperformed quantification of BM tumor burden in SMM and NDMM, and showed prognostic value in all three-disease stages. Thus, CTC assessment should be part of the diagnostic workup of MM. Early intervention in high risk SMM and undetectable MRD in NDMM may abrogate dismal outcomes associated with high CTC levels.
Puig: Amgen, Celgene, Janssen, Takeda: Consultancy; Celgene: Speakers Bureau; Celgene, Janssen, Amgen, Takeda: Research Funding; Amgen, Celgene, Janssen, Takeda and The Binding Site: Honoraria. Cedena: Janssen, Celgene and Abbvie: Honoraria. Oriol: GSK: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Oncopeptides: Consultancy, Membership on an entity's Board of Directors or advisory committees; Karyopharm: Consultancy, Membership on an entity's Board of Directors or advisory committees; Sanofi: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS/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. Sureda: Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Kite, a Gilead Company: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Sanofi: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; GSK: Consultancy, Honoraria, Speakers Bureau; Roche: Other: Support for attending meetings and/or travel; Bluebird: Membership on an entity's Board of Directors or advisory committees; Mundipharma: Consultancy; MSD: Consultancy, Honoraria, Speakers Bureau; BMS/Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Support for attending meetings and/or travel, Speakers Bureau; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Support for attending meetings and/or travel, Research Funding, Speakers Bureau. De Arriba: Amgen: Consultancy, Honoraria; Glaxo Smith Kline: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Speakers Bureau; BMS-Celgene: Consultancy, Honoraria, Speakers Bureau. Moraleda: Pfizer: Other: Educational Grants, Research Funding; Sanofi: Other: Educational Grants, Research Funding; MSD: Other: Educational Grants, Research Funding; ROCHE: Consultancy, Honoraria, Other: Educational Grants, Research Funding; Takeda: Consultancy, Honoraria, Other: Educational Grants, Research Funding; Sandoz: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Other: Educational Grants, Research Funding; Gilead: Consultancy, Honoraria, Other: Educational Grants, Research Funding; Jazz Pharmaceuticals: Consultancy, Honoraria, Other: Educational Grants, Research Funding; NovoNordisk: Other: Educational Grants, Research Funding; Janssen: Other: Educational Grants, Research Funding; Celgene: Other: Educational Grants, Research Funding; Amgen: Other: Educational Grants, Research Funding. Terpos: GSK: Honoraria, Research Funding; Genesis: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Sanofi: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; Novartis: Honoraria; Janssen-Cilag: Consultancy, Honoraria, Research Funding; BMS: Honoraria; Amgen: Consultancy, Honoraria, Research Funding. Goldschmidt: Incyte: Research Funding; Adaptive Biotechnology: Consultancy; BMS: Consultancy, Honoraria, Other: Grants and/or Provision of Investigational Medicinal Product, Research Funding; Celgene: Consultancy, Honoraria, Other: Grants and/or Provision of Investigational Medicinal Product, Research Funding; Chugai: Honoraria, Other: Grants and/or Provision of Investigational Medicinal Product, Research Funding; GSK: Honoraria; Novartis: Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Other: Grants and/or Provision of Investigational Medicinal Product, Research Funding; Johns Hopkins University: Other: Grant; Molecular Partners: Research Funding; MSD: Research Funding; Mundipharma: Research Funding; Dietmar-Hopp-Foundation: Other: Grant; Sanofi: Consultancy, Honoraria, Other: Grants and/or Provision of Investigational Medicinal Product, Research Funding; Takeda: Consultancy, Research Funding; Amgen: Consultancy, Honoraria, Other: Grants and/or Provision of Investigational Medicinal Product, Research Funding. Avet-Loiseau: Adaptive Biotechnologies: 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; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; GSK: Honoraria, Membership on an entity's Board of Directors or advisory committees; Sanofi: 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; Bristol Myers Squibb: 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. Roccaro: AstraZeneca,: Research Funding; Amgen, Celgene, Janssen, Takeda: Membership on an entity's Board of Directors or advisory committees; Associazione Italiana per la Ricerca sul Cancro (AIRC): Research Funding; European Hematology Association: Res
Background: Previous studies showed that MRD- pts after transplant may have detectable monoclonal protein through IFx, creating confusion regarding their prognostication. That said, MRD assessment in ...these pts was not performed with next generation techniques nor or in later time points. Additional discordances have been identified between multiparameter flow cytometry (MFC) and NGS, which were confirmed in recent analyses comparing NGF vs NGS.
Aim: To characterize discordances between flow cytometry vs NGS and IFx through the investigation of immature B cells sharing the same B-cell receptor immunoglobulin (BcR IG) with MM cells.
Methods: Progression-free survival (PFS) according to negative vs positive IFx was analyzed in 219 MRD- pts by MFC after transplant, enrolled in the GEM2000 and GEM2005MENOS65 trials. The same comparison was performed in 205 MRD- pts by NGF after consolidation in the GEM2012MENOS65 trial. MRD detection by NGS was compared to MFC or NGF in 140 and 104 cases, respectively. We performed NGS of BcR IG gene rearrangements (mean: 69,975 sequences) and WES (mean depth: 145x) in a total of 68 B cell samples isolated from the bone marrow (BM) of 7 MM MRD- pts by NGF after treatment (GEM2012MENOS65). These were intentionally selected to avoid contamination from MM plasma cells (PCs) during sorting of CD34 progenitors, B cell precursors, mature B cells and normal PCs. We investigated these populations for the presence of clonotypic BcR IG and somatic mutations detected in MM PCs sorted at diagnosis, using T cells as germline control. In another 10 untreated MM pts, we performed scRNA/BcRseq of total BM B cells and PCs (n=52,735), to investigate if the clonotypic BcR IG of MM PCs was detectable in other B cell stages defined by their molecular phenotype.
Results: Among 219 MRD- pts by 4 color MFC after transplant, 76 (35%) showed positive IFx and identical PFS to those with negative IFx (medians of 63 vs 66 months, p=0.96). Similarly, 23/205 (11%) MRD- pts by NGF after consolidation showed positive IFx and identical PFS to those with negative IFx (4y rates of 87% vs 78.5%, p=0.35). Thus, albeit the higher sensitivity of NGF and the later time point (consolidation), approximately 1/10 MRD- pts by NGF continued showing positive IFx, and their outcome was as favorable as that of MRD- cases in CR. We then investigated discordances between flow cytometry and NGS. Among 35 MRD- pts by 4 color MFC, 21 (60%) were MRD+ by NGS, whereas 8/44 (18%) MRD- cases by NGF were MRD+ by NGS; only one of the latter 8 pts relapsed so far. Noteworthy, 9/29 MRD- pts by MFC or NGF showed MRD levels ≥10-4 by NGS, suggesting that other factors beyond sensitivity were accounting for the discordances between MRD assessed by MFC/NGF (in the PC compartment) vs NGS (in whole BM samples).
NGS of BcR IG gene rearrangements in sorted BM cells from MRD- pts by NGF, uncovered the presence of MM clonotypes in normal PCs (4/7 pts) and in B cells (5/7 pts) at low frequencies (mean of 0.31% in both, range: 0.003% - 9.4%). These findings were confirmed by scRNA/BCRseq, which unveiled in 10/10 pts that clonotypic cells were confined mostly but not entirely within PC clusters. We next performed WES to investigate if genetic abnormalities present in MM PCs at diagnosis were detectable in the same BM cells sorted after treatment in MRD- pts. Surprisingly, 41/201 (20%) somatic mutations present in diagnostic MM PCs were detectable in CD34 progenitors (n=6/7), B-cell precursors (n=4/7), mature B cells (n=5/7) and phenotypically normal PCs (n=4/7). All somatic mutations shared by MM PCs and sorted BM normal cells were non-recurrent, and genes recurrently mutated in MM (ATM, DIS3, KRAS, LTB, MAX,) as well as copy number alterations (CNA) found in MM PCs, were undetectable in normal cells.
Conclusions: Albeit more-sensitive NGF, 11% of MRD- pts continue showing positive IFx. This should not be regarded as a false-negative result, since these pts have similar outcome to those in CR and MRD-. Our findings also suggest that, at least in some pts, discordances between NGF and NGS could be attributed to immature clonotypic cells. However, these lack most somatic mutations and CNA found in MM PCs, and therefore cannot drive disease relapse. This would explain the favorable outcome of MRD- pts by NGF despite positive NGS. From a pathogenic standpoint, our study proposes that a mutated and clonally expanded lymphopoiesis precedes secondary driver mutations or CNA leading to the expansion of MM PCs.
García-Sanz:Janssen: Honoraria, Other: Travel/accommodations/expenses; Novartis: Consultancy; Amgen: Honoraria; Gilead: Other: Research grants, Research Funding; IVS (Biomed 2-Euroclonality): Patents & Royalties: and other intellectual property; Takeda: Consultancy, Honoraria, Other: Travel/accommodations/expenses. Mateos:Janssen: 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; Seattle Genetics: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Pfizer: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Regeneron: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Roche: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Adaptive: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Oncopeptides: Honoraria, Membership on an entity’s Board of Directors or advisory committees; AbbVie: 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; Sanofi: Honoraria, Membership on an entity’s Board of Directors or advisory committees; GlaxoSmithKline: Honoraria. Chatzidimitriou:Janssen: Research Funding. San-Miguel:Bristol-Myers Squibb: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company); Celgene: Consultancy, Membership on an entity’s Board of Directors or advisory committees; Amgen: Consultancy, Membership on an entity’s Board of Directors or advisory committees; Sanofi: Consultancy, Membership on an entity’s Board of Directors or advisory committees; Takeda: Consultancy, Membership on an entity’s Board of Directors or advisory committees; Janssen: Consultancy, Membership on an entity’s Board of Directors or advisory committees; Karyopharm: Consultancy, Membership on an entity’s Board of Directors or advisory committees; GlaxoSmithKline: 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; Roche: Consultancy, Membership on an entity’s Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity’s Board of Directors or advisory committees; MSD: Consultancy, Membership on an entity’s Board of Directors or advisory committees. Paiva:Amgen: Honoraria; Janssen: Consultancy, Honoraria; Karyopharm: Consultancy, Honoraria; Kite: Consultancy; SkylineDx: Consultancy; Takeda: Consultancy, Honoraria, Research Funding; Roche: Research Funding; Adaptive: Honoraria; Sanofi: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau.
Background: Although great strides were made in the management of MM, our best chances to eradicate this malignancy may lie in preventing its progression.Most current models to predict risk of ...transformation in SMM are commonly established at diagnosis and not reevaluated over time, because some parameters such as tumor burden or genetic abnormalities require invasive bone marrow (BM) aspirates. It could be hypothesized that periodic monitoring of tumor biomarkers is needed to improve risk-stratification of SMM patients, and so would be new minimally-invasive methods that can replace those performed in BM samples. Such methods should also monitor immune profiles, to identify patients with stable tumor burden/genetics but at risk of progression due to lost immune surveillance.
Aim: Determine the level of concordance between the tumor/immune landscape in BM vs peripheral blood (PB) of SMM patients, as well as to evaluate immune profiles together with circulating tumor cell (CTC) numbers and genetic alterations every 6 months in PB, as minimally-invasive methods for identification of SMM patients at risk of developing active MM.
Methods: 300 patients are planned to be enrolled in the iMMunocell study that includes 24 sites across 8 European countries. PB samples are collected every 6 months during three years for next-generation flow (NGF) cytometry monitoring of CTCs and immune profiles. Additionally, CTCs and various immune cells are FACSorted to evaluate, every 6 months, their molecular profile in SMM patients with stable vs progressive disease. BM samples are taken at baseline and every 12 months according to patients' choice, in which the same methods described previously for PB are performed. An interim analysis was preplanned to the moment when 150 patients were enrolled.
Results: A total of 170 SMM patients were enrolled and we report here data on the first 150. Thus far, 18/150 (12%) patients progressed to MM and according to 20/20/20 criteria, 1 had low, 7 intermediate and 10 had high risk SMM. Only 7/18 cases who progressed had >20% BM plasma cells (PC) by morphology. CTCs were detectable in 107/150 (71%) patients at baseline (median of 0.001% 0% - 0.42% and 0.03 0 - 21 CTCs/µL of PB). There was no correlation (or only modestly-significant) between the percentage of CTCs and BMPC by morphology (r=0.156, p=0.065) or flow cytometry (r=0.293, p=0.02). Median CTC counts were 0.02, 0.03 and 0.11 in SMM patients with low, intermediate and high risk disease according to 20/20/20 criteria, respectively (p=0.002).
Median CTC numbers were significantly different between cases with stable vs progressive disease (0.02 vs 0.11, p=0.005). As compared to those with ≤1 CTC/µL of PB, patients with >1 CTC/uL showed significantly higher risk of transformation (8% vs 47%, p<0.001) with a median time to progression of 6 months. In addition to the 150 PB samples analyzed at baseline, another 139 specimens were processed at 6, 12 and 18 months. The fluctuation in CTC numbers every 6 months was generally low (median, 0.03 CTCs/uL; IQR, 0.003 - 0.12), though in 10% of patient-samples the absolute variation was >0.5 CTCs/uL. Data on the genetic landscape of CTCs analyzed every 6 months from baseline to disease progression will be shown at the meeting.
Immune monitoring in patient-paired PB and BM samples at baseline (n=50) uncovered that 48 of 74 innate and adaptive immune cell types measured by multidimensional flow cytometry had similar distribution. Furthermore, we found significant differences in the distribution of three CD8 T cell subsets defined by differential expression of CD28, CD127, PD1, TIGIT, in PB of SMM patients with stable vs progressive disease. In patients with longitudinal PB samples from baseline until progression to active MM (n=7), there was a significant decrease in helper effector memory CXCR3+CCR4+ and cytotoxic CD127+TIGIT+PD1+ T cells, together with a significant increase in adaptive NK cells and Tγδ CD69+ T cells.
Conclusions: This is the first study performing CTC and immune monitoring every 6 months in PB samples from patients with SMM. Our results show a significant correlation between CTC counts and stable vs progressive disease, and suggest that CTC kinetics could be complementary to the 20/20/20 criteria for real-time identification of individual SMM patients at risk of developing active MM. Beyond CTC numbers, this study is uncovering key immune cell types associated with disease progression.
Terpos:Amgen: Honoraria, Research Funding; Genesis: Honoraria, Other: travel expenses , Research Funding; Janssen: Honoraria, Other: travel expenses , Research Funding; Takeda: Honoraria, Other: travel expenses , Research Funding; Celgene: Honoraria; Medison: Honoraria. Raab:Amgen: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees, Research Funding; Heidelberg Pharma: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees. Ocio:Sanofi: Consultancy, Honoraria; Secura-Bio: Consultancy; Oncopeptides: Consultancy; Celgene: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Speakers Bureau; Amgen: Consultancy, Honoraria; MDS: Honoraria; GSK: Consultancy; Takeda: Honoraria; Asofarma: Honoraria. Martinez-Lopez:Novartis: Consultancy; Janssen-cilag: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Consultancy, Research Funding; Incyte: Consultancy, Research Funding; Janssen: Consultancy, Honoraria. de la Rubia:Amgen: Consultancy, Other: Expert Testimony; Celgene: Consultancy, Other: Expert Testimony; Janssen: Consultancy, Other: Expert Testimony; Ablynx/Sanofi: Consultancy, Other: Expert Testimony. Hajek:Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharma MAR: Consultancy, Honoraria; BMS: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Honoraria, Research Funding; Oncopeptides: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding. Ludwig:Celgene: Speakers Bureau; Janssen: Other: Advisory Boards, Speakers Bureau; Bristol Myers: Other: Advisory Boards, Speakers Bureau; Sanofi: Other: Advisory Boards, Speakers Bureau; Amgen: Other: Advisory Boards, Research Funding, Speakers Bureau; Takeda: Research Funding; Seattle Genetics: Other: Advisory Boards. Goldschmidt:Dietmar-Hopp-Foundation: Other: Grants and/or provision of Investigational Medicinal Product:; Chugai: Honoraria, Other: Grants and/or provision of Investigational Medicinal Product:, Research Funding; Incyte: Research Funding; Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other, Research Funding; Molecular Partners: Research Funding; Johns Hopkins University: Other: Grants and/or provision of Investigational Medicinal Product; Mundipharma GmbH: Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Honoraria, Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Grants and/or provision of Investigational Medicinal Product:, Research Funding; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Grants and/or provision of Investigational Medicinal Product:, Research Funding; University Hospital Heidelberg, Internal Medicine V and National Center for Tumor Diseases (NCT), Heidelberg, Germany: Current Employment; GlaxoSmithKline (GSK): Honoraria; Adaptive Biotechnology: Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Grants and/or provision of Investigational Medicinal Product, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Grants and/or provision of Investigational Medicinal Product:, Research Funding; Merck Sharp and Dohme (MSD): Research Funding. Roccaro:European Hematology Association: Research Funding; AstraZeneca: Research Funding; Transcan2-ERANET: Research Funding; Italian Association for Cancer Research (AIRC): Research Funding; Janssen: Other; Celgene: Other; Amgen: Other. San-Miguel:Amgen, BMS, Celgene, Janssen, MSD, Novartis, Takeda, Sanofi, Roche, Abbvie, GlaxoSmithKline and Karyopharm: Consultancy, Membership on an entity's Board of Directors or advisory committees. Paiva:SkylineDx: Consultancy; Takeda: Consultancy, Honoraria, Research Funding; Roche: Research Funding; Adaptive: Honoraria; Amgen: Honoraria; Janssen: Consultancy, Honoraria; Karyopharm: Consultancy, Honoraria; Kite: Consultancy; Sanofi: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau.
Background: The transformation from a normal to a cancer cell is driven by the multistep acquisition of genetic alterations. Recently, shared mutations between clonal B cells in MBL/CLL and CD34+ ...hematopoietic progenitor cells (HPC) have been identified. Similarly, a HPC origin of BRAFV600E mutations in hairy cell leukemia (HCL) has been uncovered, strengthening the notion that at least a fraction of somatic mutations may occur in CD34+ HPC before the malignant transformation of some B cell neoplasms. Since almost all WM patients have mutated MYD88L265P, it is worthy to investigate if this disease follows a similar pathogenic process than that of MBL/CLL or HCL.
Aim: Define the cellular origin of WM by comparing the genetic landscape of WM cells to that of CD34+ HPC, B cell precursors and residual normal B cells.
Methods: We used FACSorting to isolate 57 cell subsets from bone marrow (BM) aspirates of 10 WM patients: CD34+ HPC, 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 79x) was performed with 10XGenomics Exome Solution for low DNA-input due to limited numbers of some cell types. Single-cell RNA and B-cell receptor sequencing (scRNA/BCRseq) was performed 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+ HPC, B cell precursors and germinal center B cells, respectively. Upon immunization, mice from each cohort were necropsied at 5, 10 and 15 months.
Results: All 10 WM patients showed MYD88L265P and 3 had mutated CXCR4. Notably, we found MYD88L265P in B cell precursors from 1/10 cases and in residual normal B cells from 4/10 patients, which were confirmed by ASO-PCR and ddPCR. Indeed, these more sensitive methods detected MYD88L265P in B cell precursors from 6/10 cases and in residual normal B cells from 6/10 patients. CXCR4 was simultaneously mutated in B cell precursors and WM B cells from one patient. Overall, CD34+ HPC, B-cell precursors and residual normal B cells shared a median of 2 (range, 0-45; mean VAF, 0.13), 3 (range, 1-44; mean VAF, 0.168), and 6 (range, 1-56; mean VAF, 0.29) somatic mutations with WM B cells; some being found all the way from CD34+ HPC to WM B cells and PCs. Interestingly, concordance between the mutational landscape of WM B cells and PCs was <100% (median of 79%, range: 55%-100%), suggesting that not all WM B cells differentiate into PCs.
A median of 18 mutations (range, 3-26; median CCF and range, 0.72 0.07 - 1) were unique to WM cells. Importantly, clonal mutations in WM B cells were undetectable in normal cells. Thus, the few WM subclonal mutations observed in patients' lymphopoiesis could not result from contamination during FACSorting since in such cases, WM clonal mutations would become detectable in normal cells. Furthermore, copy number alterations (CNA) present in WM cells were undetectable 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. scRNA/BCRseq also uncovered transcriptional differences between clonal B cells from IgM MGUS vs WM patients (eg, proliferation, metabolism).
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+ HPC 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 MYD88L265P and CXCR4 at the B cell progenitor level. Taken together, this study suggests that in some patients, WM could develop from B cell clones carrying MYD88L265P rather than being the initiating event, and that other mutations or CNA are required for the expansion of B cells and PCs with the WM phenotype.
Motta:Roche: Honoraria; Janssen: Honoraria. Rossi:Astellas: Membership on an entity’s Board of Directors or advisory committees; Novartis: Other: Advisory board; Abbvie: Membership on an entity’s Board of Directors or advisory committees; Amgen: Honoraria; Daiichi Sankyo: Consultancy, Honoraria; Janssen: Membership on an entity’s Board of Directors or advisory committees; Celgene: Membership on an entity’s Board of Directors or advisory committees; Pfizer: Membership on an entity’s Board of Directors or advisory committees; Alexion: Membership on an entity’s Board of Directors or advisory committees; Sanofi: Honoraria; Jazz: Membership on an entity’s Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity’s Board of Directors or advisory committees. Garcia-Sanz:Takeda: Honoraria, Other: Travel, Accommodations, Expenses, Research Funding; Gilead: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses, Research Funding; Amgen: Honoraria, Other: Travel, Accommodations, Expenses, Research Funding; Janssen: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses, Research Funding; Self: Patents & Royalties: BIOMED-2 PRIMERS FOR CLONALITY ASSESSMENT; IVS technologies: Consultancy, Patents & Royalties; Novartis: Research Funding. Roccaro:Transcan2-ERANET: Research Funding; European Hematology Association: Research Funding; Amgen: Other; AstraZeneca: Research Funding; Celgene: Other; Janssen: Other; Italian Association for Cancer Research (AIRC): Research Funding. San-Miguel:Amgen, BMS, Celgene, Janssen, MSD, Novartis, Takeda, Sanofi, Roche, Abbvie, GlaxoSmithKline and Karyopharm: Consultancy, Membership on an entity’s Board of Directors or advisory committees. Paiva:Sanofi: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; SkylineDx: Consultancy; Takeda: Consultancy, Honoraria, Research Funding; Roche: Research Funding; Adaptive: Honoraria; Amgen: Honoraria; Janssen: Consultancy, Honoraria; Karyopharm: Consultancy, Honoraria; Kite: Consultancy.
Background: Although many patients with AML respond to induction chemotherapy, refractory disease is common, and relapse represents the major cause of treatment failure. Furthermore, the outcomes in ...older patients who are unable to receive intensive chemotherapy without unacceptable side effects remain dismal. Thus, the identification of novel, less toxic and effective drugs in AML is warranted. Novel mAbs have been successfully investigated in various hematological malignancies but not in AML. That notwithstanding, anti-CD38 mAbs which have demonstrated remarkable efficacy in multiple myeloma (MM), could have a potential role in AML since CD38 expression has been observed in blasts from AML patients. However, to the best of our knowledge, there are almost no data about the use of anti-CD38 mAbs in AML.
Aims: To define CD38 expression in AML, and to determine the mechanism of action and preclinical efficacy of isatuximab (SAR650984), an IgG1 anti-CD38 mAb under clinical development in MM.
Results: We used multidimensional 8-color flow cytometry to evaluate the expression of CD38 in bone marrow blasts from 111 newly diagnosed elderly AML patients. Overall, 93% of AML patients expressed CD38, with 35% of cases had bimodal (heterogenous) reactivity for CD38 whereas the remaining 58% of patients showed homogenous CD38 positive expression. Interestingly, we observed a significant correlation between the stage of blasts maturation arrest and reactivity for CD38, as the percentage of CD38+ patients progressively increased from minimally differentiated AML into subtypes without and with maturation (35% vs. 51% and 71%; p=.03).
After demonstrating that CD38 is homogenously expressed by blasts from more than half of AML patients, we measured CD38 expression in 7 AML cell lines. We selected KG-1 and MOLM-13 as cell lines representative of negative vs. bright CD38 expression, respectively. We started by analyzing if isatuximab had a direct effect on AML blasts, but after treatment with increasingly higher doses of Isatuximab (range: 0,01µg/mL - 100µg/mL) for 24h, there was no impact on cell proliferation or viability. Furthermore, while isatuximab binds to C1q, CD38 receptor density in AML blasts was insufficient to trigger complement activation on tumor cells based on the absence of C3 deposition. However, when we co-cultured CD38bright MOLM-13 blasts plus isatuximab (10µg/mL for 24h) with human leukocytes to measure ADCC and ADCP, we found that in the presence of human leukocytes, isatuximab induced a 40% increment in blast cell death; furthermore, by using sensitive FACS sorting to remove key immune cell populations from the culture, we demonstrated that the presence of NK cells was critical for the efficacy of isatuximab, whereas depletion of macrophages and T-cells had minimal impact on cell killing. Subsequently, we cultured KG-1 and MOLM-3 with NK cells isolated from six donors in the presence of isatuximab (10µg/mL for 24h) and confirmed significant (p=.03) and selective cell killing of CD38bright MOLM-13 but not CD38neg KG-1 AML blasts.
Since the efficacy of isatuximab was dependent on CD38 levels, we decided to investigate a potential synergism with all-trans retinoic acid (ATRA), as it has been described to up-regulate CD38 expression and has been investigated in non-APL AML. First, we confirmed that pre-incubation of AML cell lines with ATRA (30ng/ml for 24h) induced a 3-fold increment in CD38 expression without any effect on the viability of KG-1 and MOLM-3 cells. Then we cultured both AML lines with NK cells plus isatuximab (10µg/mL for 24h) after pre-treatment with ATRA, and observed, with selected donors, an increment in the percentage of MOLM-13 and KG-1 cell killing. Finally, we tested isatuximab (10ug/ml) ex vivo in primary samples from 11 AML patients and observed significant cell killing (median 20% of tumor lysis after 24h, p=.002) in ten out of eleven patients. Furthermore, there was a trend for higher percentage of tumor lysis in CD38+ patients compared to cases with heterogeneous CD38 expression (33% vs. 12,5%; p=0.12).
Conclusions: Using a comprehensive panel of assays, cell lines, and primary patient samples, we showed for the first time that the anti-CD38 mAb isatuximab has activity in preclinical AML models, with NK-cell mediated ADCC as the most relevant mechanism of action. CD38 may therefore represent an important and novel therapeutic target in CD38+ AML patients.
Hajek:Abbvie: Consultancy, Honoraria; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharma MAR: Consultancy, Honoraria; BMS: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Research Funding; Celgene: Consultancy, Honoraria, Research Funding. San Miguel:Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; MSD: Consultancy, Membership on an entity's Board of Directors or advisory committees; Sanofi: Consultancy, Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees. Paiva:Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Honoraria; EngMab: Research Funding; Sanofi: Consultancy, Honoraria, Research Funding; Amgen: Honoraria.
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