RESUMO
BACKGROUND: Ofatumumab is a humanized type 1 anti-CD20 monoclonal antibody. Preclinical studies show improved complement-mediated cytotoxicity (CMC) compared to rituximab in mantle cell lymphoma (MCL). This study evaluates the safety and efficacy of combining ofatumumab with HyperCVAD/MA (O-HyperCVAD) in newly diagnosed MCL. METHODS: In this single-arm phase 2 study, 37 patients were treated with the combination of O-HyperCVAD for 4 or 6 cycles, followed by high dose chemotherapy and autologous stem cell transplant. Primary objectives were overall response rate (ORR) and complete response (CR) rate at the end of therapy. Secondary objectives included minimal residual disease (MRD) negativity, progression-free survival (PFS), and overall survival (OS). RESULTS: Median age was 60 years; ORR was 86% and 73% achieved a CR by modified Cheson criteria. The MRD negativity rate was 78% after 2 cycles of therapy, increasing to 96% at the end of induction; median PFS and OS were 45.5 months and 56 months, respectively. Achieving a post-induction CR by both imaging and flow cytometry was associated with improved PFS and OS. Early MRD negativity (post-2 cycles) was also associated with an improved PFS but not OS. There were 3 deaths while on therapy, and grades 3 and 4 adverse events (AEs) were observed in 22% and 68% of the patients. CONCLUSION: The addition of ofatumumab to HyperCVAD/HD-MA led to high rates of MRD negativity by flow cytometry in patients with newly diagnosed MCL. Achieving a CR post-induction by both imaging and flow cytometry is associated with improved overall survival.
Assuntos
Anticorpos Monoclonais Humanizados , Linfoma de Célula do Manto , Adulto , Anticorpos Monoclonais Humanizados/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Humanos , Linfoma de Célula do Manto/terapia , Pessoa de Meia-Idade , Neoplasia Residual/diagnóstico , RituximabRESUMO
Previous studies have reported a beneficial effect from cytomegalovirus (CMV) reactivation after allogeneic hematopoietic stem cell transplantation (alloHCT) on immune reconstitution. We determined the CMV antigenemia level associated with increased CMV antigen-specific T cells (CASTs) at day +100 and decreased CMV reactivation after day +100. CMV reactivation and CASTs were measured with CMV antigenemia and CMV-specific major histocompatibility complex multimers. The analysis consisted of 775 CAST measurements obtained before and 30, 100, and 365 days post-alloHCT from 327 consecutive patients treated between 2008 and 2016. Detectable CASTs correlated with recipient (P < .0001) and donor (P < .0001) CMV seropositivity pre-alloHCT. CMV reactivation before day +100 was associated with a higher proportion of patients who achieved ≥3 CASTs/µL by day +100 (61% with versus 39% without reactivation, P < .001). In alloHCT recipients at high risk for CMV reactivation (R+D±) with a maximum of grade II acute graft-versus-host-disease, reactivating CMV before day +100 and achieving ≥3 versus <3 CASTs/µL at day +100 was associated with reduced CMV reactivation from day +100 to +365 (27% versus 62%, P = .04). This protective effect was observed with low-level but not high-level CMV reactivation (<5 versus ≥5/50,000 polymorphonuclear leukocytes + pp65, respectively). These findings suggest low-level CMV reactivation may be beneficial and that treatment may be delayed until progression. These findings will need validation in prospective clinical trials using CMV PCR and antigenemia assays.
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Infecções por Citomegalovirus , Transplante de Células-Tronco Hematopoéticas , Citomegalovirus , Humanos , Estudos Prospectivos , Linfócitos T , Transplante HomólogoRESUMO
The Blood and Marrow Transplant Clinical Trials Network Myeloma Intergroup Workshop on Minimal Residual Disease and Immune Profiling was convened on December 1, 2016 at the American Society of Hematology meeting to discuss the emerging data and technologies for minimal residual disease assessment and immune profiling in myeloma. Particular emphasis was placed on developing strategies to incorporate these techniques into clinical trial design. This document reviews the literature, summarizes the topics discussed in the workshop, and provides recommendations for integration of these techniques into future clinical trial design.
Assuntos
Ensaios Clínicos como Assunto , Mieloma Múltiplo/sangue , Mieloma Múltiplo/imunologia , Mieloma Múltiplo/terapia , Educação , Hematologia , Humanos , Neoplasia Residual , Guias de Prática Clínica como Assunto , Sociedades Médicas , Estados UnidosRESUMO
In the field of transplantation, flow cytometry serves a well-established role in pre-transplant crossmatching and monitoring immune reconstitution following hematopoietic stem cell transplantation. The capabilities of flow cytometers have continuously expanded and this combined with more detailed knowledge of the constituents of the immune system, their function and interaction and newly developed reagents to study these parameters have led to additional utility of flow cytometry-based analyses, particularly in the post-transplant setting. This review discusses the impact of flow cytometry on managing alloantigen reactions, monitoring opportunistic infections and graft rejection and gauging immunosuppression in the context of solid organ transplantation.
Assuntos
Citometria de Fluxo , Rejeição de Enxerto/imunologia , Transplante de Células-Tronco Hematopoéticas , Tolerância Imunológica , Isoantígenos/imunologia , Infecções Oportunistas/imunologia , Transplante de Órgãos , Animais , Separação Celular , Rejeição de Enxerto/prevenção & controle , Histocompatibilidade/imunologia , Teste de Histocompatibilidade , Humanos , Monitorização Imunológica/métodosRESUMO
Broadly speaking, cell tracking dyes are fluorescent compounds that bind stably to components on or within the cells so the fate of the labeled cells can be followed. Their staining should be bright and homogeneous without affecting cell function. For purposes of monitoring cell proliferation, each time a cell divides the intensity of cell tracking dye should diminish equally between daughter cells. These dyes can be grouped into two different classes. Protein reactive dyes label cells by reacting covalently but non-selectively with intracellular proteins. Carboxyfluorescein diacetate succinimidyl ester (CFSE) is the prototypic general protein label. Membrane intercalating dyes label cells by partitioning non-selectively and non-covalently within the plasma membrane. The PKH membrane dyes are examples of lipophilic compounds whose chemistry allows for their retention within biological membranes without affecting cellular growth, viability, or proliferation when used properly. Here we provide considerations based for labeling cell lines and peripheral blood mononuclear cells using both classes of dyes. Examples from optimization experiments are presented along with critical aspects of the staining procedures to help mitigate common risks. Of note, we present data where a logarithmically growing cell line is labeled with both a protein dye and a membrane tracking dye to compare dye loss rates over 6days. We found that dual stained cells paralleled dye loss of the corresponding single stained cells. The decrease in fluorescence intensity by protein reactive dyes, however, was more rapid than that with the membrane reactive dyes, indicating the presence of additional division-independent dye loss.
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Proliferação de Células , Fluoresceínas , Corantes Fluorescentes , Coloração e Rotulagem , Succinimidas , Humanos , Corantes Fluorescentes/química , Fluoresceínas/química , Succinimidas/química , Coloração e Rotulagem/métodos , Rastreamento de Células/métodos , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/metabolismo , Animais , Membrana Celular/metabolismo , Membrana Celular/químicaRESUMO
High dimensional studies that include proliferation dyes face two inherent challenges in panel design. First, the more rounds of cell division to be monitored based on dye dilution, the greater the starting intensity of the labeled parent cells must be in order to distinguish highly divided daughter cells from background autofluorescence. Second, the greater their starting intensity, the more difficult it becomes to avoid spillover of proliferation dye signal into adjacent spectral channels, with resulting limitations on the use of other fluorochromes and ability to resolve dim signals of interest. In the third and fourth editions of this series, we described the similarities and differences between protein-reactive and membrane-intercalating dyes used for general cell tracking, provided detailed protocols for optimized labeling with each dye type, and summarized characteristics to be tested by the supplier and/or user when validating either dye type for use as a proliferation dye. In this fifth edition, we review: (a) Fundamental assumptions and critical controls for dye dilution proliferation assays; (b) Methods to evaluate the effect of labeling on cell growth rate and test the fidelity with which dye dilution reports cell division; and. (c) Factors that determine how many daughter generations can be accurately included in proliferation modeling. We also provide an expanded section on spectral characterization, using data collected for three protein-reactive dyes (CellTrace™ Violet, CellTrace™ CFSE, and CellTrace™ Far Red) and three membrane-intercalating dyes (PKH67, PKH26, and CellVue® Claret) on three different cytometers to illustrate typical decisions and trade-offs required during multicolor panel design. Lastly, we include methods and controls for assessing regulatory T cell potency, a functional assay that incorporates the "know your dye" and "know your cytometer" principles described herein.
Assuntos
Rastreamento de Células , Corantes Fluorescentes , Citometria de Fluxo/métodos , Proliferação de Células/fisiologia , Divisão Celular , Rastreamento de Células/métodosRESUMO
PURPOSE: There is compelling evidence that CD4+ and CD8+T cells are dysfunctional in multiple myeloma, compromising their ability to control disease progression. Pre-clinical models suggest that exercise represents a non-pharmacologic means to reduce immune exhaustion, but no studies to date have examined the relationship between an exercise intervention and biomarkers of immune exhaustion in multiple myeloma patients. PATIENTS AND METHODS: The current study includes 24 multiple myeloma patients who participated in a six-month physical activity intervention, consisting of supervised strength training (n = 12) and unsupervised home-based walking arms (n = 12). Comprehensive flow cytometry was utilized to assess the frequency of CD4+ and CD8+T cells and subpopulations expressing the markers of exhaustion PD-1, TIGIT, TIM3 and/or LAG3. Ratios of exhausted to non-exhausted cell populations, and percentages of exhausted to total populations of the same lineage, were calculated for the baseline and final timepoints. RESULTS: Eighteen of 20 exhaustion measures were lower at the end of the intervention than at baseline, and several were significantly or borderline significantly reduced in the entire sample or in one of the arms. The entire sample saw improvements in the ratios of CD4+ TIGIT+ to non-exhausted CD4+ (0.7 [0.6] to 0.6 [0.4], P = .04) and CD8+ PD1+ to non-exhausted CD8+ (1.8 [2.6] to 1.5 [2.0], P = .06), and in total exhausted CD8+ as a percent of total CD8+ (72.9 [21.9] to 68.3 [19.6], P < .01). CONCLUSIONS: This pilot study suggests that physical activity induces changes in MM patients' immune systems, potentially rendering a less exhausted T cell state.
Assuntos
Linfócitos T CD8-Positivos , Exercício Físico , Mieloma Múltiplo , Humanos , Mieloma Múltiplo/terapia , Mieloma Múltiplo/imunologia , Masculino , Feminino , Pessoa de Meia-Idade , Idoso , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Biomarcadores , Receptor de Morte Celular Programada 1/metabolismo , Terapia por Exercício/métodos , Proteína do Gene 3 de Ativação de Linfócitos , Exaustão das Células TRESUMO
Haploidentical (Haplo) allogeneic HCTs (alloHCT) have been used more frequently over the last decade as survival is similar to HLA-matched related donor (MRD) alloHCTs. We aimed to identify donor and recipient immune signatures before alloHCT that are associated with clinically meaningful outcomes in MRD vs Haplo alloHCT recipients. This retrospective cohort study of 165 MRD (n = 132) and Haplo (n = 33) alloHCT recipients and their related donors between 2007-2019 with paired peripheral blood samples immunophenotyped for T-cell, B-cell, NK cell and dendritic cell (DC) subsets. Immune cells were quantified before alloHCT in donors and recipients; calculations of immune cell ratios were classified as high, intermediate, and low and analyzed with alloHCT outcomes. Haplo donors were younger than MRD donors (median: 35 vs 51 years), whereas Haplo recipients were older than MRD recipients (median: 68 vs 54 years), were more likely to have a Karnofsky Performance Score ≤ 70 (76% vs 57%), 3+ comorbidities (54% vs 47%), and were in complete remission prior to alloHCT (58% vs 42%). In MRD alloHCT, a lower ratio of CD4+ to CD8+ effector memory cells in the donor was associated with lower 4-yr overall survival (OS; 25% vs 61%; P = .009), lower 4-yr progression free survival (PFS; 25% vs 58%; P = .014) and higher incidence of 1-yr transplant-related mortality (TRM; 39% vs 7%; P = .009) in recipients. A higher ratio of CD8+ effector memory to total NK cells measured in MRD recipients was associated with a higher incidence of grade II-IV aGvHD (63% vs 37%; P = .004) but was not statistically significant for III-IV aGvHD (23% vs 12%). In Haplo alloHCT, a lower ratio of total T-regulatory to CD4+ central memory cells in the donor was associated with lower 4-yr PFS (22% vs 60%; P = .0091). A higher ratio of CD4+ effector memory to CD8+ effector memory cells measured in Haplo recipients pre-alloHCT was associated with lower 4-yr OS (25% vs 88%; P = .0039). In both MRD and Haplo recipients, a higher ratio of CD4+ naïve to CD4+ central memory cells was associated with a higher incidence of grade II-IV aGvHD (64% vs 38%; P = .04). Evaluation of pre-alloHCT immune signatures of the donor and recipient may influence clinically meaningful patient outcomes in both MRD and Haplo transplants.
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Antígenos HLA , Transplante de Células-Tronco Hematopoéticas , Doadores de Tecidos , Transplante Haploidêntico , Humanos , Pessoa de Meia-Idade , Masculino , Adulto , Feminino , Estudos Retrospectivos , Doadores de Tecidos/estatística & dados numéricos , Antígenos HLA/imunologia , Idoso , Transplante Homólogo , Resultado do Tratamento , Doença Enxerto-Hospedeiro/imunologia , Adulto JovemRESUMO
PURPOSE: Prognostic Immunophenotyping in Myeloma Response (PRIMeR) is an ancillary study of minimal residual disease (MRD) assessment for multiple myeloma by next-generation multiparameter flow cytometry (MFC). Patients were enrolled on a three-arm randomized control trial (Blood and Marrow Transplants Clinical Trials Network 0702 Stem Cell Transplant for Myeloma in Combination of Novel Agents [STaMINA]; ClinicalTrials.gov identifier: NCT01109004). METHODS: Four hundred and thirty-five patients consented to the MRD panel, which included 10 monoclonal antibodies measured via six-color MFC. MRD was measured at baseline/preautologous hematopoietic cell transplant (BL/preAutoHCT), premaintenance (PM), and 1 year (Y1) after AutoHCT with a sensitivity of 10-5 to 10-6. The primary objective was to assess MRD-negative (MRDneg) at 1 year after AutoHCT and progression-free survival and overall survival (PFS/OS). RESULTS: Similar to the STaMINA results, at a median follow-up of 70 months, there was no significant difference in PFS/OS by treatment arm in the PRIMeR patients. MRDneg at all three time points was associated with significantly improved PFS, and MRDneg at Y1 had significantly longer OS. Multivariate analysis of PFS, adjusting for disease risk and treatment arm, demonstrated hazard ratios (HRs) in MRD-positive patients compared with MRDneg patients at BL, PM, and Y1 of 1.55 (P = .0074), 1.83 (P = .0007), and 3.61 (P < .0001), respectively. Corresponding HRs for OS were 1.19 (P = .48), 0.88 (P = .68), and 3.36 (P < .001). Patients with sustained MRDneg or who converted to MRDneg by Y1 had similar PFS/OS. CONCLUSION: To our knowledge, this first, prospective US cooperative group, multicenter study demonstrates that MRDneg at Y1 after AutoHCT with lenalidomide maintenance is prognostic for improved 6-year PFS and OS. Serial MRD measurements may direct trials to test how further therapy may improve long-term PFS and OS.
Assuntos
Transplante de Células-Tronco Hematopoéticas , Lenalidomida , Mieloma Múltiplo , Neoplasia Residual , Transplante Autólogo , Humanos , Lenalidomida/uso terapêutico , Lenalidomida/administração & dosagem , Mieloma Múltiplo/mortalidade , Mieloma Múltiplo/terapia , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Masculino , Pessoa de Meia-Idade , Feminino , Idoso , Adulto , Quimioterapia de Manutenção , Citometria de Fluxo , Intervalo Livre de ProgressãoRESUMO
BACKGROUND: Daratumumab is an anti-CD38 immunotherapeutic drug that has increasingly been used to treat patients with heavily pre-treated and relapsed/refractory multiple myeloma. In so doing, the detection of CD38 antigen on plasma cells by flow cytometry is impeded. We hypothesized that alternative markers can be used in place or in addition to CD38 when detecting plasma cells post-treated with daratumumab. METHODS: A total of 16 alternative markers were tested using 22 bone marrow aspirates from patients with plasma cell neoplasm. The ability of selected markers to discern plasma cells from other hematopoietic cells were evaluated. The stability of tested markers when stored at 4 or 25°C after T = 0, 24, 48, and 72 h was also established. Finally, selected markers were incorporated into a panel used for monitoring multiple myeloma measurable residual disease to test their utility to identify plasma cells in the presence of daratumumab and/or elotuzumab (anti-CD319) drugs. RESULTS: Out of the 16 tested markers, CD319, CD54, CD229, CD317, and p63 were expressed by >90% of the plasma cells. Only CD319, CD54, and CD229 achieved 100% detection sensitivity. Further analysis showed that CD319 was better than CD229 and CD54 at resolving plasma cells from background hematopoietic cells, with CD54 being the worst (resolution metric, mean ± SD: CD319 [2.04 ± 0.86]; CD229 [1.47 ± 0.45]; and CD54 [1.22 ± 0.60]). CD229 was expressed by >90% of T lymphocytes, whereas CD319 was expressed preferentially by the CD8+ T cells and less frequently in CD4+ T cells. Additionally, CD229 was found on >60% of B and NK cells, as well as minor subsets of monocytes and granulocytes. CD319 was expressed on most NK cells and a minor subset of B cells, granulocytes, and monocytes. Even though CD229 and CD319 were expressed by different leukocyte subsets, their expression levels were highest on plasma cells. The expression of CD138 on plasma cells was significantly lower after storage at 4°C, while the expression levels of CD38, CD229, and CD319 remained stable at 4 or 25°C. Using limiting dilution experiments, the treatment of cells with daratumumab severely impeded the detection of CD38 antigen on plasma cells, whereas elotuzumab treatment did not block detection of CD319 on plasma cells. CONCLUSIONS: CD319 is a suitable alternative to CD38 for identifying plasma cells. Our results showed that a panel used for monitoring multiple myeloma measurable residual disease could be modified by using CD319 alone or in combination with CD38 to detect PCs in daratumumab or elotuzumab treated patients.
Assuntos
Biomarcadores Tumorais/sangue , Mieloma Múltiplo/tratamento farmacológico , Neoplasias de Plasmócitos/sangue , Família de Moléculas de Sinalização da Ativação Linfocitária/sangue , ADP-Ribosil Ciclase 1/antagonistas & inibidores , Adolescente , Adulto , Anticorpos Monoclonais/efeitos adversos , Anticorpos Monoclonais/sangue , Anticorpos Monoclonais Humanizados/efeitos adversos , Anticorpos Monoclonais Humanizados/sangue , Feminino , Citometria de Fluxo/métodos , Regulação Neoplásica da Expressão Gênica/genética , Granulócitos/metabolismo , Granulócitos/patologia , Humanos , Imunofenotipagem/métodos , Molécula 1 de Adesão Intercelular/sangue , Masculino , Pessoa de Meia-Idade , Monócitos/metabolismo , Monócitos/patologia , Mieloma Múltiplo/sangue , Mieloma Múltiplo/patologia , Neoplasias de Plasmócitos/patologia , Plasmócitos/efeitos dos fármacos , Plasmócitos/patologia , Adulto JovemRESUMO
PROBLEM: Previous studies identified circulating CD14+ HLA-DRlo/- monocytic cells as an immune suppressive subset in solid malignancies, such as prostate, renal cell carcinoma, and pancreatic cancer. Such monocytic cells have been implicated not only in tumour progression but also as a potential barrier for immunotherapy. This study examined the relationship between the frequency of circulating monocytic cells and epithelial ovarian cancer (EOC) progression pre- and post-frontline chemotherapy, defined by disease stage, which is a leading prognostic factor for this malignancy. METHOD OF STUDY: Incident cases of 236 women with EOC were recruited and comprehensive flow cytometry was utilized to assess the frequency of peripheral blood CD33+ CD11b+ HLA-DR-/low CD14+ CD15- monocytic cells, henceforth termed CD14+ HLA-DRlo/- monocytic cells, prior to and after completion of frontline chemotherapy. Multivariable odds ratios (OR) were used to estimate the association between CD14+ HLA-DRlo/- monocytic cell percentages and disease stage. Wilcoxon signed-rank tests evaluated changes in these monocytic cell levels pre- and post-chemotherapy in a patient subset (n = 70). RESULTS: Patients with elevated frequencies of circulating CD14+ HLA-DRlo/- monocytic cells at diagnosis were at 3.33-fold greater odds of having advanced stage (III/IV) EOC (CI: 1.04-10.64), with a significant trend in increasing CD14+ HLA-DRlo/- monocytic cell levels (P = .04). There was a 2.02% median decrease of these monocytic cells post-chemotherapy among a subset of patients with advanced stage disease (P < .0001). CONCLUSION: These findings support the potential clinical relevance of CD14+ HLA-DRlo/- monocytic cells in EOC for prognosis and may indicate a non-invasive biomarker to measure disease progression.
Assuntos
Células Epiteliais/patologia , Imidas/imunologia , Neoplasias Ovarianas/imunologia , Polifosfatos/imunologia , Idoso , Biomarcadores , Carcinogênese , Progressão da Doença , Feminino , Antígenos HLA-DR/metabolismo , Humanos , Tolerância Imunológica , Receptores de Lipopolissacarídeos/metabolismo , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Neoplasias Ovarianas/diagnóstico , PrognósticoRESUMO
PURPOSE: Increased ß-adrenergic receptor (ß-AR) signaling has been shown to promote the creation of an immunosuppressive tumor microenvironment (TME). Preclinical studies have shown that abrogation of this signaling pathway, particularly ß2-AR, provides a more favorable TME that enhances the activity of anti-PD-1 checkpoint inhibitors. We hypothesize that blocking stress-related immunosuppressive pathways would improve tumor response to immune checkpoint inhibitors in patients. Here, we report the results of dose escalation of a nonselective ß-blocker (propranolol) with pembrolizumab in patients with metastatic melanoma. PATIENTS AND METHODS: A 3 + 3 dose escalation study for propranolol twice a day with pembrolizumab (200 mg every 3 weeks) was completed. The primary objective was to determine the recommended phase II dose (RP2D). Additional objectives included safety, antitumor activity, and biomarker analyses. Responders were defined as patients with complete or partial response per immune-modified RECIST at 6 months. RESULTS: Nine patients with metastatic melanoma received increasing doses of propranolol in cohorts of 10, 20, and 30 mg twice a day. No dose-limiting toxicities were observed. Most common treatment-related adverse events (TRAEs) were rash, fatigue, and vitiligo, observed in 44% patients. One patient developed two grade ≥3 TRAEs. Objective response rate was 78%. While no significant changes in treatment-associated biomarkers were observed, an increase in IFNγ and a decrease in IL6 was noted in responders. CONCLUSIONS: Combination of propranolol with pembrolizumab in treatment-naïve metastatic melanoma is safe and shows very promising activity. Propranolol 30 mg twice a day was selected as RP2D in addition to pembrolizumab based on safety, tolerability, and preliminary antitumor activity.
Assuntos
Anticorpos Monoclonais Humanizados/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Melanoma/tratamento farmacológico , Propranolol/efeitos adversos , Neoplasias Cutâneas/tratamento farmacológico , Antagonistas Adrenérgicos beta/administração & dosagem , Antagonistas Adrenérgicos beta/efeitos adversos , Adulto , Idoso , Anticorpos Monoclonais Humanizados/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Feminino , Humanos , Masculino , Melanoma/diagnóstico , Melanoma/imunologia , Melanoma/secundário , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Propranolol/administração & dosagem , Critérios de Avaliação de Resposta em Tumores Sólidos , Neoplasias Cutâneas/diagnóstico , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/patologia , Evasão Tumoral/efeitos dos fármacos , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/imunologiaRESUMO
BACKGROUND: Recent advances in therapeutic interventions have dramatically improved complete response rates in patients with multiple myeloma (MM). The ability to identify residual myeloma cells (e.g., measurable residual disease [MRD]) can provide valuable information pertaining to patient's depth of response to therapy and risk of relapse. Multiparametric flow cytometry is an excellent technique to monitor MRD and has been demonstrated to correlate with patient outcome post-treatment. To achieve the high sensitivity (one abnormal cell in 105 -106 cells) required for MRD evaluation, millions of cells have to be acquired and conventional immunophenotyping protocols are unable to attain these numbers, indicating the needs for alternative flow cytometric staining procedures. A bulk, "Pre-lysis" method is the consensus approach for staining large number of cells, requires two red blood cell lysis steps, and can adversely affect epitope density. In this study, we tested the "Pooled-tube" and "Dextran Sedimentation" staining procedures and correlated them with the "Pre-lysis" method as potential alternative approaches. METHODS: A total of 22 bone marrow aspirates from patients with plasma cell (PC) dyscrasia were processed in parallel using the "Pre-lysis," "Pooled-tube," and "Dextran Sedimentation" techniques. Stain indices were calculated and compared to assess their impacts on staining performance for each antibody used in the consensus panel. The recovery of normal and abnormal PCs, mast cells, and B cell precursors was enumerated and compared after their counts were normalized using fluorescent beads. The limit of blank, limit of detection, and lower limit of quantification were established using serial dilution experiments. RESULTS: The staining performances of CD19 PECy7, CD27 BV510, CD81 APCH7, and CD138 BV421 were improved using the "Pooled-tube" method when compared to "Pre-lysis." "Pre-lysis" was better at resolving CD56 using clone C5.9 but our results demonstrated similar improvement can also be achieved by "Pooled-tube" when alternative CD56 PE clones were used. "Dextran sedimentation" yielded similar staining results when compared to "Pre-lysis" for all the markers analyzed. The "Pooled-tube" method, when normalized to "Pre-lysis," recovered higher numbers of total PCs (1.2 ± 0.2 times higher; p = .049), normal PCs (1.4 ± 0.26; p = .007), mast cells (1.46 ± 0.27; p = .003), and B cell precursors (1.42 ± 0.3; p = .011), but not abnormal PCs (1.09 ± 0.2; p = .352). There was no evidence that the recovery of cells was different between "Pre-lysis" versus "Dextran Sedimentation." All three flow cytometric assays achieved a minimum sensitivity of 10-5 and approached that of 10-6 for detecting rare events. CONCLUSION: Both "Pooled-tube" and "Dextran Sedimentation" staining procedures were comparable to the "Pre-lysis" method and are suitable high sensitivity flow cytometric approaches that can be used to process bone marrow samples for MM MRD testing.
Assuntos
Citometria de Fluxo , Mieloma Múltiplo/diagnóstico , Mieloma Múltiplo/patologia , Idoso , Idoso de 80 Anos ou mais , Biópsia por Agulha , Medula Óssea/imunologia , Medula Óssea/patologia , Separação Celular/métodos , Separação Celular/normas , Citometria de Fluxo/métodos , Citometria de Fluxo/normas , Humanos , Imunofenotipagem/métodos , Imunofenotipagem/normas , Pessoa de Meia-Idade , Monitorização Fisiológica/métodos , Monitorização Fisiológica/normas , Mieloma Múltiplo/terapia , Metástase Neoplásica , Neoplasia Residual , Plasmócitos/imunologia , Plasmócitos/patologia , Recidiva , Sensibilidade e EspecificidadeRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
This is the first longitudinal study of immune profiles and autologous hematopoietic cell transplant (AHCT) survival in B-cell non-Hodgkin lymphoma (B-NHL) patients and the effect of plerixafor mobilization on immune reconstitution in this population. A comprehensive immunophenotyping panel was performed in 104 consecutive adult B-NHL patients (58% diffuse large B cell and 42% mantle cell) who received AHCT (1/2008-11/2014), at a median of 28 days pre-AHCT (N = 104) and Day +100 (N = 83) post-AHCT. Median follow-up post-AHCT was 61 months (range: 8-120 months). Compared to patients mobilized with filgrastim and plerixafor, patients mobilized with filgrastim alone had a higher proportion of CD4+ naïve (p = 0.006) and CD8+ central memory T-cells (p = 0.006) pre-AHCT. For patients transplanted in complete remission (CR), a higher proportion of CD8+ effector memory T-cells pre-AHCT was associated with worse progression-free survival (PFS; p < 0.01) and overall survival (OS; p < 0.01). A higher ratio of CD8:CD4+ central memory T-cells pre-AHCT was associated with worse PFS (p < 0.0001) and OS (p = 0.0034). This same ratio measured post-AHCT among patients in CR on Day +100 was associated with worse and OS (p = 0.008) but not PFS (p = not significant). These immune subsets are complementary biomarkers which identify patients transplanted in CR who have poor survival prognoses and may warrant further clinical interventions.
Assuntos
Transplante de Células-Tronco Hematopoéticas , Compostos Heterocíclicos , Linfoma Difuso de Grandes Células B , Linfoma de Célula do Manto , Adulto , Mobilização de Células-Tronco Hematopoéticas , Humanos , Estudos Longitudinais , Linfoma Difuso de Grandes Células B/terapia , Linfoma de Célula do Manto/terapia , Transplante AutólogoRESUMO
Myeloid derived suppressor cells (MDSC) are a heterogeneous group of immature cells that accumulate in the peripheral blood and tumor microenvironment and are barriers to cancer therapy. MDSCs serve as prognostic biomarkers and are targets for therapy. On the basis of surface markers, three subsets of MDSCs have been defined in humans: granulocytic, monocytic, and early stage (e-MDSC). The markers attributed to e-MDSCs overlap with those of basophils, which are rare circulating myeloid cells with unrecognized roles in cancer. Thus, we asked whether e-MDSCs in circulation and the tumor microenvironment include basophils. On average, 58% of cells with e-MDSC surface markers in blood and 36% in ascites from patients with ovarian cancer were basophils based on CD123high expression and cytology, whereas cells with immature features were rare. Circulating and ascites basophils did not suppress proliferation of stimulated T cells, a key feature of MDSCs. Increased accumulation of basophils and basogranulin, a marker of basophil degranulation, were observed in ascites compared to serum in patients with newly diagnosed ovarian cancer. Basophils recruited to the tumor microenvironment may exacerbate fluid accumulation by their release of proinflammatory granular constituents that promote vascular leakage. No significant correlation was observed between peripheral basophil counts and survival in patients with ovarian cancer. Our results suggest that studies in which e-MDSCs were defined solely by surface markers should be reevaluated to exclude basophils. Both immaturity and suppression are criteria to define e-MDSCs in future studies.
Assuntos
Ascite/patologia , Basófilos/patologia , Biomarcadores Tumorais/sangue , Leucócitos Mononucleares/patologia , Células Supressoras Mieloides/patologia , Neoplasias Ovarianas/patologia , Microambiente Tumoral , Estudos de Casos e Controles , Feminino , Humanos , Pessoa de Meia-Idade , Neoplasias Ovarianas/sangue , Neoplasias Ovarianas/cirurgia , Prognóstico , Estudos Prospectivos , Estudos Retrospectivos , Taxa de Sobrevida , Células Tumorais CultivadasRESUMO
Cell-tracking reagents such as the green-fluorescent protein labeling dye CFSE and the red-fluorescent lipophilic membrane dye PKH26 are commonly used to monitor cell proliferation by flow cytometry in heterogeneous cell populations responding to immune stimuli. Both reagents stain cells with a bright homogeneous fluorescence, which is partitioned between daughter cells during each cell division. Because daughter cell fluorescence intensities are approximately halved after each division, the intensity of a cell relative to its intensity at the time of staining provides information about how many divisions it has undergone. Knowing how many rounds of division have occurred and the relative number of cells in each daughter generation, one can back-calculate the number of cells in the original population (i.e., cells present at the time of stimulus) that went on to respond by proliferating. Using this information, the precursor cell frequencies and extent of expansion to a specific antigen or mitogen of interest can be calculated. Concurrently, the phenotype of the cells can be determined, as well as their ability to bind antigen or synthesize cytokines, providing more detailed characterization of all cells responding to the antigen, not just effector cells. In multiparameter flow cytometric experiments to simultaneously analyze antigen-specific tetramer binding, cytokine production and T-cell proliferation, we found that only approximately half of the cells that exhibited specific binding to influenza tetramer also proliferated, as measured by dye dilution, and synthesized IFNgamma in response to antigen. We expect the advent of new cell tracking dyes emitting from the violet to the near infrared combined with the increasing number of lasers and detectors on contemporary flow cytometers to further expand the usefulness of this approach to characterization of complex antigen-driven immunological responses.
Assuntos
Antígenos/imunologia , Técnica de Diluição de Corante , Citometria de Fluxo/métodos , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD8-Positivos/citologia , Proliferação de Células , Fluoresceínas/metabolismo , Humanos , Compostos Orgânicos/metabolismo , Reprodutibilidade dos Testes , Coloração e Rotulagem , Succinimidas/metabolismoRESUMO
In the third edition of this series, we described protocols for labeling cell populations with tracking dyes, and addressed issues to be considered when combining two different tracking dyes with other phenotypic and viability probes for the assessment of cytotoxic effector activity and regulatory T cell functions. We summarized key characteristics of and differences between general protein and membrane labeling dyes, discussed determination of optimal staining concentrations, and provided detailed labeling protocols for both dye types. Examples of the advantages of two-color cell tracking were provided in the form of protocols for: (a) independent enumeration of viable effector and target cells in a direct cytotoxicity assay; and (b) an in vitro suppression assay for simultaneous proliferation monitoring of effector and regulatory T cells.The number of commercially available fluorescent cell tracking dyes has expanded significantly since the last edition, with new suppliers and/or new spectral properties being added at least annually. In this fourth edition, we describe evaluations to be performed by the supplier and/or user when characterizing a new cell tracking dye and by the user when selecting one for use in multicolor proliferation monitoring. These include methods for: (a) Assessment of the dye's spectral profile on the laboratory's flow cytometer(s) to optimize compatibility with other employed fluorochromes and minimize compensation problems; (b) Evaluating the effect of labeling on cell growth rate;
Assuntos
Proliferação de Células , Citometria de Fluxo , Sondas Moleculares , Divisão Celular , Linhagem Celular , Rastreamento de Células , Citotoxicidade Imunológica , Interpretação Estatística de Dados , Citometria de Fluxo/métodos , Humanos , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Coloração e RotulagemRESUMO
Previously, we have successfully targeted the mannose receptor (MR) expressed on monocyte-derived dendritic cells (DCs) using a fully human MR-specific antibody, B11, as a vehicle to deliver whole protein tumor antigens such as the human chorionic gonadotropin hormone (hCGbeta). Since MRs play a role in bridging innate immunity with adaptive immunity we have explored several toll-like receptor (TLR)-specific ligands that may synergize with MR targeting and be applicable as adjuvants in the clinic. We demonstrate that antigen-specific helper and cytolytic T cells from both healthy donors and cancer patients were effectively primed with B11-hCGbeta-treated autologous DCs when a combination of one or several TLR ligands is used. Specifically, concomitant signaling of DCs via TLR3 with dsRNA (poly I:C) and DC TLR 7/8 with Resiquimod (R-848), respectively, elicited efficient antigen presentation-mediated by MR-targeting. We demonstrate that MR and TLRs contribute towards maturation and activation of DCs by a mechanism that may be driven by a combination of adjuvant and antibody vaccines that specifically deliver antigenic targets to DCs.
Assuntos
Anticorpos/imunologia , Vacinas Anticâncer/imunologia , Células Dendríticas/imunologia , Imunidade Celular/imunologia , Receptores Toll-Like/imunologia , Antígenos de Neoplasias/imunologia , Compartimento Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Membrana Celular/imunologia , Gonadotropina Coriônica Humana Subunidade beta/imunologia , Citocinas/biossíntese , Células Dendríticas/efeitos dos fármacos , Epitopos/imunologia , Humanos , Imidazóis/farmacologia , Imunidade Celular/efeitos dos fármacos , Lectinas Tipo C/imunologia , Ligantes , Receptor de Manose , Lectinas de Ligação a Manose/imunologia , Fenótipo , Poli I-C/farmacologia , Receptores de Superfície Celular/imunologia , Linfócitos T Citotóxicos/efeitos dos fármacos , Linfócitos T Citotóxicos/imunologia , Células Th1/imunologiaRESUMO
The advent of contemporary digital instrumentation has enhanced both the potential and the complexity of flow cytometric experiments, allowing for the detailed dissection of immune cell subsets and their functions. The use of cell tracking labels such as PKH26 and CFSE has been important in observing such cellular functions, but their visible emission characteristics have limited the design of such analyses. As the demand for multiparametric flow cytometry intensifies, it will become increasingly important to utilize a broader range of cell tracking reagents to optimize the measurement of fluorescence signals and to provide flexibility in the use of commercially available fluorochrome - antibody combinations. We report on the evaluation of three lipophilic membrane dyes, CellVue Lavender, CellVue Plum and CellVue NIR780; with fluorescence emissions in the violet, far-red and near infrared wavelength regions, respectively. These reagents are similar to established tracking dyes such as PKH26 and CFSE in terms of staining procedure, membrane stability, optimal concentration, and lack of effect on cellular proliferation. The CellVue dyes however, exhibit different spectral characteristics than existing tracking compounds, and capitalize upon the increased number of lasers incorporated into commercially available instrumentation; thus permitting measurement of labeled populations in underexploited regions of the spectrum.