RESUMO
Biological hallmarks of splenic marginal zone lymphoma (SMZL) remain poorly described. Herein, we performed in-depth SMZL characterization through multimodal single-cell analyses of paired blood/spleen samples. The 3'-single-cell RNA-sequencing, Cellular Indexing of Transcriptomes and Epitopes by sequencing, and 5'-V(D)J single-cell RNA-sequencing datasets were integrated to characterize SMZL transcriptome profiles, including B-cell receptor and T-cell receptor repertoires. Hyperexpanded B-cell clones in the spleen were at a memory-like stage, whereas recirculating tumor B-cells in blood encompassed multiple differentiation stages, indicating an unexpected desynchronization of the B-cell maturation program in SMZL cells. Spatial transcriptomics showed the enrichment of T-effector and T-follicular helper (TFH) signatures in the nodular subtype of SMZL. This latter also exhibited gene-based cell-cell interactions suggestive of dynamic crosstalk between TFH and cancer cells in transcriptomics, further substantiated by using imaging mass cytometry. Our findings provide a comprehensive high-resolution description of SMZL biological hallmarks and characterize, for the first time in situ, inter- and intra-patient heterogeneity at both transcriptomic and protein levels. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.
Assuntos
Linfoma de Zona Marginal Tipo Células B , Análise de Célula Única , Neoplasias Esplênicas , Transcriptoma , Humanos , Neoplasias Esplênicas/genética , Neoplasias Esplênicas/patologia , Neoplasias Esplênicas/metabolismo , Linfoma de Zona Marginal Tipo Células B/genética , Linfoma de Zona Marginal Tipo Células B/patologia , Linfoma de Zona Marginal Tipo Células B/metabolismo , Linfoma de Zona Marginal Tipo Células B/imunologia , Perfilação da Expressão Gênica/métodos , Masculino , Feminino , Pessoa de Meia-Idade , Linfócitos B/patologia , Linfócitos B/metabolismo , Idoso , Baço/patologia , Baço/imunologia , Baço/metabolismoRESUMO
BACKGROUND: One of the first clinical observations of ibrutinib activity in the treatment of chronic lymphocytic leukemia (CLL) is a rapid decline in lymph nodes size. This phenomenon is accompanied by an hyperlymphocytosis, either transient or prolonged, which is associated with distinct clinical responses and thus has an impact on long-term outcomes. Understanding which factors determine distinct disease courses upon ibrutinib treatment remains a scientific challenge. METHODS AND FINDINGS: From 2016 to 2021, we conducted a longitudinal and observational study in 2 cohorts of patients with chronic lymphocytic leukemia (CLL) (cohort 1, n = 41; cohort 2, n = 81). These cohorts reflect the well-known clinical features of CLL patients, such as Male/Female sex ratio of 2/1, a median age of 70 years at diagnosis, and include patients in first-line therapy (27%) or relapsed/refractory patients (73%). Blood cell counts were followed for each patient during 2 years of ibrutinib treatment. In addition, immunophenotyping and whole-body magnetic resonance imaging (MRI) were assessed in patients from cohort 1. These data were integrated in a newly built mathematical model, inspired by previous mathematical works on CLL treatment and combining dynamical and statistical models, leading to the identification of biological mechanisms associated with the 2 types of clinical responses. This multidisciplinary approach allowed to identify baseline parameters that dictated lymphocytes kinetics upon ibrutinib treatment. Indeed, ibrutinib-induced lymphocytosis defined 2 CLL patient subgroups, transient hyperlymphocytosis (tHL) or prolonged hyperlymphocytosis (pHL), that can be discriminated, before the treatment, by absolute counts of CD4+ T lymphocytes (p = 0.026) and regulatory CD4 T cells (p = 0.007), programmed cell death protein 1 PD1 (p = 0.022) and CD69 (p = 0.03) expression on B leukemic cells, CD19/CD5high/CXCR4low level (p = 0.04), and lymph node cellularity. We also pinpointed that the group of patients identified by the transient hyperlymphocytosis has lower duration response and a poor clinical outcome. The mathematical approach led to the reproduction of patient-specific dynamics and the estimation of associated patient-specific biological parameters, and highlighted that the differences between the 2 groups were mainly due to the production of leukemic B cells in lymph node compartments, and to a lesser extent to T lymphocytes and leukemic B cell egress into bloodstream. Access to additional data, especially longitudinal MRI data, could strengthen the conclusions regarding leukemic B cell dynamics in lymph nodes and the relevance of 2 distinct groups of patients. CONCLUSIONS: Altogether, our multidisciplinary study provides a better understanding of ibrutinib response and highlights new pharmacodynamic parameters before and along ibrutinib treatment. Since our results highlight a reduced duration response and outcome in patients with transient hyperlymphocytosis, our approach provides support for managing ibrutinib therapy after 3 months of treatment. TRIAL REGISTRATION: ClinicalTrials.gov NCT02824159.
Assuntos
Adenina , Leucemia Linfocítica Crônica de Células B , Piperidinas , Humanos , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/patologia , Adenina/análogos & derivados , Adenina/uso terapêutico , Piperidinas/uso terapêutico , Masculino , Feminino , Idoso , Pessoa de Meia-Idade , Estudos Prospectivos , Modelos Teóricos , Idoso de 80 Anos ou mais , Estudos Longitudinais , Pirimidinas/uso terapêutico , Resultado do Tratamento , Pirazóis/uso terapêutico , Pirazóis/farmacologia , Imageamento por Ressonância Magnética , Inibidores de Proteínas Quinases/uso terapêuticoRESUMO
The Bruton tyrosine kinase (BTK) inhibitor ibrutinib is widely used for treatment of patients with relapsed/refractory or treatment-naïve chronic lymphocytic leukemia (CLL). A prominent effect of ibrutinib is to disrupt the retention of CLL cells from supportive lymphoid tissues, by altering BTK-dependent adhesion and migration. To further explore the mechanism of action of ibrutinib and its potential impact on non-leukemic cells, we quantified multiple motility and adhesion parameters of human primary CLL cells and non-leukemic lymphoid cells. In vitro, ibrutinib affected CCL19-, CXCL12- and CXCL13-evoked migration behavior of CLL cells and non-neoplastic lymphocytes, by reducing both motility speed and directionality. De-phosphorylation of BTK induced by ibrutinib in CLL cells was associated with defective polarization over fibronectin and inability to assemble the immunological synapse upon B-cell receptor engagement. In patients' samples collected during a 6-month monitoring of therapy, chemokine-evoked migration was repressed in CLL cells and marginally reduced in T cells. This was accompanied by profound modulation of the expression of chemokine receptors and adhesion molecules. Remarkably, the relative expression of the receptors governing lymph node entry (CCR7) versus exit (S1PR1) stood out as a reliable predictive marker of the clinically relevant treatment-induced lymphocytosis. Together, our data reveal a multifaceted modulation of motility and adhesive properties of ibrutinib on both CLL leukemic cell and T-cell populations and point to intrinsic differences in CLL recirculation properties as an underlying cause for variability in treatment response.
Assuntos
Adenina/análogos & derivados , Leucemia Linfocítica Crônica de Células B , Piperidinas , Humanos , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Movimento Celular , Tecido Linfoide , LinfócitosRESUMO
γδ T lymphocytes represent â¼1% of human peripheral blood mononuclear cells and even more cells in most tissues of vertebrates. Although they have important anticancer functions, most current single-cell RNA sequencing (scRNA-seq) studies do not identify γδ T lymphocytes because their transcriptomes at the single-cell level are unknown. Here we show that high-resolution clustering of large scRNA-seq datasets and a combination of gene signatures allow the specific detection of human γδ T lymphocytes and identification of their T cell receptor (TCR)Vδ1 and TCRVδ2 subsets in large datasets from complex cell mixtures. In t-distributed stochastic neighbor embedding plots from blood and tumor samples, the few γδ T lymphocytes appear collectively embedded between cytotoxic CD8 T and NK cells. Their TCRVδ1 and TCRVδ2 subsets form close yet distinct subclusters, respectively neighboring NK and CD8 T cells because of expression of shared and distinct cytotoxic maturation genes. Similar pseudotime maturation trajectories of TCRVδ1 and TCRVδ2 γδ T lymphocytes were discovered, unveiling in both subsets an unattended pool of terminally differentiated effector memory cells with preserved proliferative capacity, a finding confirmed by in vitro proliferation assays. Overall, the single-cell transcriptomes of thousands of individual γδ T lymphocytes from different CMV+ and CMV- donors reflect cytotoxic maturation stages driven by the immunological history of donors. This landmark study establishes the rationale for identification, subtyping, and deep characterization of human γδ T lymphocytes in further scRNA-seq studies of complex tissues in physiological and disease conditions.
Assuntos
Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Subpopulações de Linfócitos T/imunologia , Adulto , Sequência de Bases , Linfócitos T CD8-Positivos/imunologia , Proliferação de Células/fisiologia , Células Cultivadas , Humanos , Memória Imunológica/imunologia , Células Matadoras Naturais/imunologia , Leucócitos Mononucleares/imunologia , Análise de Sequência de RNA/métodos , Transcriptoma/imunologiaRESUMO
This single-arm, open-label, phase 1b study evaluated the maximum tolerated dose (MTD) of venetoclax when given with obinutuzumab and its safety and tolerability in patients with relapsed/refractory (R/R) or previously untreated (first line [1L]) chronic lymphocytic leukemia (CLL). Venetoclax dose initially was escalated (100-400 mg) in a 3 + 3 design to define MTD combined with standard-dose obinutuzumab. Patients received venetoclax (schedule A) or obinutuzumab (schedule B) first to compare safety and determine dose/schedule for expansion. Venetoclax-obinutuzumab was administered for 6 cycles, followed by venetoclax monotherapy until disease progression (R/R) or fixed duration 1-year treatment (1L). Fifty R/R and 32 1L patients were enrolled. No dose-limiting toxicities were observed. Safety, including incidence of tumor lysis syndrome (TLS), did not differ between schedules (2 laboratory TLSs per schedule). Schedule B and a 400-mg dose of venetoclax were chosen for expansion. The most common grade 3-4 adverse event was neutropenia (R/R, 58% of patients; 1L, 53%). Rates of grade 3-4 infections were 29% (R/R) and 13% (1L); no fatal infections occurred in 1L. All infusion-related reactions were grade 1-2, except for 2 grade 3 events. No clinical TLS was observed. Overall best response rate was 95% in R/R (complete response [CR]/CR with incomplete marrow recovery [CRi], 37%) and 100% in 1L (CR/CRi, 78%) patients. Rate of undetectable (<10-4) minimal residual disease (uMRD) in peripheral blood for R/R and 1L patients, respectively, was 64% and 91% ≥3 months after last obinutuzumab dose. Venetoclax and obinutuzumab therapy had an acceptable safety profile and elicited durable responses and high rates of uMRD. This trial was registered at www.clinicaltrials.gov as #NCT01685892.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Monoclonais Humanizados/administração & dosagem , Anticorpos Monoclonais Humanizados/efeitos adversos , Compostos Bicíclicos Heterocíclicos com Pontes/administração & dosagem , Compostos Bicíclicos Heterocíclicos com Pontes/efeitos adversos , Feminino , Humanos , Masculino , Dose Máxima Tolerável , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/tratamento farmacológico , Sulfonamidas/administração & dosagem , Sulfonamidas/efeitos adversosRESUMO
BACKGROUND: Eradication of minimal residual disease (MRD), at the end of Fludarabine-Cyclophosphamide-Rituximab (FCR) treatment, is a validated surrogate marker for progression-free and overall survival in chronic lymphocytic leukaemia. But such deep responses are also associated with severe immuno-depletion, leading to infections and the development of secondary cancers. METHODS: We assessed, blood MRD and normal immune cell levels at the end of treatment, in 162 first-line FCR patients, and analysed survival and adverse event. RESULTS: Multivariate Landmark analysis 3 months after FCR completion identified unmutated IGHV status (HR, 2.03, p = 0.043), the level of MRD reached (intermediate versus low, HR, 2.43, p = 0.002; high versus low, HR, 4.56, p = 0.002) and CD4 > 200/mm3 (HR, 3.30, p < 0.001) as factors independently associated with progression-free survival (PFS); neither CD8 nor NK counts were associated with PFS. The CD4 count was associated with PFS irrespective of IGHV mutational status, but only in patients with detectable MRD (HR, 3.51, p = 0.0004, whereas it had no prognostic impact in MRD < 10- 4 patients: p = 0.6998). We next used a competitive risk model to investigate whether immune cell subsets could be associated with the risk of infection and found no association between CD4, CD8 and NK cells and infection. CONCLUSIONS: Consolidation/maintenance trials based on detectable MRD after FCR should investigate CD4 T-cell numbers both as a selection and a response criterion, and consolidation treatments should target B-cell/T-cell interactions.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Linfócitos T CD4-Positivos/patologia , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Linfócitos T CD4-Positivos/efeitos dos fármacos , Ciclofosfamida/efeitos adversos , Ciclofosfamida/uso terapêutico , Feminino , Seguimentos , Humanos , Região Variável de Imunoglobulina/genética , Imunossupressores/efeitos adversos , Imunossupressores/uso terapêutico , Leucemia Linfocítica Crônica de Células B/diagnóstico , Leucemia Linfocítica Crônica de Células B/imunologia , Leucemia Linfocítica Crônica de Células B/patologia , Contagem de Linfócitos , Masculino , Pessoa de Meia-Idade , Mutação , Neoplasia Residual , Prognóstico , Rituximab/efeitos adversos , Rituximab/uso terapêutico , Análise de Sobrevida , Resultado do Tratamento , Vidarabina/efeitos adversos , Vidarabina/análogos & derivados , Vidarabina/uso terapêuticoRESUMO
Alteration in the DNA replication, repair or recombination processes is a highly relevant mechanism of genomic instability. Despite genomic aberrations manifested in hematologic malignancies, such a defect as a source of biomarkers has been underexplored. Here, we investigated the prognostic value of expression of 82 genes involved in DNA replication-repair-recombination in a series of 99 patients with chronic lymphocytic leukemia without detectable 17p deletion or TP53 mutation. We found that expression of the POLN gene, encoding the specialized DNA polymerase ν (Pol ν) correlates with time to relapse after first-line therapy with fludarabine. Moreover, we found that POLN was the only gene up-regulated in primary patients' lymphocytes when exposed in vitro to proliferative and pro-survival stimuli. By using two cell lines that were sequentially established from the same patient during the course of the disease and Pol ν knockout mouse embryonic fibroblasts, we reveal that high relative POLN expression is important for DNA synthesis and cell survival upon fludarabine treatment. These findings suggest that Pol ν could influence therapeutic resistance in chronic lymphocytic leukemia. (Patients' samples were obtained from the CLL 2007 FMP clinical trial registered at: clinicaltrials.gov identifer: 00564512).
Assuntos
DNA Polimerase Dirigida por DNA/genética , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Leucêmica da Expressão Gênica , Leucemia Linfocítica Crônica de Células B/genética , Proteína Supressora de Tumor p53/genética , Vidarabina/análogos & derivados , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Linhagem Celular Tumoral , DNA Polimerase Dirigida por DNA/metabolismo , Progressão da Doença , Perfilação da Expressão Gênica , Humanos , Leucemia Linfocítica Crônica de Células B/diagnóstico , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/mortalidade , Camundongos , Mutação , Prognóstico , Modelos de Riscos Proporcionais , Vidarabina/farmacologia , Vidarabina/uso terapêuticoRESUMO
Recombinant human IL-15 (rhIL-15) is one of the most promising cytokines for antitumor immunotherapy. In physiology IL-15 trans-presentation by accessory cells leads to pleiotropic activities, including activation of immune cells, such as NK cells. NK cells are largely involved in Ab-dependent cellular cytotoxicity mediated by therapeutic mAbs, such as rituximab, in chronic lymphocytic leukemia (CLL). Nevertheless, in CLL, Ab-dependent cellular cytotoxicity is relatively impaired by the low E:T ratio (NK/B leukemic cells). Thus, any strategy leading to an increase in NK cell number and activation status can offer new strategies for CLL treatment. To this end, we evaluated the effect of rhIL-15 on autologous NK cell stimulation in CLL samples. We show that rhIL-15 induces NK cell activation and proliferation, leading to improved B leukemic cell depletion. This phenomenon is significantly increased in the presence of anti-CD20 mAbs. In addition, the greater effect of obinutuzumab versus rituximab suggests a cooperative role between rhIL-15 signaling and CD16 signaling in the induction of NK cell proliferation. Moreover, rhIL-15-induced proliferation of autologous NK cells is strictly dependent on their interaction with B leukemic cells, identified in this study as new accessory cells for rhIL-15 trans-presentation. Thus, rhIL-15 is able to promote NK cell-based activity in Ab immunotherapy of CLL.
Assuntos
Anticorpos Monoclonais Murinos/farmacologia , Anticorpos Monoclonais/farmacologia , Antineoplásicos/farmacologia , Interleucina-15/farmacologia , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Leucemia Linfocítica Crônica de Células B/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Monoclonais/uso terapêutico , Anticorpos Monoclonais Murinos/uso terapêutico , Antineoplásicos/uso terapêutico , Proliferação de Células/efeitos dos fármacos , Feminino , Humanos , Imunoterapia , Interleucina-15/uso terapêutico , Células Matadoras Naturais/metabolismo , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Depleção Linfocítica , Masculino , Pessoa de Meia-Idade , Receptores de IgG/metabolismo , Proteínas Recombinantes/farmacologia , RituximabRESUMO
CD8+ CTLs are thought to play a role in the control of follicular lymphoma (FL). Yet, the link between CTL tissue distribution, activation status, ability to kill FL cells in vivo, and disease progression is still elusive. Pretreatment lymph nodes from FL patients were analyzed by IHC (n = 80) or by 3-color confocal microscopy (n = 10). IHC revealed a rich infiltrate of CD8+ granzyme B+ (GrzB) cells in FL interfollicular spaces. Accordingly, confocal microscopy showed an increased number of CD3+CD8+GrzB+ CTLs and a brighter GrzB staining in individual CTL in FL samples compared with reactive lymph nodes. CTLs did not penetrate tumor nodules. In 3-dimensional (3-D) image reconstructions, CTLs were detected at the FL follicle border where they formed lytic synapse-like structures with FL B cells and with apoptotic cells, suggesting an in situ cytotoxic function. Finally, although GrzB expression in CTLs did not correlate with risk factors, high GrzB content correlated with prolonged progression free-survival (PFS) after rituximab-combined chemotherapy. Our results show the recruitment of armed CTLs with a tumor-controlling potential into FL lymph nodes and suggest that CTL-associated GrzB expression could influence PFS in FL patients having received rituximab-combined chemotherapy.
Assuntos
Imageamento Tridimensional , Linfonodos/patologia , Linfoma Folicular/epidemiologia , Linfoma Folicular/patologia , Linfócitos T Citotóxicos/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Monoclonais Murinos/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Biomarcadores Tumorais/metabolismo , Complexo CD3/metabolismo , Antígenos CD8/metabolismo , Ciclofosfamida/administração & dosagem , Doxorrubicina/administração & dosagem , Feminino , Granzimas/metabolismo , Humanos , Linfoma Folicular/tratamento farmacológico , Masculino , Microscopia Confocal/métodos , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Prednisona/administração & dosagem , Prognóstico , Fatores de Risco , Rituximab , Linfócitos T Citotóxicos/metabolismo , Vincristina/administração & dosagemRESUMO
Rituximab (RTX), a monoclonal antibody directed against the CD20 protein, is a drug commonly used in the treatment of B-cell-derived lymphoid neoplasias and of antibody-mediated autoimmune diseases. In addition to cell- and complement-mediated B-cell depletion, RTX is thought to inhibit B-cell survival and proliferation through negative regulation of canonical signaling pathways involving Akt, ERK, and mammalian target of rapamycin. However, surprisingly, although B-cell receptor (BCR) signaling has been considered critical for normal and more recently, for neoplastic B cells, the hypothesis that RTX could target BCR has never been investigated. Using follicular lymphoma cell lines as models, as well as normal B cells, we show here, for the first time, that pretreatment with RTX results in a time-dependent inhibition of the BCR-signaling cascade involving Lyn, Syk, PLC gamma 2, Akt, and ERK, and calcium mobilization. The inhibitory effect of RTX correlates with decrease of raft-associated cholesterol, complete inhibition of BCR relocalization into lipid raft microdomains, and down-regulation of BCR immunoglobulin expression. Thus, RTX-mediated alteration of BCR expression, dynamics, and signaling might contribute to the immunosuppressive activity of the drug.
Assuntos
Anticorpos Monoclonais/farmacologia , Colesterol/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo , Transdução de Sinais/efeitos dos fármacos , Anticorpos Monoclonais Murinos , Western Blotting , Cálcio/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Citometria de Fluxo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Microdomínios da Membrana/efeitos dos fármacos , Microdomínios da Membrana/metabolismo , Fosfolipase C gama/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 6/genética , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Interferência de RNA , Rituximab , Quinase Syk , Fatores de Tempo , Quinases da Família src/metabolismoAssuntos
Biomarcadores Tumorais/genética , Genes de Cadeia Pesada de Imunoglobulina/genética , Leucemia Linfocítica Crônica de Células B/genética , Mutação , RNA Nucleolar Pequeno/genética , Idoso , Feminino , Perfilação da Expressão Gênica/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Região Variável de Imunoglobulina/genética , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Prognóstico , RNA Neoplásico/genéticaRESUMO
In human blood, 1% to 5% of lymphocytes are gammadelta T cells; they mostly express the gammadelta T-cell receptor (TCR)Vgamma9, recognize nonpeptide phosphoantigens (PAgs) produced by microbes and tumor cells, and mediate different modes of lytic activities directed against tumor target cells. Antibody-dependent cell-mediated cytotoxicity (ADCC) mediated by cytolytic lymphoid cells is essential for the clinical activity of anticancer monoclonal antibodies (mAbs), but whether PAgs affect ADCC by gammadelta T cells is unknown. Here we report that, in association with the CD20(+)-specific mAb rituximab (RTX), the synthetic PAg bromohydrin pyrophosphate (BrHPP) increased TCRVgamma9(+) cell binding to CD20(+) lymphoma cells in vitro. This combination activated phospho-ZAP70 and phospho-ERK1/2 signaling in TCRVgamma9(+) cells and strongly enhanced their ADCC activity. We obtained similar results with BrHPP in the context of the mAbs alemtuzumab and trastuzumab. Furthermore, BrHPP enhanced RTX-mediated depletion of CD20(+) cells in vitro from peripheral blood mononuclear cells of healthy subjects and enhanced ADCC by gammadelta T cells from patients with chronic lymphocytic leukemia. In cynomolgus macaques, a regimen combining RTX, BrHPP, and IL2 activated TCRVgamma9(+) lymphocytes and enhanced B-cell depletion from blood and lymph nodes. Thus, the combination with BrHPP PAg is able to improve the efficacy of cancer immunotherapy by therapeutic mAbs.
Assuntos
Anticorpos Monoclonais/administração & dosagem , Citotoxicidade Celular Dependente de Anticorpos/efeitos dos fármacos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Difosfatos/farmacologia , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Adulto , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais Murinos , Protocolos de Quimioterapia Combinada Antineoplásica/química , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Linfócitos B/imunologia , Linfócitos B/metabolismo , Células Cultivadas , Difosfatos/administração & dosagem , Difosfatos/imunologia , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Humanos , Interleucina-2/administração & dosagem , Leucemia Linfocítica Crônica de Células B/sangue , Leucemia Linfocítica Crônica de Células B/imunologia , Macaca fascicularis , Masculino , Fosfatos/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Rituximab , Linfócitos T/imunologia , Linfócitos T/metabolismo , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/imunologiaRESUMO
High-definition transcriptomic studies through single-cell RNA sequencing (scRNA-Seq) have revealed the heterogeneity and functionality of the various microenvironments across numerous solid tumors. Those pioneer studies have highlighted different cellular signatures correlated with clinical response to immune checkpoint inhibitors. scRNA-Seq offers also a unique opportunity to unravel the intimate heterogeneity of the ecosystems across different lymphoma entities. In this review, we will first cover the basics and future developments of the technology, and we will discuss its input in the field of translational lymphoma research, from determination of cell-of-origin and functional diversity, to monitoring of anti-cancer targeted drugs response and toxicities, and how new improvements in both data collection and interpretation will further foster precision medicine in the upcoming years.
Assuntos
Perfilação da Expressão Gênica , Linfoma/genética , Análise de Célula Única , Transcriptoma , Biomarcadores Tumorais , Terapia Combinada , Biologia Computacional/métodos , Perfilação da Expressão Gênica/métodos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Linfoma/diagnóstico , Linfoma/patologia , Linfoma/terapia , Anotação de Sequência Molecular , Medicina de Precisão , Prognóstico , Análise de Célula Única/métodos , Resultado do TratamentoRESUMO
γδ T lymphocytes diverge from conventional T CD8 lymphocytes for ontogeny, homing, and antigen specificity, but whether their differentiation in tumors also deviates was unknown. Using innovative analyses of our original and ~150 published single-cell RNA sequencing datasets validated by phenotyping of human tumors and murine models, here we present the first high-resolution view of human γδ T cell differentiation in cancer. While γδ T lymphocytes prominently encompass TCRVγ9 cells more differentiated than T CD8 in healthy donor's blood, a different scenario is unveiled in tumors. Solid tumors and lymphomas are infiltrated by a majority of TCRVγnon9 γδ T cells which are quantitatively correlated and remarkably aligned with T CD8 for differentiation, exhaustion, gene expression profile, and response to immune checkpoint therapy. This cancer-wide association is critical for developing cancer immunotherapies.
Assuntos
Neoplasias , Transcriptoma , Animais , Linfócitos T CD8-Positivos , Diferenciação Celular , Humanos , Linfócitos do Interstício Tumoral , Camundongos , Neoplasias/genética , Receptores de Antígenos de Linfócitos T gama-delta/genética , Subpopulações de Linfócitos TRESUMO
BACKGROUND: Ibrutinib, an irreversible Bruton Tyrosine Kinase (BTK) inhibitor, has revolutionized Chronic Lymphocytic Leukemia (CLL) treatment, but resistances to ibrutinib have emerged, whether related or not to BTK mutations. Patterns of CLL evolution under ibrutinib therapy are well characterized for the leukemic cells but not for their microenvironment. METHODS: Here, we addressed this question at the single cell level of both transcriptome and immune-phenotype. The PBMCs from a CLL patient were monitored during ibrutinib treatment using Cellular Indexing of Transcriptomes and Epitopes by sequencing (CITE-Seq) technology. RESULTS: This unveiled that the short clinical relapse of this patient driven by BTK mutation is associated with intraclonal heterogeneity in B leukemic cells and up-regulation of common signaling pathways induced by ibrutinib in both B leukemic cells and immune cells. This approach also pinpointed a subset of leukemic cells present before treatment and highly enriched during progression under ibrutinib. These latter exhibit an original gene signature including up-regulated BCR, MYC-activated, and other targetable pathways. Meanwhile, although ibrutinib differentially affected the exhaustion of T lymphocytes, this treatment enhanced the T cell cytotoxicity even during disease progression. CONCLUSIONS: These results could open new alternative of therapeutic strategies for ibrutinib-refractory CLL patients, based on immunotherapy or targeting B leukemic cells themselves.
RESUMO
BACKGROUND AND OBJECTIVE: Ibrutinib is used for the treatment of chronic lymphocytic leukemia and other lymphoid malignancies. The aim of this work is to develop a population pharmacokinetic model for ibrutinib and its dihydrodiol metabolite to quantify pharmacokinetic inter- and intra-individual variability, to evaluate the impact of several covariates on ibrutinib pharmacokinetic parameters, and to examine the relationship between exposure and clinical outcome. METHODS: Patients treated with ibrutinib were included in the study and followed up for 2 years. Pharmacokinetic blood samples were taken from months 1 to 12 after inclusion. Ibrutinib and dihydrodiol-ibrutinib concentrations were assessed using ultra-performance liquid chromatography tandem mass spectrometry. A population pharmacokinetic model was developed using NONMEM version 7.4. RESULTS: A total of 89 patients and 1501 plasma concentrations were included in the pharmacokinetic analysis. The best model consisted in two compartments for each molecule. Absorption was described by a sequential zero first-order process and a lag time. Ibrutinib was either metabolised into dihydrodiol-ibrutinib or excreted through other elimination routes. A link between the dosing compartment and the dihydrodiol-ibrutinib central compartment was added to assess for high first-pass hepatic metabolism. Ibrutinib clearance had 67% and 47% inter- and intra-individual variability, respectively, while dihydrodiol-ibrutinib clearance had 51% and 26% inter- and intra-individual variability, respectively. Observed ibrutinib exposure is significantly higher in patients carrying one copy of the cytochrome P450 3A4*22 variant (1167 ng.h/mL vs 743 ng.h/mL, respectively, p = 0.024). However, no covariates with a clinically relevant effect on ibrutinib or dihydrodiol-ibrutinib exposure were identified in the PK model. An external evaluation of the model was performed. Clinical outcome was expressed as the continuation or discontinuation of ibrutinib therapy 1 year after treatment initiation. Patients who had treatment discontinuation because of toxicity had significantly higher ibrutinib area under the curve (p = 0.047). No association was found between cessation of therapy due to disease progression and ibrutinib area under the curve in patients with chronic lymphocytic leukemia. For the seven patients with mantle cell lymphoma studied, an association trend was observed between disease progression and low exposure to ibrutinib. CONCLUSIONS: We present the first population pharmacokinetic model describing ibrutinib and dihydrodiol-ibrutinib concentrations simultaneously. Large inter-individual variability and substantial intra-individual variability were estimated and could not be explained by any covariate. Higher plasma exposure to ibrutinib is associated with cessation of therapy due to the occurrence of adverse events within the first year of treatment. The association between disease progression and ibrutinib exposure in patients with mantle cell lymphoma should be further investigated. TRIAL REGISTRATION: ClinicalTrials.gov no. NCT02824159.
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Adenina/análogos & derivados , Leucemia Linfocítica Crônica de Células B , Piperidinas/farmacocinética , Adenina/farmacocinética , Adulto , Humanos , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , NaftalenosRESUMO
Acute myeloid leukemia (AML) cell lines treated by genotoxic agents or by Tumor Necrosis Factor alpha (TNFalpha) acquire potent cytotoxicity towards myeloid cells through activation of granzyme B (GrB)/perforin (PFN) system. Here we first extend this observation to another death receptor activator, Fas Ligand (FasL). Moreover, we analyzed GrB induction signalling pathway in TNFalpha- and FasL-stimulated AML cells. The effects of TNFalpha and FasL on GrB expression were specifically mediated by p38MAPK (Mitogen-activated-protein-kinase) activation. Otherwise, TNFalpha and FasL stimulation led to radical oxygen species (ROS) generation and ASK1 (Apoptosis-signal-regulating-kinase-1) activation. Endogenous activation of ASK1 by either H2O2 or thioredoxin (Trx) reductase inhibition had the same effects as TNFalpha and FasL on GrB up regulation. Altogether, our results suggest that TNFalpha- and FasL-stimulated AML cell lytic induction is regulated by a signalling pathway involving sequentially, ROS generation, Trx oxidation, ASK1 activation, p38MAPK stimulation and GrB induction at mRNA and protein levels.
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Proteína Ligante Fas/farmacologia , Granzimas/biossíntese , Granzimas/genética , Leucemia Mieloide Aguda/patologia , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologia , Animais , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Ativação Enzimática/efeitos dos fármacos , Indução Enzimática/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Granzimas/metabolismo , Humanos , Peróxido de Hidrogênio/farmacologia , MAP Quinase Quinase Quinase 5/metabolismo , Glicoproteínas de Membrana/metabolismo , Camundongos , Perforina , Proteínas Citotóxicas Formadoras de Poros/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
In a recent study, we described that UV-C irradiation resulted in redox-dependent activation and relocalization of A-SMase (acid sphingomyelinase) to the external surface of raft membrane microdomains, hydrolysis of SM (sphingomyelin) associated with the plasma membrane outer leaflet, ceramide generation and apoptosis. In the present study, we have investigated the influence of PKCzeta (protein kinase Czeta), an atypical form of PKC on this pathway. This study shows that PKCzeta overexpression resulted in the abrogation of UV-C-induced A-SMase translocation and activation into the raft microdomains, lack of ceramide generation and apoptosis inhibition. Moreover, PKCzeta overexpression resulted in a decrease in UV-C-induced ROS (reactive oxygen species) production, which correlated with increased gene expression level of various antioxidant enzymes, including TRx (thioredoxin), TR (thioredoxin reductase) 1, TR2 and peroxiredoxin 1/TPx2 (thioredoxin peroxidase 2). Importantly, enforced TPx2 gene expression inhibited UV-C-induced A-SMase translocation. Finally, PKCzeta inhibition led to a significant reduction in TPx2 protein expression. Altogether, these results suggest that PKCzeta interferes with the UV-activated sphingolipid signalling pathway by regulating the TRx system. These findings may have important consequences for UV-induced carcinogenesis and resistance to phototherapy.
Assuntos
Apoptose/efeitos da radiação , Ceramidas/biossíntese , Proteína Quinase C/metabolismo , Esfingomielina Fosfodiesterase , Animais , Linhagem Celular , Humanos , Peróxido de Hidrogênio/metabolismo , Microdomínios da Membrana/metabolismo , Oxidantes/metabolismo , Proteína Quinase C/genética , Ratos , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/fisiologia , Esfingomielina Fosfodiesterase/antagonistas & inibidores , Esfingomielina Fosfodiesterase/metabolismo , Tiorredoxinas/metabolismo , Raios UltravioletaRESUMO
In the tumoral micro-environment (TME) of chronic lymphocytic leukemia (CLL), nurse-like cells (NLC) are tumor-associated macrophages which play a critical role in the survival and chemoresistance of tumoral cells. This pro-survival activity is known to involve soluble factors, but few data are available on the relative role of cells cross-talk. Here, we used a transcriptome-based approach to systematically investigate the expression of various receptor/ligand pairs at the surface of NLC/CLL cells. Their relative contribution to CLL survival was assessed both by fluorescent microscopy to identify cellular interactions and by the use of functional tests to measure the impact of uncoupling these pairs with blocking monoclonal antibodies. We found for the first time that lymphocyte function-associated antigen 3 (LFA-3), expressed in CLL at significantly higher levels than in healthy donor B-cells, and CD2 expressed on NLC, were both key for the specific pro-survival signals delivered by NLC. Moreover, we found that NLC/CLL interactions induced the shedding of soluble LFA-3. Importantly, in an exploratory cohort of 60 CLL patients receiving frontline immunochemotherapy, increased levels of soluble LFA-3 were found to correlate with shorter overall survival. Altogether, these data suggest that LFA-3/CD2 interactions promote the survival of CLL cells in the tumor microenvironment.
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The hMutS alpha (hMSH2-hMSH6) protein heterodimer plays a critical role in the detection of DNA mispairs in the mismatch repair (MMR) process. We recently reported that hMutS alpha proteins were degraded by the ubiquitin-proteasome pathway in a cell-type-dependent manner, indicating that one or several regulator(s) may interfere with hMutS alpha protein ubiquitination and degradation. On the other hand, we and others have shown that protein kinase C (PKC) is involved as a positive regulator of MMR activity. Here, we provide evidence that the atypical PKC zeta regulates ubiquitination, degradation, and levels of hMutS alpha proteins. Using both PKC zeta-transfected U937 and PKC zeta siRNA-transfected MRC-5 cell lines, we found that PKC zeta protein expression was correlated with that of hMutS alpha as well as with MMR activity, but was inversely correlated with hMutS alpha protein ubiquitination and degradation. Interestingly, PKC zeta interacts with hMSH2 and hMSH6 proteins and phosphorylates both. Moreover, in an in vitro assay PKCzeta mediates phosphorylation events decreasing hMutS alpha protein degradation via the ubiquitin-proteasome pathway. Altogether, our results indicate that PKC zeta modulates hMutS alpha stability and protein levels, and suggest a role for PKC zeta in genome stability by regulating MMR activity.