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OBJECTIVE: We investigated the performance of enzyme linked immunospot (ELISpot) assay for the diagnosis of invasive aspergillosis (IA) in high-risk patients with hematologic malignancies. METHODS: We prospectively enrolled two cohorts of patients undergoing intensive myelosuppressive or immunosuppressive treatments at high risk for IA. ELISpot was performed to detect Aspergillus-specific T cells producing Interleukin-10. RESULTS: In the discovery cohort, a derived cut-off of 40 spot forming cells (SFCs)/106 PBMCs has shown to correctly classify IA cases with a sensitivity and specificity of 89.5% and 88.6%, respectively. This cut-off is lowered to 25 SFC when considering the subset of possible IA patients, with sensitivity and specificity of 76% and 93%, respectively. The application of the 40 SFCs cut-off to the validation cohort resulted in a positivity rate of 83.3% in proven/probable cases and a negativity rate of 92.5% in possible/non-IA cases. Adopting the 25 SCFs cut-off, the assay resulted positive in 83.3% of proven/probable cases while it resulted negative in 66.7% of possible/non-IA cases. CONCLUSIONS: ELISpot shows promises in the diagnosis of IA and the possibility to use two distinct cut-offs with similar diagnostic performances according to patients' different pre-test probability of infection can widen its use in patients at risk.
Assuntos
ELISPOT , Humanos , ELISPOT/métodos , Masculino , Feminino , Pessoa de Meia-Idade , Idoso , Adulto , Estudos Prospectivos , Aspergilose/diagnóstico , Aspergilose/imunologia , Interleucina-10/imunologia , Neoplasias Hematológicas/imunologia , Neoplasias Hematológicas/diagnóstico , Sensibilidade e Especificidade , Linfócitos T/imunologiaAssuntos
Herpesvirus Humano 8 , Linfo-Histiocitose Hemofagocítica , Sarcoma de Kaposi , Humanos , Linfo-Histiocitose Hemofagocítica/diagnóstico , Linfo-Histiocitose Hemofagocítica/virologia , Diagnóstico Diferencial , Sarcoma de Kaposi/diagnóstico , Sarcoma de Kaposi/virologia , Herpesvirus Humano 8/isolamento & purificação , Masculino , Síndrome da Liberação de Citocina/diagnóstico , Pessoa de Meia-IdadeRESUMO
Acute myeloid leukemia (AML) with nucleophosmin (NPM1) genetic mutations is the most common subtype in adult patients. Refractory or relapsed disease in unfit patients or after allogeneic hematopoietic stem cell transplantation (allo-HSCT) has a poor prognosis. NPM1-mutated protein, stably expressed on tumor cells but not on normal tissues, may serve as an ideal target for NPM1-mutated AML immunotherapy. The study aim was to investigate the feasibility of producing mutated-NPM1-specific cytotoxic T cells (CTLs) suitable for somatic cell therapy to prevent or treat hematologic relapse in patients with NPM1-mutated AML. T cells were expanded or primed from patient or donor peripheral blood mononuclear cells by NPM1-mutated protein-derived peptides, and tested for leukemia antigen-targeted cytotoxic activity, cytokine production and hematopoietic precursor inhibitory effect. We found that mutated-NPM1-specific CTLs, displaying specific cytokine production and high-level cytotoxicity against patients' leukemia blasts, and limited inhibitory activity in clonogenic assays, could be obtained from both patients and donors. The polyfunctional mutated-NPM1-specific CTLs included both CD8+ and CD4+ T cells endowed with strong lytic capacity. Our results suggest that mutated-NPM1-targeted CTLs may be a useful therapeutic option to control low-tumor burden relapse following conventional chemotherapy in older NPM1-mutated AML patients or eradicate persistent MRD after HSCT.
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Acute myeloid leukemia is a heterogeneous hematopoietic malignancy, characterized by uncontrolled clonal proliferation of abnormal myeloid progenitor cells, with poor outcomes. The internal tandem duplication (ITD) mutation of the Fms-like receptor tyrosine kinase 3 (FLT3) (FLT3-ITD) represents the most common genetic alteration in AML, detected in approximately 30% of AML patients, and is associated with high leukemic burden and poor prognosis. Therefore, this kinase has been regarded as an attractive druggable target for the treatment of FLT3-ITD AML, and selective small molecule inhibitors, such as quizartinib, have been identified and trialled. However, clinical outcomes have been disappointing so far due to poor remission rates, also because of acquired resistance. A strategy to overcome resistance is to combine FLT3 inhibitors with other targeted therapies. In this study, we investigated the preclinical efficacy of the combination of quizartinib with the pan PI3K inhibitor BAY-806946 in FLT3-ITD cell lines and primary cells from AML patients. We show here that BAY-806946 enhanced quizartinib cytotoxicity and, most importantly, that this combination increases the ability of quizartinib to kill CD34+ CD38-leukemia stem cells, whilst sparing normal hematopoietic stem cells. Because constitutively active FLT3 receptor tyrosine kinase is known to boost aberrant PI3K signaling, the increased sensitivity of primary cells to the above combination can be the mechanistic results of the disruption of signaling by vertical inhibition.
Assuntos
Leucemia Mieloide Aguda , Fosfatidilinositol 3-Quinases , Humanos , Fosfatidilinositol 3-Quinases/genética , Benzotiazóis/farmacologia , Benzotiazóis/uso terapêutico , Mutação , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/uso terapêutico , Tirosina Quinase 3 Semelhante a fms/genética , Tirosina Quinase 3 Semelhante a fms/uso terapêutico , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêuticoRESUMO
Multiple Myeloma (MM) typically originates from underlying precursor conditions, known as Monoclonal Gammopathy of Undetermined Significance (MGUS) and Smoldering Multiple Myeloma (SMM). Validated risk factors, related to the main features of the clonal plasma cells, are employed in the current prognostic models to assess long-term probabilities of progression to MM. In addition, new prognostic immunologic parameters, measuring protective MM-specific T-cell responses, could help to identify patients with shorter time-to-progression. In this report, we described a novel Multi-antigenic Myeloma-specific (MaMs) T-cell assay, based on ELISpot technology, providing simultaneous evaluation of T-cell responses towards ten different MM-associated antigens. When performed during long-term follow-up (mean 28 months) of 33 patients with either MGUS or SMM, such deca-antigenic myeloma-specific immunoassay allowed to significantly distinguish between stable vs. progressive disease (p < 0.001), independently from the Mayo Clinic risk category. Here, we report the first clinical experience showing that a wide (multi-antigen), standardized (irrespective to patients' HLA), MM-specific T-cell assay may routinely be applied, as a promising prognostic tool, during the follow-up of MGUS/SMM patients. Larger studies are needed to improve the antigenic panel and further explore the prognostic value of MaMs test in the risk assessment of patients with monoclonal gammopathies.
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The trajectory of B cell development goes through subsequent steps governed by complex genetic programs, strictly regulated by multiple transcription factors. Interferon regulatory factor 4 (IRF4) regulates key points from pre-B cell development and receptor editing to germinal center formation, class-switch recombination and plasma cell differentiation. The pleiotropic ability of IRF4 is mediated by its "kinetic control", allowing different IRF4 expression levels to activate distinct genetic programs due to modulation of IRF4 DNA-binding affinity. IRF4 is implicated in B cell malignancies, acting both as tumor suppressor and as tumor oncogene in different types of precursors and mature B cell neoplasia. Here, we summarize the complexity of IRF4 functions related to different DNA-binding affinity, multiple IRF4-specific target DNA motif, and interactions with transcriptional partners. Moreover, we describe the unique role of IRF4 in acute leukemias and B cell mature neoplasia, focusing on pathogenetic implications and possible therapeutic strategies in multiple myeloma and chronic lymphocytic leukemia.
Assuntos
Centro Germinativo , Neoplasias , Humanos , Diferenciação Celular , Fatores Reguladores de Interferon/genética , Fatores Reguladores de Interferon/metabolismo , DNA/metabolismoAssuntos
Leucemia Mieloide Aguda , Células-Tronco de Sangue Periférico , Humanos , Adulto , Estudos de Viabilidade , Estaurosporina/uso terapêutico , Leucemia Mieloide Aguda/terapia , Leucemia Mieloide Aguda/tratamento farmacológico , Tirosina Quinase 3 Semelhante a fms/genética , Mutação , Inibidores de Proteínas Quinases/uso terapêuticoRESUMO
Multiple Myeloma (MM) is a malignant growth of clonal plasma cells, typically arising from asymptomatic precursor conditions, namely monoclonal gammopathy of undetermined significance (MGUS) and smoldering MM (SMM). Profound immunological dysfunctions and cytokine deregulation are known to characterize the evolution of the disease, allowing immune escape and proliferation of neoplastic plasma cells. In the past decades, several studies have shown that the immune system can recognize MGUS and MM clonal cells, suggesting that anti-myeloma T cell immunity could be harnessed for therapeutic purposes. In line with this notion, chimeric antigen receptor T cell (CAR-T) therapy is emerging as a novel treatment in MM, especially in the relapsed/refractory disease setting. In this review, we focus on the pivotal contribution of T cell impairment in the immunopathogenesis of plasma cell dyscrasias and, in particular, in the disease progression from MGUS to SMM and MM, highlighting the potentials of T cell-based immunotherapeutic approaches in these settings.
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Gamopatia Monoclonal de Significância Indeterminada , Mieloma Múltiplo , Paraproteinemias , Mieloma Múltiplo Latente , Progressão da Doença , Humanos , Gamopatia Monoclonal de Significância Indeterminada/patologia , Mieloma Múltiplo/patologia , Mieloma Múltiplo/terapia , Paraproteinemias/terapia , Linfócitos T/patologiaRESUMO
In recent years, the introduction of new drugs targeting Bruton's tyrosine kinase (BTK) has allowed dramatic improvement in the prognosis of patients with chronic lymphocytic leukemia (CLL) and other B-cell neoplasms. Although these small molecules were initially considered less immunosuppressive than chemoimmunotherapy, an increasing number of reports have described the occurrence of unexpected opportunistic fungal infections, in particular invasive aspergillosis (IA). BTK represents a crucial molecule in several signaling pathways depending on different immune receptors. Based on a variety of specific off-target effects on innate immunity, namely on neutrophils, monocytes, pulmonary macrophages, and nurse-like cells, ibrutinib has been proposed as a new host factor for the definition of probable invasive pulmonary mold disease. The role of platelets in the control of fungal growth, through granule-dependent mechanisms, was described in vitro almost two decades ago and is, so far, neglected by experts in the field of clinical management of IA. In the present study, we confirm the antifungal role of platelets, and we show, for the first time, that the exposure to BTK inhibitors impairs several immune functions of platelets in response to Aspergillus fumigatus, i.e., the ability to adhere to conidia, activation (as indicated by reduced expression of P-selectin), and direct killing activity. In conclusion, our experimental data suggest that antiplatelet effects of BTK inhibitors may contribute to an increased risk for IA in CLL patients.
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Aspergilose , Infecções Fúngicas Invasivas , Leucemia Linfocítica Crônica de Células B , Tirosina Quinase da Agamaglobulinemia/metabolismo , Antifúngicos/farmacologia , Antifúngicos/uso terapêutico , Aspergilose/tratamento farmacológico , Aspergilose/metabolismo , Plaquetas/metabolismo , Humanos , Infecções Fúngicas Invasivas/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Inibidores de Proteínas Quinases/uso terapêuticoAssuntos
Anemia/complicações , Leucemia Mieloide Aguda/patologia , Neutropenia/complicações , Nucleofosmina/genética , Pancitopenia/complicações , Trombocitopenia/complicações , Adulto , Idoso , Feminino , Seguimentos , Humanos , Leucemia Mieloide Aguda/etiologia , Leucemia Mieloide Aguda/metabolismo , Masculino , Pessoa de Meia-Idade , Prognóstico , Estudos RetrospectivosRESUMO
Along with the evolution of immunophenotypic and molecular diagnostics, the assessment of Minimal Residual Disease (MRD) has progressively become a keystone in the clinical management of hematologic malignancies, enabling valuable post-therapy risk stratifications and guiding risk-adapted therapeutic approaches. However, specific prognostic values of MRD in different hematological settings, as well as its appropriate clinical uses (basically, when to measure it and how to deal with different MRD levels), still need further investigations, aiming to improve standardization and harmonization of MRD monitoring protocols and MRD-driven therapeutic strategies. Currently, MRD measurement in hematological neoplasms with bone marrow involvement is based on advanced highly sensitive methods, able to detect either specific genetic abnormalities (by PCR-based techniques and next-generation sequencing) or tumor-associated immunophenotypic profiles (by multiparametric flow cytometry, MFC). In this review, we focus on the growing clinical role for MFC-MRD diagnostics in hematological malignancies-from acute myeloid and lymphoblastic leukemias (AML, B-ALL and T-ALL) to chronic lymphocytic leukemia (CLL) and multiple myeloma (MM)-providing a comparative overview on technical aspects, clinical implications, advantages and pitfalls of MFC-MRD monitoring in different clinical settings.
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The C-terminal aminoacidic sequence from NPM1-mutated protein, absent in normal human tissues, may serve as a leukemia-specific antigen and can be considered an ideal target for NPM1-mutated acute myeloid leukemia (AML) immunotherapy. Different in silico instruments and in vitro/ex vivo immunological platforms have identified the most immunogenic epitopes from NPM1-mutated protein. Spontaneous development of endogenous NPM1-mutated-specific cytotoxic T cells has been observed in patients, potentially contributing to remission maintenance and prolonged survival. Genetically engineered T cells, namely CAR-T or TCR-transduced T cells, directed against NPM1-mutated peptides bound to HLA could prospectively represent a promising therapeutic approach. Although either adoptive or vaccine-based immunotherapies are unlikely to be highly effective in patients with full-blown leukemia, these strategies, potentially in combination with immune-checkpoint inhibitors, could be promising in maintaining remission or preemptively eradicating persistent measurable residual disease, mainly in patients ineligible for allogeneic hematopoietic stem cell transplant (HSCT). Alternatively, neoantigen-specific donor lymphocyte infusion derived from healthy donors and targeting NPM1-mutated protein to selectively elicit graft-versus-leukemia effect may represent an attractive option in subjects experiencing post-HSCT relapse. Future studies are warranted to further investigate dynamics of NPM1-mutated-specific immunity and explore whether novel individualized immunotherapies may have potential clinical utility in NPM1-mutated AML patients.
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Antígenos de Neoplasias/imunologia , Leucemia Mieloide Aguda/imunologia , Proteínas Nucleares/genética , Linfócitos T/imunologia , Animais , Humanos , Imunoterapia/métodos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/terapia , Mutação , Proteínas Nucleares/imunologia , NucleofosminaRESUMO
Acute myeloid leukemia (AML) carrying inv(16)/t(16;16), resulting in fusion transcript CBFB-MYH11, belongs to the favorable-risk category. However, even if most patients obtain morphological complete remission after induction, approximately 30% of cases eventually relapse. While well-established clinical features and concomitant cytogenetic/molecular lesions have been recognized to be relevant to predict prognosis at disease onset, the independent prognostic impact of measurable residual disease (MRD) monitoring by quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR), mainly in predicting relapse, actually supersedes other prognostic factors. Although the ELN Working Party recently indicated that patients affected with CBFB-MYH11 AML should have MRD assessment at informative clinical timepoints, at least after two cycles of intensive chemotherapy and after the end of treatment, several controversies could be raised, especially on the frequency of subsequent serial monitoring, the most significant MRD thresholds (most commonly 0.1%) and on the best source to be analyzed, namely, bone marrow or peripheral blood samples. Moreover, persisting low-level MRD positivity at the end of treatment is relatively common and not predictive of relapse, provided that transcript levels remain stably below specific thresholds. Rising MRD levels suggestive of molecular relapse/progression should thus be confirmed in subsequent samples. Further prospective studies would be required to optimize post-remission monitoring and to define effective MRD-based therapeutic strategies.
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The Philadelphia-negative myeloproliferative neoplasms (MPNs) are malignancies of the hematopoietic stem cell (HSC) arising as a consequence of clonal proliferation driven by somatically acquired driver mutations in discrete genes (JAK2, CALR, MPL). In recent years, along with the advances in molecular characterization, the role of immune dysregulation has been achieving increasing relevance in the pathogenesis and evolution of MPNs. In particular, a growing number of studies have shown that MPNs are often associated with detrimental cytokine milieu, expansion of the monocyte/macrophage compartment and myeloid-derived suppressor cells, as well as altered functions of T cells, dendritic cells and NK cells. Moreover, akin to solid tumors and other hematological malignancies, MPNs are able to evade T cell immune surveillance by engaging the PD-1/PD-L1 axis, whose pharmacological blockade with checkpoint inhibitors can successfully restore effective antitumor responses. A further interesting cue is provided by the recent discovery of the high immunogenic potential of JAK2V617F and CALR exon 9 mutations, that could be harnessed as intriguing targets for innovative adoptive immunotherapies. This review focuses on the recent insights in the immunological dysfunctions contributing to the pathogenesis of MPNs and outlines the potential impact of related immunotherapeutic approaches.
Assuntos
Células-Tronco Hematopoéticas/imunologia , Imunoterapia/métodos , Inflamação/imunologia , Transtornos Mieloproliferativos/terapia , Linfócitos T/imunologia , Microambiente Tumoral/imunologia , Calreticulina/genética , Calreticulina/imunologia , Calreticulina/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Humanos , Inflamação/genética , Janus Quinase 2/genética , Janus Quinase 2/imunologia , Janus Quinase 2/metabolismo , Mutação/imunologia , Transtornos Mieloproliferativos/genética , Transtornos Mieloproliferativos/imunologia , Cromossomo Filadélfia , Linfócitos T/metabolismo , Microambiente Tumoral/genéticaRESUMO
Nucleophosmin (NPM1) gene mutations rarely occur in non-acute myeloid neoplasms (MNs) with <20% blasts. Among nearly 10,000 patients investigated so far, molecular analyses documented NPM1 mutations in around 2% of myelodysplastic syndrome (MDS) cases, mainly belonging to MDS with excess of blasts, and 3% of myelodysplastic/myeloproliferative neoplasm (MDS/MPN) cases, prevalently classified as chronic myelomonocytic leukemia. These uncommon malignancies are associated with an aggressive clinical course, relatively rapid progression to overt acute myeloid leukemia (AML) and poor survival outcomes, raising controversies on their classification as distinct clinico-pathologic entities. Furthermore, fit patients with NPM1-mutated MNs with <20% blasts could benefit most from upfront intensive chemotherapy for AML rather than from moderate intensity MDS-directed therapies, although no firm conclusion can currently be drawn on best therapeutic approaches, due to the limited available data, obtained from small and mainly retrospective series. Caution is also suggested in definitely diagnosing NPM1-mutated MNs with blast count <20%, since NPM1-mutated AML cases frequently present dysplastic features and multilineage bone marrow cells showing abnormal cytoplasmic NPM1 protein delocalization by immunohistochemical staining, therefore belonging to NPM1-mutated clone regardless of blast morphology. Further prospective studies are warranted to definitely assess whether NPM1 mutations may become sufficient to diagnose AML, irrespective of blast percentage.
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Crise Blástica/genética , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Mutação/genética , Proteínas Nucleares/genética , Animais , Modelos Animais de Doenças , Hematopoese/genética , Humanos , NucleofosminaRESUMO
Infections represent a cause of morbidity and mortality in patients affected by chronic lymphocytic leukemia (CLL). Introduction of new drugs in CLL clinical practice has showed impressive efficacy, in particular those targeting BTK. Among the consistent clinical data, an increasing number of reports describing the occurrence of unexpected opportunistic fungal infections has been reported during treatment with ibrutinib in the first 6 months of treatment. The reason underlying manifestations of invasive fungal infections in patients treated with ibrutinib is still under investigation. Our study aimed to understand the impact of BTK inhibition on immune response to fungal infection mediated by macrophages and CD14+ monocytic population obtained from CLL patients. Exposure to ibrutinib and acalabrutinib reduced signaling pathways activated by Aspergillus fumigatus determining an exacerbation of an immunosuppressive signature, a reduction of phagocytosis and a significant deficit in the secretion of inflammatory cytokines either in macrophages and monocytes isolated from CLL patients and healthy donors. These effects lead to a failure in completely counteracting conidia germination. In addition we investigated the biological effects of ibrutinib on monocyte counterpart in patients who were undergoing therapy. A significant impairment in cytokine secretion and a deficit of phagocytosis in circulating monocytes were detected after 3 months of treatment. Thus, our results uncover modifications in the innate response in CLL patients induced by ibrutinib that may impair the immunological response to fungal infection. KEYPOINTS: â¢BTK inhibition affects a productive immune response of CLL-associated macrophages (NLC) during Aspergillus fumigatus infection.â¢Reduction of TNF-α secretion and phagocytosis are detected in monocytes isolated from CLL patients during ibrutinib therapy.
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Adenina/análogos & derivados , Tirosina Quinase da Agamaglobulinemia/antagonistas & inibidores , Antineoplásicos/uso terapêutico , Aspergilose/imunologia , Aspergillus fumigatus/fisiologia , Leucemia Linfocítica Crônica de Células B/imunologia , Macrófagos/imunologia , Piperidinas/uso terapêutico , Adenina/farmacologia , Adenina/uso terapêutico , Antineoplásicos/farmacologia , Humanos , Imunidade Inata , Imunomodulação , Macrófagos/microbiologia , Fagocitose , Piperidinas/farmacologia , Transdução de Sinais , Células Tumorais Cultivadas , Fator de Necrose Tumoral alfa/metabolismoAssuntos
Anemia Aplástica/epidemiologia , Tuberculose Latente/epidemiologia , Leucemia Mieloide Aguda/epidemiologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/epidemiologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Anemia Aplástica/tratamento farmacológico , Antituberculosos/uso terapêutico , Vacina BCG , Terapia Combinada , Comorbidade , Neutropenia Febril/epidemiologia , Transplante de Células-Tronco Hematopoéticas , Humanos , Testes de Liberação de Interferon-gama , Isoniazida/uso terapêutico , Tuberculose Latente/diagnóstico por imagem , Tuberculose Latente/tratamento farmacológico , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/terapia , Pessoa de Meia-Idade , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Estudos Retrospectivos , Adulto JovemRESUMO
Both human immunodeficiency virus (HIV) infection and acute myeloid leukemia (AML) may be considered relatively uncommon disorders in the general population, but the precise incidence of AML in people living with HIV infection (PLWH) is uncertain. However, life expectancy of newly infected HIV-positive patients receiving anti-retroviral therapy (ART) is gradually increasing, rivaling that of age-matched HIV-negative individuals, so that the occurrence of AML is also expected to progressively increase. Even if HIV is not reported to be directly mutagenic, several indirect leukemogenic mechanisms, mainly based on bone marrow microenvironment disruption, have been proposed. Despite a well-controlled HIV infection under ART should no longer be considered per se a contraindication to intensive chemotherapeutic approaches, including allogeneic hematopoietic stem cell transplantation, in selected fit patients with AML, survival outcomes are still generally unsatisfactory. We discussed several controversial issues about pathogenesis and clinical management of AML in PLWH, but few evidence-based answers may currently be provided, due to the limited number of cases reported in the literature, mainly as case reports or small retrospective case series. Prospective multicenter clinical trials are warranted to more precisely investigate epidemiology and cytogenetic/molecular features of AML in PLWH, but also to standardize and further improve its therapeutic management.