RESUMO
Monoclonal antibodies are standard therapeutics for several cancers including the anti-CD20 antibody rituximab for B cell non-Hodgkin lymphoma (NHL). Rituximab and other antibodies are not curative and must be combined with cytotoxic chemotherapy for clinical benefit. Here we report the eradication of human NHL solely with a monoclonal antibody therapy combining rituximab with a blocking anti-CD47 antibody. We identified increased expression of CD47 on human NHL cells and determined that higher CD47 expression independently predicted adverse clinical outcomes in multiple NHL subtypes. Blocking anti-CD47 antibodies preferentially enabled phagocytosis of NHL cells and synergized with rituximab. Treatment of human NHL-engrafted mice with anti-CD47 antibody reduced lymphoma burden and improved survival, while combination treatment with rituximab led to elimination of lymphoma and cure. These antibodies synergized through a mechanism combining Fc receptor (FcR)-dependent and FcR-independent stimulation of phagocytosis that might be applicable to many other cancers.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Antígeno CD47/imunologia , Linfoma não Hodgkin/imunologia , Linfoma não Hodgkin/terapia , Fagocitose , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais Murinos , Linfócitos B/imunologia , Linhagem Celular Tumoral , Humanos , Linfoma não Hodgkin/diagnóstico , Camundongos , Receptores Fc/imunologia , Rituximab , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Chimeric antigen receptor (CAR) T-cell therapy has had considerable success in the treatment of B-cell malignancies. Targeting the B-lineage marker CD19 has brought great advances to the treatment of acute lymphoblastic leukemia and B-cell lymphomas. However, relapse remains an issue in many cases. Such relapse can result from downregulation or loss of CD19 from the malignant cell population or expression of alternate isoforms. Consequently, there remains a need to target alternative B-cell antigens and diversify the spectrum of epitopes targeted within the same antigen. CD22 has been identified as a substitute target in cases of CD19-negative relapse. One anti-CD22 antibody-clone m971-targets a membrane-proximal epitope of CD22 and has been widely validated and used in the clinic. Here, we have compared m971-CAR with a novel CAR derived from IS7, an antibody that targets a central epitope on CD22. The IS7-CAR has superior avidity and is active and specific against CD22-positive targets, including B-acute lymphoblastic leukemia patient-derived xenograft samples. Side-by-side comparisons indicated that while IS7-CAR killed less rapidly than m971-CAR in vitro, it remains efficient in controlling lymphoma xenograft models in vivo. Thus, IS7-CAR presents a potential alternative candidate for the treatment of refractory B-cell malignancies.
Assuntos
Leucemia-Linfoma Linfoblástico de Células Precursoras , Receptores de Antígenos Quiméricos , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico , Humanos , Antígenos CD19 , Epitopos , Imunoterapia Adotiva , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , RecidivaRESUMO
The BCR consists of surface-bound Ig and a heterodimeric signaling unit comprised of CD79A and CD79B. Upon cognate Ag recognition, the receptor initiates important signals for B cell development and function. The receptor also conveys Ag-independent survival signals termed tonic signaling. Although the requirement of a CD79A/CD79B heterodimer for BCR complex assembly and surface expression is well established based on mice models, few studies have investigated this in human mature B cells. In this study, we found that human tonsillar B cells with high surface expression of IgM or IgG had potentiated BCR signaling compared with BCRlow cells, and high IgM expression in germinal center B cells was associated with reduced apoptosis. We explored the mechanism for IgM surface expression by CRISPR/Cas9-induced deletion of CD79A or CD79B in four B lymphoma cell lines. Deletion of either CD79 protein caused loss of surface IgM in all cell lines and reduced fitness in three. From two cell lines, we generated stable CD79A or CD79B knockout clones and demonstrated that loss of CD79A or CD79B caused a block in N-glycan maturation and accumulation of immature proteins, compatible with retention of BCR components in the endoplasmic reticulum. Rescue experiments with CD79B wild-type restored surface expression of CD79A and IgM with mature glycosylation, whereas a naturally occurring CD79B G137S mutant disrupting CD79A/CD79B heterodimerization did not. Our study highlights that CD79A and CD79B are required for surface IgM expression in human B cells and illuminates the importance of the IgM expression level for signaling and fitness.
Assuntos
Linfócitos B , Receptores de Antígenos de Linfócitos B , Humanos , Animais , Camundongos , Receptores de Antígenos de Linfócitos B/genética , Contagem de Células , Centro Germinativo , Imunoglobulina M , Antígenos CD79/genéticaRESUMO
CAR T cells targeting the B lymphocyte antigen CD19 have led to remarkable clinical results in B cell leukemia and lymphoma but eliminate all B lineage cells, leading to increased susceptibility to severe infections. As malignant B cells will express either immunoglobulin (Ig) light chain κ or λ, we designed a second-generation CAR targeting Igκ, IGK CAR. This construct demonstrated high target specificity but displayed reduced efficacy in the presence of serum IgG. Since CD19 CAR is insensitive to serum IgG, we designed various combinatorial CAR constructs in order to maintain the CD19 CAR T cell efficacy, but with IGK CAR target selectivity. The Kz-19BB design, combining CD19 CAR containing a 4-1BB costimulatory domain with an IGK CAR containing a CD3zeta stimulatory domain, maintained the target specificity of IgK CAR and was resistant to the presence of soluble IgG. Our results demonstrate that a combinatorial CAR approach can improve target selectivity and efficacy.
Assuntos
Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos Quiméricos/metabolismo , Antígenos CD19/metabolismo , Linfócitos B/metabolismo , Antígenos CD28/metabolismo , Linhagem Celular Tumoral , Humanos , Imunoterapia Adotiva , Linfoma/metabolismo , Receptores de Antígenos Quiméricos/química , Linfócitos T/metabolismoRESUMO
CD40 expression is required for germinal center (GC) formation and function, but the kinetics and magnitude of signaling following CD40 engagement remain poorly characterized in human B cells undergoing GC reactions. Here, differences in CD40 expression and signaling responses were compared across differentiation stages of mature human tonsillar B cells. A combination of mass cytometry and phospho-specific flow cytometry was used to quantify protein expression and CD40L-induced signaling in primary human naïve, GC, and memory B cells. Protein expression signatures of cell subsets were quantified using viSNE and Marker Enrichment Modeling (MEM). This approach revealed enriched expression of CD40 protein in GC B cells, compared to naïve and memory B cells. Despite this, GC B cells responded to CD40L engagement with lower phosphorylation of NFκB p65 during the first 30 min following CD40L activation. Before CD40L stimulation, GC B cells expressed higher levels of suppressor protein IκBα than naïve and memory B cells. Following CD40 activation, IκBα was rapidly degraded and reached equivalently low levels in naïve, GC, and memory B cells at 30 min following CD40L. Quantifying CD40 signaling responses as a function of bound ligand revealed a correlation between bound CD40L and degree of induced NFκB p65 phosphorylation, whereas comparable IκBα degradation occurred at all measured levels of CD40L binding. These results characterize cell-intrinsic signaling differences that exist in mature human B cells undergoing GC reactions. © 2019 International Society for Advancement of Cytometry.
Assuntos
Linfócitos B/fisiologia , Antígenos CD40/metabolismo , Ligante de CD40/metabolismo , Centro Germinativo/citologia , Memória Imunológica , Linfócitos B/citologia , Linfócitos B/metabolismo , Ligante de CD40/fisiologia , Células Cultivadas , Centro Germinativo/imunologia , Centro Germinativo/metabolismo , Humanos , NF-kappa B/metabolismo , Fosforilação , Transdução de Sinais/imunologiaRESUMO
Kinases downstream of B-cell antigen receptor (BCR) represent attractive targets for therapy in non-Hodgkin lymphoma (NHL). As clinical responses vary, improved knowledge regarding activation and regulation of BCR signaling in individual patients is needed. Here, using phosphospecific flow cytometry to obtain malignant B-cell signaling profiles from 95 patients representing 4 types of NHL revealed a striking contrast between chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL) tumors. Lymphoma cells from diffuse large B-cell lymphoma patients had high basal phosphorylation levels of most measured signaling nodes, whereas follicular lymphoma cells represented the opposite pattern with no or very low basal levels. MCL showed large interpatient variability in basal levels, and elevated levels for the phosphorylated forms of AKT, extracellular signal-regulated kinase, p38, STAT1, and STAT5 were associated with poor outcome. CLL tumors had elevated basal levels for the phosphorylated forms of BCR-signaling nodes (Src family tyrosine kinase, spleen tyrosine kinase [SYK], phospholipase Cγ), but had low α-BCR-induced signaling. This contrasted MCL tumors, where α-BCR-induced signaling was variable, but significantly potentiated as compared with the other types. Overexpression of CD79B, combined with a gating strategy whereby signaling output was directly quantified per cell as a function of CD79B levels, confirmed a direct relationship between surface CD79B, immunoglobulin M (IgM), and IgM-induced signaling levels. Furthermore, α-BCR-induced signaling strength was variable across patient samples and correlated with BCR subunit CD79B expression, but was inversely correlated with susceptibility to Bruton tyrosine kinase (BTK) and SYK inhibitors in MCL. These individual differences in BCR levels and signaling might relate to differences in therapy responses to BCR-pathway inhibitors.
Assuntos
Regulação Neoplásica da Expressão Gênica , Leucemia Linfocítica Crônica de Células B/genética , Linfoma Folicular/genética , Linfoma Difuso de Grandes Células B/genética , Linfoma de Célula do Manto/genética , Fosfoproteínas/genética , Receptores de Antígenos de Linfócitos B/genética , Tirosina Quinase da Agamaglobulinemia , Antígenos CD79/genética , Antígenos CD79/metabolismo , Diagnóstico Diferencial , Citometria de Fluxo , Humanos , Imunoglobulina M/genética , Leucemia Linfocítica Crônica de Células B/diagnóstico , Leucemia Linfocítica Crônica de Células B/metabolismo , Leucemia Linfocítica Crônica de Células B/patologia , Linfoma Folicular/diagnóstico , Linfoma Folicular/metabolismo , Linfoma Folicular/patologia , Linfoma Difuso de Grandes Células B/diagnóstico , Linfoma Difuso de Grandes Células B/metabolismo , Linfoma Difuso de Grandes Células B/patologia , Linfoma de Célula do Manto/diagnóstico , Linfoma de Célula do Manto/metabolismo , Linfoma de Célula do Manto/patologia , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosfolipase C gama/genética , Fosfolipase C gama/metabolismo , Fosfoproteínas/metabolismo , Fosforilação , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT1/metabolismo , Fator de Transcrição STAT5/genética , Fator de Transcrição STAT5/metabolismo , Transdução de Sinais , Análise de Célula Única , Quinase Syk/genética , Quinase Syk/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Quinases da Família src/genética , Quinases da Família src/metabolismoRESUMO
Patients with mantle cell lymphoma (MCL) generally have a dismal prognosis. Intensified induction treatment with rituximab and high dose cytarabine (R_HDAC), and consolidation with high-dose therapy with autologous stem cell support has resulted in 10-year overall survival (OS) higher than 60%. However, the clinical course varies. Diagnostic tools capable of stratifying patients include the MCL International Prognostic Index (MIPI), gene expression-based proliferation signature, Ki-67 proliferation index or tumour cell morphology. Here, we tested the performance of a newly developed Nanostring-based RNA expression-based proliferation assay (MCL35) on formalin-fixed paraffin-embedded tumour tissue from younger patients recruited in or treated according to Nordic MCL protocols compared to the prognosticators listed above. Seventy-four patients were included and the assay performed well in all cases except four, which had inadequate RNA quality. The patients were evenly distributed in the MCL35 low-, intermediate- and high-risk categories. MCL35 low- and intermediate- risk groups had overlapping progression-free survival (PFS), while patients in the high-risk category had significantly inferior PFS. Combining MCL35 with MIPI or the MIPI-C (MIPI with the addition of binary Ki67 score +/-30%) showed a better discrimination than either assessment alone. In conclusion, the MCL35 assay alone or combined with MIPI or MIPI-C scores can identify patients who still have a dismal outcome despite intensified treatment.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Linfoma de Célula do Manto/tratamento farmacológico , Linfoma de Célula do Manto/genética , Adulto , Idoso , Biópsia , Proliferação de Células , Feminino , Perfilação da Expressão Gênica/métodos , Transplante de Células-Tronco Hematopoéticas , Humanos , Estimativa de Kaplan-Meier , Linfoma de Célula do Manto/diagnóstico , Linfoma de Célula do Manto/patologia , Masculino , Pessoa de Meia-Idade , Prognóstico , RNA Neoplásico/genética , Fatores de RiscoRESUMO
Adoptive cell therapy with T-cell receptor (TCR)-engineered T cells represents a powerful method to redirect the immune system against tumours. However, although TCR recognition is restricted to a specific peptide-MHC (pMHC) complex, increasing numbers of reports have shown cross-reactivity and off-target effects with severe consequences for the patients. This demands further development of strategies to validate TCR safety prior to clinical use. We reasoned that the desired TCR signalling depends on correct pMHC recognition on the outside and a restricted clustering on the inside of the cell. Since the majority of the adverse events are due to TCR recognition of the wrong target, we tested if blocking the signalling would affect the binding. By over-expressing the c-SRC kinase (CSK), a negative regulator of LCK, in redirected T cells, we showed that peripheral blood T cells inhibited anti-CD3/anti-CD28-induced phosphorylation of ERK, whereas TCR proximal signalling was not affected. Similarly, overexpression of CSK together with a therapeutic TCR prevented pMHC-induced ERK phosphorylation. Downstream effector functions were also almost completely blocked, including pMHC-induced IL-2 release, degranulation and, most importantly, target cell killing. The lack of effector functions contrasted with the unaffected TCR expression, pMHC recognition, and membrane exchange activity (trogocytosis). Therefore, co-expression of CSK with a therapeutic TCR did not compromise target recognition and binding, but rendered T cells incapable of executing their effector functions. Consequently, we named these redirected T cells "dummy T cells" and propose to use them for safety validation of new TCRs prior to therapy.
Assuntos
Células Apresentadoras de Antígenos/imunologia , Complexo Principal de Histocompatibilidade/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/imunologia , Quinases da Família src/metabolismo , Proteína Tirosina Quinase CSK , Morte Celular , Células Cultivadas , Humanos , Fosforilação , Ligação Proteica , Receptores de Antígenos de Linfócitos T/antagonistas & inibidores , Receptores de Antígenos de Linfócitos T/genética , Transdução de Sinais , Linfócitos T/citologia , Quinases da Família src/genéticaRESUMO
Patients with common variable immunodeficiency (CVID) constitute a clinically and immunologically heterogeneous group characterized by B-cell dysfunction with hypogammaglobulinemia and defective immunoglobulin class switch of unknown etiology. Current classification systems are insufficient to achieve precise disease management. Characterization of signaling pathways essential for B-cell differentiation and class switch could provide new means to stratify patients. We evaluated constitutive and induced signaling by phospho-specific flow cytometry in 26 CVID patients and 18 healthy blood donors. Strong responses were induced both in CVID and healthy donor B cells upon activation. In contrast, constitutive phosphorylation levels of STAT3,-5,-6, Erk, PLC-γ and Syk were significantly increased in CVID B cells only. Hierarchical clustering revealed a subgroup of CVID patients with elevated constitutive phosphorylation of Syk and PLC-γ. All these patients had non-infectious complications, indicating that a distinct phosphorylation pattern of kinases in B cells identifies a clinically important subgroup of CVID patients.
Assuntos
Subpopulações de Linfócitos B/imunologia , Imunodeficiência de Variável Comum/imunologia , Fosforilação/imunologia , Fosfotransferases/imunologia , Adulto , Idoso , Feminino , Humanos , Switching de Imunoglobulina/imunologia , Ativação Linfocitária/imunologia , Masculino , Pessoa de Meia-Idade , Receptores de Antígenos de Linfócitos B/imunologia , Transdução de Sinais/imunologia , Adulto JovemRESUMO
Common variable immunodeficiency (CVID) is defined by hypogammaglobulinemia and B-cell dysfunction, with significant clinical and immunological heterogeneity. Severe non-infectious complications, such as autoimmunity, granulomatous disease and splenomegaly, constitute a major cause of morbidity in CVID patients. T cells are generally regarded important for development of these clinical features. However, while T-cell abnormalities have been found in CVID patients, functional characteristics of T cells corresponding to well-defined clinical subtypes have not been identified. As common γ-chain cytokines play important roles in survival and differentiation of T cells, characterization of their signaling pathways could reveal functional differences of clinical relevance. We characterized CVID T cells functionally by studies of cytokine-induced signaling, and correlated the findings to defined clinical subtypes. Peripheral blood T cells from 29 CVID patients and 19 healthy donors were analyzed for i) phenotype, ii) cytokine-induced (interleukin (IL)-2, IL-4, IL-7 and IL-21) phosphorylation of signal transducer and activator of transcription (STAT) 3, STAT5 and STAT6, and iii) T-helper (Th)1/Th2 polarization. Expression of IL-4 receptor and downstream signaling molecules was measured. A subgroup of CVID patients (n = 7) was identified by impaired IL-4-induced p-STAT6 in naive and memory CD4 and CD8 T cells. This corresponded to patients with the largest accumulation of severe (non-infectious) complications. The signaling defect persisted over years and was not due to constitutively activated p-STAT6. The CD4 T cells were strongly Th1-skewed, but IL-4 signaling was impaired independently of Th status. However, IL-4Rα and Janus kinase (JAK) 1 mRNA levels were significantly lower than in normal donors, providing a likely mechanism for the defective IL-4-induced p-STAT6 and Th1-bias. In conclusion, we identified a subgroup of CVID patients with defective IL-4 signaling in T cells, with severe clinical features of inflammation and autoimmunity.
Assuntos
Imunodeficiência de Variável Comum/imunologia , Imunodeficiência de Variável Comum/metabolismo , Interleucina-4/metabolismo , Transdução de Sinais , Linfócitos T/imunologia , Linfócitos T/metabolismo , Adulto , Biomarcadores , Imunodeficiência de Variável Comum/complicações , Imunodeficiência de Variável Comum/diagnóstico , Feminino , Expressão Gênica , Humanos , Imunofenotipagem , Janus Quinase 1/metabolismo , Masculino , Pessoa de Meia-Idade , Receptor de Morte Celular Programada 1/genética , Receptor de Morte Celular Programada 1/metabolismo , Fator de Transcrição STAT6/metabolismo , Índice de Gravidade de Doença , Células Th1/imunologia , Células Th1/metabolismo , Células Th2/imunologia , Células Th2/metabolismoRESUMO
Vitamin A is an essential anti-infective agent with pleiotropic effects on cells of the immune system. The goal of the present study was to unravel the impact of the vitamin A metabolite retinoic acid (RA) on B-cell survival related both to normal B-cell homeostasis and to the detrimental effects imposed by DNA-damaging agents. By combining RA with Toll-like receptor 9 (TLR9) ligands, we show that RA prevents spontaneous, irradiation- and doxorubicin-induced apoptosis of human B cells in an RA receptor-dependent manner. RA-mediated survival involved up-regulation of the anti-apoptotic protein myeloid cell leukemia 1 (MCL1) at the transcriptional level, and knock down of MCL1 by small interfering RNA partially reversed the effects of RA. To ensure that the combination of TLR9-ligands and RA would not promote the survival of malignant B cells, the combined effects of stimulation with RA and TLR9 ligands was assessed on cells from patients with B-cell malignancies. In contrast to the effects on normal B cells, the combination of TLR9 stimulation and RA neither enhanced the MCL1 levels nor inhibited the death of malignant B cells challenged by DNA-damaging agents. Taken together, the present results reveal a vital role of MCL1 in RA-mediated survival of normal B cells. Moreover, the findings suggest that RA in combination with TLR9 ligands might be useful adjuvants in the treatment of B-cell malignancies by selectively protecting normal and not malignant B cells from DNA-damage-induced cell death.
Assuntos
Linfócitos B/efeitos dos fármacos , Leucemia de Células B/tratamento farmacológico , Tretinoína/farmacologia , Apoptose/efeitos dos fármacos , Linfócitos B/fisiologia , Sobrevivência Celular/efeitos dos fármacos , Dano ao DNA , Doxorrubicina/toxicidade , Feminino , Homeostase/efeitos dos fármacos , Humanos , Leucemia de Células B/patologia , Ativação Linfocitária/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Radiação , Receptor Toll-Like 9/metabolismo , Tretinoína/química , Células Tumorais Cultivadas , Vitamina A/químicaRESUMO
The tumour microenvironment influences outcome in patients with follicular lymphoma (FL), but its impact on transformation is less studied. We investigated the prognostic significance of the tumour microenvironment on transformation and survival in FL patients treated in the rituximab era. We examined diagnostic and transformed biopsies from 52 FL patients using antibodies against CD3, CD4, CD8, CD21 (CR2), CD57 (B3GAT1), CD68, FOXP3, TIA1, PD-1 (PDCD1), PD-L1 (CD274) and PAX5. Results were compared with a second cohort of 40 FL patients without signs of transformation during a minimum of five years observation time. Cell numbers and localization were semi-quantitatively assessed. Better developed CD21+ follicular dendritic cell (FDC) meshworks at diagnosis was a negative prognostic factor for overall survival (OS), progression-free survival (PFS) and time to transformation (TTT) in patients with subsequently transformed FL. Remnants of FDC meshworks at transformation were associated with shorter OS and PFS from transformation. High degrees of intrafollicular CD68+ and PD-L1+ macrophage infiltration, high total area scores and an extrafollicular/diffuse pattern of FOXP3+ T cells and high intrafollicular scores of CD4+ T cells at diagnosis were associated with shorter TTT. Scores of several T-cell subset markers from the combined patient cohorts were predictive for transformation, especially CD4 and CD57.
Assuntos
Transformação Celular Neoplásica , Linfoma Folicular/mortalidade , Linfoma Folicular/patologia , Microambiente Tumoral , Adulto , Idoso , Antineoplásicos/uso terapêutico , Biomarcadores , Biópsia , Feminino , Seguimentos , Humanos , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Linfoma Folicular/tratamento farmacológico , Linfoma Folicular/metabolismo , Masculino , Pessoa de Meia-Idade , Gradação de Tumores , Prognóstico , Modelos de Riscos Proporcionais , Rituximab/uso terapêutico , Fatores de Tempo , Microambiente Tumoral/imunologia , Adulto JovemRESUMO
Transformation of follicular lymphoma (FL) to a more aggressive disease is associated with rapid progression and death. Existing molecular markers for transformation are few and their clinical impact is limited. Here, we report on a whole-genome study of DNA copy numbers and gene expression profiles in serial FL biopsies. We identified 698 genes with high correlation between gene expression and copy number, and the molecular network most enriched for these cis-associated genes. This network includes 14 cis-associated genes directly related to the nuclear factor κB (NF-κB) pathway. For each of these 14 genes, the correlated NF-κB target genes were identified and corresponding expression scores were defined. The scores for 6 of the cis-associated NFκB pathway genes (BTK, IGBP1, IRAK1, ROCK1, TMED7-TICAM2, and TRIM37) were significantly associated with transformation. The results suggest that genes regulating B-cell survival and activation are involved in transformation of FL.
Assuntos
Transformação Celular Neoplásica/genética , Linfoma Folicular/genética , Transcriptoma , Dosagem de Genes , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Humanos , Linfoma Folicular/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Prognóstico , Integração de SistemasRESUMO
Multiple myeloma (MM) is considered an incurable B cell malignancy, although many patients can benefit from high-dose therapy with autologous stem cell transplantation (ASCT) as a first-line treatment. In non-Hodgkin lymphoma (NHL), ASCT is usually performed after relapse with curative intent. Disease progression is often associated with increased angiogenesis, in which endothelial progenitor cells (EPC) may have a central role. Here, we investigated the clinical impact of EPC levels in peripheral blood stem cell (PBSC) autografts for MM and NHL patients who received ASCT. EPC were identified by flow cytometry as aldehyde dehydrogenase(hi) CD34(+) vascular endothelial growth factor receptor 2(+) CD133(+) cells in both MM and NHL autografts. In MM, there was a positive correlation between EPC percentage and serum (s)-ß2-microglobulin levels (r(2) = .371, P = .002). Unlike for NHL patients, MM patients with high numbers of infused EPC (EPC cells per kilogram) during ASCT had significant shorter progression-free survival (PFS) (P = .035), overall survival (P = .044) and time to next treatment (P = .009). In multivariate analysis, EPC cells per kilogram was a significant independent negative prognostic indicator of PFS (P = .03). In conclusion, the presence of high number of EPC in PBSC grafts is associated with adverse prognosis after ASCT in MM.
Assuntos
Células Endoteliais/metabolismo , Mieloma Múltiplo , Neovascularização Patológica , Transplante de Células-Tronco , Antígeno AC133 , Adulto , Idoso , Antígenos CD/metabolismo , Antígenos CD34/metabolismo , Autoenxertos , Intervalo Livre de Doença , Seguimentos , Glicoproteínas/metabolismo , Humanos , Pessoa de Meia-Idade , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/mortalidade , Mieloma Múltiplo/patologia , Mieloma Múltiplo/terapia , Neovascularização Patológica/metabolismo , Neovascularização Patológica/mortalidade , Neovascularização Patológica/patologia , Neovascularização Patológica/terapia , Peptídeos/metabolismo , Valor Preditivo dos Testes , Taxa de Sobrevida , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Follicular lymphoma (FL) is currently incurable using conventional chemotherapy or immunotherapy regimes, compelling new strategies. Advances in high-throughput sequencing technologies that can reveal oncogenic pathways have stimulated interest in tailoring therapies toward actionable somatic mutations. However, for mutation-directed therapies to be most effective, the mutations must be uniformly present in evolved tumor cells as well as in the self-renewing tumor-cell precursors. Here, we show striking intratumoral clonal diversity within FL tumors in the representation of mutations in the majority of genes as revealed by whole exome sequencing of subpopulations. This diversity captures a clonal hierarchy, resolved using immunoglobulin somatic mutations and IGH-BCL2 translocations as a frame of reference and by comparing diagnosis and relapse tumor pairs, allowing us to distinguish early versus late genetic eventsduring lymphomagenesis. We provide evidence that IGH-BCL2 translocations and CREBBP mutations are early events, whereas MLL2 and TNFRSF14 mutations probably represent late events during disease evolution. These observations provide insight into which of the genetic lesions represent suitable candidates for targeted therapies.
Assuntos
Evolução Clonal/genética , DNA de Neoplasias/genética , Linfoma Folicular/genética , Mutação/fisiologia , Células Clonais/metabolismo , Células Clonais/patologia , Progressão da Doença , Exoma/genética , Frequência do Gene , Genoma Humano/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Imunofenotipagem , Linfoma Folicular/patologia , Taxa de Mutação , Reação em Cadeia da Polimerase , RecidivaRESUMO
Defects in T-cell function in patients with cancer might influence their capacity to mount efficient antitumor immune responses. Here, we identified highly reduced IL-4-, IL-10-, and IL-21-induced phosphorylation of STAT6 and STAT3 in tumor-infiltrating T cells (TILs) in follicular lymphoma (FL) tumors, contrasting other non-Hodgkin lymphoma TILs. By combining phospho-protein-specific flow cytometry with several T-cell markers, we identified that CD4(+)CD45RO(+)CD62L(-) FL TILs were largely nonresponsive to cytokines, in contrast to the corresponding autologous peripheral blood subset. We observed differential expression of the inhibitory receptor PD-1 in FL TILs and peripheral blood T cells. Furthermore, CD4(+)PD-1(hi) FL TILs, containing T(FH) and non-T(FH) cells, had lost their cytokine responsiveness, whereas PD-1 TILs had normal cytokine signaling. However, this phenomenon was not tumor specific, because tonsil T cells were similar to FL TILs. FL tumor cells were negative for PD-1 ligands, but PD-L1(+) histiocytes were found within the T cell-rich zone of the neoplastic follicles. Disruption of the microenvironment and in vitro culture of FL TILs could restore cytokine signaling in the PD-1(hi) subset. Because FL TILs in vivo probably receive suppressive signals through PD-1, this provides a rationale for testing PD-1 Ab in combination with immunotherapy in patients with FL.
Assuntos
Linfócitos T CD4-Positivos/citologia , Citocinas/metabolismo , Linfoma Folicular/patologia , Receptor de Morte Celular Programada 1/imunologia , Receptor de Morte Celular Programada 1/metabolismo , Transdução de Sinais/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Separação Celular/métodos , Células Cultivadas , Criança , Selectina E/metabolismo , Citometria de Fluxo/métodos , Histiócitos/metabolismo , Histiócitos/patologia , Humanos , Interleucina-10/metabolismo , Interleucina-4/metabolismo , Interleucinas/metabolismo , Antígenos Comuns de Leucócito/metabolismo , Linfoma Folicular/imunologia , Linfoma Folicular/metabolismo , Tonsila Palatina/citologia , Tonsila Palatina/imunologia , Microambiente Tumoral/imunologiaAssuntos
Transformação Celular Neoplásica , Regulação Neoplásica da Expressão Gênica , Imunoterapia , Linfoma Folicular , Transformação Celular Neoplásica/imunologia , Transformação Celular Neoplásica/patologia , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/imunologia , Humanos , Linfoma Folicular/imunologia , Linfoma Folicular/terapia , Masculino , Pessoa de Meia-Idade , Estudos ProspectivosRESUMO
Pathomechanisms that activate oncogenic B-cell receptor (BCR) signaling in diffuse large B-cell lymphoma (DLBCL), are largely unknown. Kelch-like family member 6 (KLHL6) encoding a substrate-adapter for Cullin-3-RING E3 ubiquitin-ligase (CRL) with poorly established targets is recurrently mutated in DLBCL. By applying high-throughput protein interactome screens and functional characterization, we discovered that KLHL6 regulates BCR by targeting its signaling subunits CD79A and CD79B. Loss of physiological KLHL6 expression pattern was frequent among the MCD/C5-like activated B-cell DLBCLs and was associated with higher CD79B levels and dismal outcome. Mutations in the BTB domain of KLHL6 disrupted its localization and heterodimerization, and increased surface BCR levels and signaling, whereas Kelch domain mutants had the opposite effect. Malfunctions of KLHL6 mutants extended beyond proximal BCR signaling with distinct phenotypes from KLHL6 silencing. Collectively, our findings uncover how recurrent mutations in KLHL6 alter BCR signaling and induce actionable phenotypic characteristics in DLBCL.
RESUMO
Several gene-expression signatures predict survival in diffuse large B-cell lymphoma (DLBCL), but the lack of practical methods for genome-scale analysis has limited translation to clinical practice. We built and validated a simple model using one gene expressed by tumor cells and another expressed by host immune cells, assessing added prognostic value to the clinical International Prognostic Index (IPI). LIM domain only 2 (LMO2) was validated as an independent predictor of survival and the "germinal center B cell-like" subtype. Expression of tumor necrosis factor receptor superfamily member 9 (TNFRSF9) from the DLBCL microenvironment was the best gene in bivariate combination with LMO2. Study of TNFRSF9 tissue expression in 95 patients with DLBCL showed expression limited to infiltrating T cells. A model integrating these 2 genes was independent of "cell-of-origin" classification, "stromal signatures," IPI, and added to the predictive power of the IPI. A composite score integrating these genes with IPI performed well in 3 independent cohorts of 545 DLBCL patients, as well as in a simple assay of routine formalin-fixed specimens from a new validation cohort of 147 patients with DLBCL. We conclude that the measurement of a single gene expressed by tumor cells (LMO2) and a single gene expressed by the immune microenvironment (TNFRSF9) powerfully predicts overall survival in patients with DLBCL.
Assuntos
Biomarcadores Tumorais/genética , Genes Neoplásicos , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/mortalidade , Microambiente Tumoral/genética , Proteínas Adaptadoras de Transdução de Sinal , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/fisiologia , Criança , Estudos de Coortes , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/fisiologia , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Predisposição Genética para Doença , Testes Genéticos , Humanos , Proteínas com Domínio LIM , Linfoma Difuso de Grandes Células B/diagnóstico , Metaloproteínas/genética , Metaloproteínas/fisiologia , Pessoa de Meia-Idade , Neoplasias/genética , Neoplasias/patologia , Prognóstico , Proteínas Proto-Oncogênicas , Análise de Sobrevida , Microambiente Tumoral/imunologia , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/genética , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/fisiologia , Adulto JovemRESUMO
Lymphoma is the fifth most common cancer in the USA. Most lymphomas are classified as non-Hodgkin's lymphoma, and nearly 95% of these cancers are of B-cell origin. B-cell receptor (BCR) surface expression and BCR functional signaling are critical for survival and proliferation of both healthy B cells, as well as most B-lymphoma cells. Agents that inhibit various components of the BCR signaling pathway, as well as parallel signaling pathways, are currently in clinical trials for the treatment of various lymphoma subtypes, including those targeting isoforms of PI3K, mTOR and BTK. In this review, we describe the signaling pathways in healthy mature B cells, the aberrant signaling in lymphomatous B cells and the rationale for clinical trials of agents targeting these pathways as well as the results of clinical trials to date. We propose that the entry into a kinase inhibitor era of lymphoma therapy will be as transformative for our patients as the advent of the antibody or chemotherapy era before it.