CD137 (4-1BB)-activating receptor represents a promising cancer immunotherapeutic target. Yet, the cellular program driven by CD137 and its role in cancer immune surveillance remain unresolved. Using T cell-specific deletion and agonist antibodies, we found that CD137 modulates tumor infiltration of CD8+-exhausted T (Tex) cells expressing PD1, Lag-3, and Tim-3 inhibitory receptors. T cell-intrinsic, TCR-independent CD137 signaling stimulated the proliferation and the terminal differentiation of Tex precursor cells through a mechanism involving the RelA and cRel canonical NF-κB subunits and Tox-dependent chromatin remodeling. While Tex cell accumulation induced by prophylactic CD137 agonists favored tumor growth, anti-PD1 efficacy was improved with subsequent CD137 stimulation in pre-clinical mouse models. Better understanding of T cell exhaustion has crucial implications for the treatment of cancer and infectious diseases. Our results identify CD137 as a critical regulator of Tex cell expansion and differentiation that holds potential for broad therapeutic applications.

Cytogenetic abnormalities (CAs) are known to be the preponderant prognostic factor in multiple myeloma. Our team has recently developed a prognostic score based on 6 CAs, with which del(1p32) appears to be the second worst abnormality after del(17p). This study aimed to confirm the adverse effect of 1p32 deletion in patients with newly diagnosed multiple myeloma (NDMM). Among 2551 patients with newly diagnosed multiple myeloma, 11% were harboring del(1p32). Their overall survival (OS) was significantly inferior compared with patients without del(1p32) (median OS: 49 months vs 124 months). Likewise, progression-free survival was significantly shorter. More importantly, biallelic del(1p32) conferred a dramatically poorer prognosis than a monoallelic del(1p32) (median OS: 25 months vs 60 months). As expected, the OS of patients with del(1p32) significantly decreased when this abnormality was associated with other high-risk CAs [del(17p), t(4;14), or gain(1q)]. In the multivariate analysis, del(1p32) appeared as a negative prognostic factor; after adjustment for age and treatment, the risk of progression was 1.3 times higher among patients harboring del(1p32), and the risk of death was 1.9 times higher. At the dawn of risk-adapted treatment strategies, we have confirmed the adverse effect of del(1p32) in multiple myeloma and the relevance of its assessment at diagnosis.

Primary plasma cell leukemia (pPCL) is an aggressive form of multiple myeloma (MM) that has not benefited from recent therapeutic advances in the field. Because it is very rare and heterogeneous, it remains poorly understood at the molecular level. To address this issue, we performed DNA and RNA sequencing of sorted plasma cells from a large cohort of 90 newly diagnosed pPCL and compared with MM. We observed that pPCL presents a specific genomic landscape with a high prevalence of t(11;14) (about half) and high-risk genomic features such as del(17p), gain 1q, and del(1p32). In addition, pPCL displays a specific transcriptome when compared with MM. We then wanted to characterize specifically pPCL with t(11;14). We observed that this subentity displayed significantly fewer adverse cytogenetic abnormalities. This translated into better overall survival when compared with pPCL without t(11;14) (39.2 months vs 17.9 months, P = .002). Finally, pPCL with t(11;14) displayed a specific transcriptome, including differential expression of BCL2 family members. This study is the largest series of patients with pPCL reported so far.

CD8(+) T cells within the tumor microenvironment (TME) are exposed to various signals that ultimately determine functional outcomes. Here, we examined the role of the co-activating receptor CD226 (DNAM-1) in CD8(+) T cell function. The absence of CD226 expression identified a subset of dysfunctional CD8(+) T cells present in peripheral blood of healthy individuals. These cells exhibited reduced LFA-1 activation, altered TCR signaling, and a distinct transcriptomic program upon stimulation. CD226(neg) CD8(+) T cells accumulated in human and mouse tumors of diverse origin through an antigen-specific mechanism involving the transcriptional regulator Eomesodermin (Eomes). Despite similar expression of co-inhibitory receptors, CD8(+) tumor-infiltrating lymphocyte failed to respond to anti-PD-1 in the absence of CD226. Immune checkpoint blockade efficacy was hampered in Cd226(-/-) mice. Anti-CD137 (4-1 BB) agonists also stimulated Eomes-dependent CD226 loss that limited the anti-tumor efficacy of this treatment. Thus, CD226 loss restrains CD8(+) T cell function and limits the efficacy of cancer immunotherapy.

Background: Primary plasma cell leukemia (pPCL) is a rare and aggressive form of multiple myeloma (MM) with an extremely poor prognosis and distinct biological and clinical features. Because of its low incidence and its heterogeneity, biological knowledge about pPCL is lacking especially molecular process responsible for its aggressiveness. Here, we took advantage of a large series of pPCL to describe the genomic and transcriptomic landscape of pPCL, to identify potential driver mutations and pathways, and to determine their clinical impacts. Methods: To address these issues, we performed a targeted DNA sequencing and a RNA sequencing of sorted bone marrow plasma cells collected at the time of diagnosis from 96 patients with pPCL between 2014 and 2020. We compared their genomic profiles with those of 907 MM at diagnosis previously obtained in our laboratory and their transcriptomic profiles with those of 300 MM at diagnosis obtained from the IFM2009/DFCI trial (NCT01191060). Copy number aberrations (CNA), single nucleotide variants (SNV), translocations, mutations, gene expression (GE) and gene set enrichment were analyzed and correlated with clinical information (overall survival and progression-free survival). Results: Genome analysis highlighted a specific genomic profile of pPCL. Indeed, hyperdiploid karyotypes were less frequent in pPCL compared with MM (20% vs 57%, p<0,001). We found a high prevalence of translocations involving the heavy chain locus (IGH) in pPCL with higher incidences of t(11;14) (51% vs 23%, p<0,001) and t(14;16) (14% vs 3%, p<0,001), but an identical incidence of t(4;14) (11% vs 10%, p=0,7). pPCL presented more adverse cytogenetic abnormalities such as del(17p) (30% vs 9,5%, p<0,001), 1q gain (53% vs 32%, p<0,001) and del(1p32) (24% vs 9%, p<0,001). Among the 246 recurrently mutated genes in MM, mutations of TP53 (21% vs 5%, p<0,001) and IRF4 (11% vs 4%, p<0,005) were significantly more frequent in pPCL. Furthermore, pPCL presented high-risk genomic features with an increased proportion of Double Hit profiles (27% vs 5%, p<0,001) with more bi-allelic inactivation of TP53 (17% vs 3%, p<0,001) and more amp1q on the background of International Staging System III (11% vs 5%, p<0,005). Interestingly, by comparing genomic profiles from pPCL with and without t(11;14) we found two distinctive patterns. Indeed pPCL with t(11;14) showed more TP53 mutations and more bi-allelic inactivation of TP53. While pPCL without t(11;14) showed more adverse cytogenetic abnormalities such as trisomy 21, 1q gains and del(1p32). These results suggest two distinctive oncogenic mechanisms. RNA-seq analysis showed also a specific transcriptional landscape of pPCL. Indeed, unsupervised hierarchical clustering of gene expression profiles demonstrated two distinct clusters between pPCL and MM. Gene set enrichment analysis identified a significantly higher expression of genes involved in MYC Targets and G2M checkpoint, and a significantly lower expression of genes involved in P53 pathway, hypoxia and TNF alpha signaling via NF-κB. Furthermore, pPCL with and without t(11;14) presented two distinct transcriptomic patterns, in particular for genes implicated in the apoptotic machinery. Three members of the BCL2 family were differentially expressed with BCL2 and PMAIP1 [NOXA] significantly overexpressed and BCL2L1 significantly underexpressed in pPCL with t(11;14). Median PFS and OS of patients with pPCL were respectively at 11 and 15 months. Presence of TP53 mutations was associated with a significantly lower PFS (4 months, p<0,05) and OS (5 months, p<0,05). Neither the IgH translocations nor the ploidy status predicted for survival. Conclusion: To our knowledge, we present the study on the largest series of patients with pPCL. Our results provide new information on both genomic and transcriptomic landscape of pPCL. Despite their heterogeneity, pPCL present a specific mutational landscape with high prevalence of t(11;14) and high-risk genomic features. These results help to better understand oncogenicity and the aggressive behavior of pPCL and support the use of new treatment strategies such as BCL2 inhibitor (Venetoclax) for pPCL with t(11;14). Disclosures Perrot: Amgen, BMS/Celgene, Janssen, Sanofi, Takeda: Consultancy, Honoraria, Research Funding. Hulin:Celgene/Bristol-Myers Squibb, Janssen, GlaxoSmithKline, and Takeda: Honoraria.

Although anti-PD-1 and anti-CTLA-4 based immune checkpoint blockade (ICB) has represented a turning point in cancer care, clinical responses are observed only in a fraction of cancer patients. Most research focuses on the identification of additional inhibitory receptors restraining the anti-tumor functions of CD8+ T cells. By contrast, herein, we found that loss of the activating receptor CD226 (DNAM-1) was a critical mechanism affecting CD8+ T cell responsiveness to TCR stimulation. Using cancer patients’ samples and preclinical mouse models, we discovered that dysfunctional CD226-negative CD8+ T cells progressively accumulated in the tumor microenvironment through a mechanism involving the T-box transcription factor Eomesodermin (EOMES). More importantly, we demonstrated that CD226-negative tumor infiltrating lymphocytes had reduced anti-tumor functions and failed to respond to ICB. Altogether, our results revealed that CD226 loss is a critical immune escape mechanism restraining CD8+ T cell function and potentially affecting the therapeutic efficacy of cancer immunotherapy.

To the editor:Multiple myeloma (MM) is characterized by a large diversity of genetic abnormalities.[1][1][⇓][2]-[3][3] They can be classified in 3 categories: copy number changes, mutations, and translocations involving mainly the IGH gene at 14q32. Translocations are usually balanced

Key Points There is no correlation between ctDNA and bone marrow for MRD by NGS using only immunoglobulin gene rearrangements in myeloma patients.

The introduction of novel agents has led to major improvements in clinical outcomes for patients with multiple myeloma. To shorten evaluation times for new treatments, health agencies are currently examining minimal residual disease (MRD) as a surrogate end point in clinical trials. We assessed the prognostic value of MRD, measured during maintenance therapy by next-generation sequencing (NGS). MRD negativity was defined as the absence of tumor plasma cell within 1 000 000 bone marrow cells (<10-6). Data were analyzed from a recent clinical trial that evaluated the role of transplantation in newly diagnosed myeloma patients treated with lenalidomide, bortezomib, and dexamethasone (RVD). MRD negativity was achieved at least once during maintenance in 127 patients (25%). At the start of maintenance therapy, MRD was a strong prognostic factor for both progression-free survival (adjusted hazard ratio, 0.22; 95% confidence interval, 0.15-0.34; P < .001) and overall survival (adjusted hazard ratio, 0.24; 95% confidence interval, 0.11-0.54; P = .001). Patients who were MRD negative had a higher probability of prolonged progression-free survival than patients with detectable residual disease, regardless of treatment group (RVD vs transplant), cytogenetic risk profile, or International Staging System disease stage at diagnosis. These results were similar after completion of maintenance therapy. Our findings confirm the value of MRD status, as determined by NGS, as a prognostic biomarker in multiple myeloma, and suggest that this approach could be used to adapt treatment strategies in future clinical trials.

Background: Recent reports support the prognostic importance of minimal residual disease (MRD) levels in multiple myeloma (MM) patients and suggest that novel methods for MRD assessment can play a role in the evolving MM treatment paradigm (Martinez-Lopez et al., Blood 2014). The application of next-generation sequencing (NGS)-based MRD assessment has been previously demonstrated in multiple lymphoid malignancies (Faham et al., Blood 2012; Ladetto et al., Leukemia 2013). NGS-based MRD assessment requires a diagnostic sample for initial identification of the myeloma clonotype. In order for this MRD assessment approach to be clinically practical, it must allow for analysis of a diverse set of diagnostic samples. In this study, we assessed the rate of myeloma clonotype identification in 6 sample types at diagnosis: bone marrow (BM) aspirate slides, RNA extracted from CD138+ plasma cells, methanol-fixed BM cells, BM mononuclear cells, RBC-lysed BM cells, and DNA extracted from small numbers of CD138+ plasma cells. Methods: Baseline samples were collected from 606 patients with MM. The following samples were provided at baseline: bone marrow aspirate (BMA) slides (164), RNA extracted from CD138+ plasma cells (402), methanol-fixed BM cells (30), BMA cell preparations using a Ficoll protocol (13), BMA cell preparations using an RBC lysis protocol (19), and DNA extracted from small numbers of CD138+ plasma cells (5). Samples with sufficient input DNA (>15ng) were included in the analysis, although this requirement was waived for samples from CD138+ cells. The Ficoll BMA cell preparations were divided into the mononuclear cell fraction and the lower Ficoll fraction, which is typically comprised of granulocytes and erythrocytes. Identification of myeloma clonotypes was performed using Sequenta's LymphoSIGHT™ method. Briefly, using universal primer sets, we amplified immunoglobulin heavy chain (IGH) and light chain (IGK) variable, diversity, and joining gene segments from genomic DNA. Amplified products were sequenced and analyzed using standardized algorithms for clonotype determination. Myeloma-specific clonotypes were identified for each patient based on their high-frequency (>5%) within the B-cell repertoire. Results: The NGS assay identified a high-frequency myeloma clonotype in 555/606 (92%) of patients with MM. Myeloma clonotype identification rates were 141/164 (86%) in BMA slides, 375/402 (93%) in RNA extracted from CD138+ plasma cells, 30/30 (100%) in methanol cell preparations, 13/13 (100%) in Ficoll cell preparations, 18/19 (95%) in RBC lysis cell preparations, and 5/5 (100%) using small amounts of input CD138+ DNA (approximately 5000 cells). These applicability rates are consistent with previous reports of sequencing applicability in MM patients. In thirteen patients, we investigated the potential loss of myeloma-specific clonotypes due to Ficoll cell preparation. The variation in myeloma cell loss was typically low but ranged from essentially no loss to the loss of more than 90% of the myeloma cells in the PBMC of one patient compared to the RBC lysis preparation. The myeloma cells were detected in the typically discarded lower layer of the Ficoll preparation which explained the loss. Conclusions: These results suggest that sequencing based MRD analysis is applicable in >90% of patients with MM. Multiple sample types, including archived BMA slides, can be used for identification of the myeloma clonotype. Further evaluation and optimization of sample processing methods is ongoing to enable application of the sequencing method for clinical MRD assessment in MM patients. Disclosures Zheng: Sequenta, Inc.: Employment, Equity Ownership. Faham:Sequenta, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Munshi:Celgene: Consultancy; Onyx: Consultancy; Janssen: Consultancy; Sanofi-Aventi: Consultancy; Oncopep: Consultancy, Equity Ownership, Patents & Royalties.