Knowledge about the mechanism of action (MoA) of monoclonal antibodies (mAb) is required to understand which patients with multiple myeloma (MM) benefit the most from a given mAb, alone or in ...combination therapy. Although there is considerable research about daratumumab, knowledge about other anti-CD38 mAbs remains scarce.
We performed a comprehensive analysis of the MoA of isatuximab.
Isatuximab induces internalization of CD38 but not its significant release from MM cell surface. In addition, we uncovered an association between levels of CD38 expression and different MoA: (i) Isatuximab was unable to induce direct apoptosis on MM cells with CD38 levels closer to those in patients with MM, (ii) isatuximab sensitized CD38
MM cells to bortezomib plus dexamethasone in the presence of stroma, (iii) antibody-dependent cellular cytotoxicity (ADCC) was triggered by CD38
and CD38
tumor plasma cells (PC), (iv) antibody-dependent cellular phagocytosis (ADCP) was triggered only by CD38
MM cells, whereas (v) complement-dependent cytotoxicity could be triggered in less than half of the patient samples (those with elevated levels of CD38). Furthermore, we showed that isatuximab depletes CD38
B-lymphocyte precursors and natural killer (NK) lymphocytes
-the latter through activation followed by exhaustion and eventually phagocytosis.
This study provides a framework to understand response determinants in patients treated with isatuximab based on the number of MoA triggered by CD38 levels of expression, and for the design of effective combinations aimed at capitalizing disrupted tumor-stroma cell protection, augmenting NK lymphocyte-mediated ADCC, or facilitating ADCP in CD38
MM patients.
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Persistence of chemoresistant minimal residual disease (MRD) plasma cells (PCs) is associated with inferior survival in multiple myeloma (MM). Thus, characterization of the minor MRD subclone may ...represent a unique model to understand chemoresistance, but to our knowledge, the phenotypic and genetic features of the MRD subclone have never been investigated. Here, we compared the antigenic profile of MRD vs diagnostic clonal PCs in 40 elderly MM patients enrolled in the GEM2010MAS65 study and showed that the MRD subclone is enriched in cells overexpressing integrins (CD11a/CD11c/CD29/CD49d/CD49e), chemokine receptors (CXCR4), and adhesion molecules (CD44/CD54). Genetic profiling of MRD vs diagnostic PCs was performed in 12 patients; 3 of them showed identical copy number alterations (CNAs), in another 3 cases, MRD clonal PCs displayed all genetic alterations detected at diagnosis plus additional CNAs that emerged at the MRD stage, whereas in the remaining 6 patients, there were CNAs present at diagnosis that were undetectable in MRD clonal PCs, but also a selected number of genetic alterations that became apparent only at the MRD stage. The MRD subclone showed significant downregulation of genes related to protein processing in endoplasmic reticulum, as well as novel deregulated genes such as ALCAM that is prognostically relevant in MM and may identify chemoresistant PCs in vitro. Altogether, our results suggest that therapy-induced clonal selection could be already present at the MRD stage, where chemoresistant PCs show a singular phenotypic signature that may result from the persistence of clones with different genetic and gene expression profiles. This trial was registered at www.clinicaltrials.gov as #NCT01237249.
•We report for the first time the biological features of MRD cells in MM and unravel that clonal selection is already present at the MRD stage.•MRD cells show a singular phenotypic signature that may result from persisting clones with different genetic and gene expression profiles.
Patients with multiple myeloma (MM) carrying standard- or high-risk cytogenetic abnormalities (CAs) achieve similar complete response (CR) rates, but the later have inferior progression-free survival ...(PFS). This questions the legitimacy of CR as a treatment endpoint and represents a biological conundrum regarding the nature of tumor reservoirs that persist after therapy in high-risk MM. We used next-generation flow (NGF) cytometry to evaluate measurable residual disease (MRD) in MM patients with standard- vs high-risk CAs (n = 300 and 90, respectively) enrolled in the PETHEMA/GEM2012MENOS65 trial, and to identify mechanisms that determine MRD resistance in both patient subgroups (n = 40). The 36-month PFS rates were higher than 90% in patients with standard- or high-risk CAs achieving undetectable MRD. Persistent MRD resulted in a median PFS of ∼3 and 2 years in patients with standard- and high-risk CAs, respectively. Further use of NGF to isolate MRD, followed by whole-exome sequencing of paired diagnostic and MRD tumor cells, revealed greater clonal selection in patients with standard-risk CAs, higher genomic instability with acquisition of new mutations in high-risk MM, and no unifying genetic event driving MRD resistance. Conversely, RNA sequencing of diagnostic and MRD tumor cells uncovered the selection of MRD clones with singular transcriptional programs and reactive oxygen species–mediated MRD resistance in high-risk MM. Our study supports undetectable MRD as a treatment endpoint for patients with MM who have high-risk CAs and proposes characterizing MRD clones to understand and overcome MRD resistance. This trial is registered at www.clinicaltrials.gov as #NCT01916252.
•Achieving undetectable MRD overcomes the dismal prognosis of transplant-eligible multiple myeloma patients with high risk cytogenetics.•Characterization of MRD cells reveals greater clonal selection in standard-risk MM and ROS-mediated drug resistance in high-risk MM.
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Multiple myeloma (MM) patients undergo repetitive bone marrow (BM) aspirates for genetic characterization. Circulating tumor cells (CTCs) are detectable in peripheral blood (PB) of virtually all MM ...cases and are prognostic, but their applicability for noninvasive screening has been poorly investigated. Here, we used next-generation flow (NGF) cytometry to isolate matched CTCs and BM tumor cells from 53 patients and compared their genetic profile. In eight cases, tumor cells from extramedullary (EM) plasmacytomas were also sorted and whole-exome sequencing was performed in the three spatially distributed tumor samples. CTCs were detectable by NGF in the PB of all patients with MM. Based on the cancer cell fraction of clonal and subclonal mutations, we found that ~22% of CTCs egressed from a BM (or EM) site distant from the matched BM aspirate. Concordance between BM tumor cells and CTCs was high for chromosome arm-level copy number alterations (≥95%) though not for translocations (39%). All high-risk genetic abnormalities except one t(4;14) were detected in CTCs whenever present in BM tumor cells. Noteworthy, ≥82% mutations present in BM and EM clones were detectable in CTCs. Altogether, these results support CTCs for noninvasive risk-stratification of MM patients based on their numbers and genetic profile.
Background: Genetic characterization is becoming relevant to predict risk of progression in smoldering MM and is fundamental to estimate survival in active MM. Thus, patients undergo multiple bone ...marrow (BM) aspirates for genetic screening that beyond painful, may not be fully representative due to patchy BM involvement, spatial genomic heterogeneity, or extramedullary disease. Accordingly, cell-free DNA has been investigated and showed high concordance with BM aspirates, but information is typically restricted to a few recurrent mutations since comprehensive genetic characterization (eg. whole-exome sequencing, WES) is applicable to <25% of MM patients (those with ≥10% tumor DNA). By contrast, CTCs are detectable in virtually all smoldering and active MM patients and their numbers are prognostically relevant, but their applicability for non-invasive genetic characterization of MM has been poorly investigated.
Aim: To compare the genetic landscape of CTCs vs matched BM clonal plasma cells (PCs) and extramedullary (EM) plasmacytomas, and validate standardized assays for CTCs' detection, isolation and genetic characterization.
Methods: We used EuroFlow next-generation flow (NGF) cytometry to detect and isolate peripheral blood (PB) CTCs and matched BM clonal PCs from 38 MM patients (25 at diagnosis and 13 at relapse). In 8 cases, clonal PCs from EM plasmacytomas were also FACSorted. PB T cells were always used as matched germline control. In the training set, we performed custom WES (preceded by triplicates of whole-genome amplification) in matched CTCs, BM and EM clonal PCs from the 8 patients with all three spatially distributed clones. Only those mutations present in 2/3 libraries analyzed per sample were considered positive. In the validation set, we compared mutations, copy number alterations (CNA) and translocations present in CTCs and BM clonal PCs using the Chromium Exome Solution for low DNA-input (n=8), and solely CNA using the Affymetrix CytoScan HD platform (n=22). Read mapping, variant and structural calling were performed with the Multisample Exome (Dreamgenics) and LongRanger (10XGenomics) pipelines. The Chromosome Analysis Suite software (Affymetrix) was used to analyze CNA. Only those mutations with ≥10% variant allele frequency (VAF) and CNA larger than 1Mb were considered.
Results: In the training set, 193/226 (85%) and 231/269 (86%) of total mutations present in BM and EM clonal PCs, respectively, were detectable on CTCs. All MM recurrent mutations (eg. BRAF) found in BM or EM clonal PCs were present in CTCs. Of note, there were 39 mutations in EM plasmacytomas that were detectable in CTCs but absent in BM clonal PCs. Furthermore, up to 50 mutations were present in CTCs while undetectable in BM clonal PCs (n=44) or EM plasmacytomas (n=6).
After showing that CTCs harbor most mutations present in both medullary and extramedullary disease and even unveil mutations undetectable in single BM aspirates or individual EM plasmacytomas, we sought to evaluate the performance of standardized assays suitable to screen mutations and/or CNA from low cell numbers (ie. CTCs). Using 10XGenomics, 250/266 (94%) of total mutations and 17/17 (100%) of MM recurrent mutations present in BM clonal PCs were detectable on CTCs (eg. KRAS, BRAF, TP53 or FAM46C). The VAF of private mutations ranged between 0.1 and 0.3, suggesting these were subclonal in their respective spatial regions. Using 10XGenomics, 101/119 (85%) CNA and 2/2 (100%) IgH Tx present in BM clonal PCs were detectable in CTCs. Using the Cytoscan HD, there was 100% concordance between CNA in CTCs and BM clonal PCs, both at the chromosomal arm and interstitial levels.
All mutations in TP53 were detectable in CTCs. Furthermore, +1q, del(1p), del(17p) or t(4;14) were always detected in CTCs whenever present in BM clonal PCs, and confirmed by FISH. Conversely, such comprehensive genetic characterization unveiled innumerous CNA and translocations not tested by routine FISH panels eg. MYC amplification or t(6;14).
Conclusions: Using two different standardized methods, we showed in the largest series in which CTCs were genetically characterized, that these are a reliable surrogate of MM patients' genetic landscape inside and outside the BM. Because NGF is broadly used, quantification, isolation and genetic characterization of CTCs may emerge as an optimal and standardized approach for non-invasive risk-stratification of MM patients.
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. 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: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene, Janssen, Sanofi and Takeda: 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.
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.
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