RESUMO
High-risk cytogenetics and minimal residual disease (MRD) after chemoimmunotherapy (CIT) predict unfavorable outcome in chronic lymphocytic leukemia (CLL). This phase 2 study investigated risk-adapted CIT in treatment-naïve CLL (NCT01145209). Patients with high-risk cytogenetics received induction with fludarabine, cyclophosphamide, and ofatumumab. Those without high-risk cytogenetics received fludarabine and ofatumumab. After induction, MRD positive (MRD+) patients received 4 doses of ofatumumab consolidation. MRD negative (MRD-) patients had no intervention. Of 28 evaluable for response, all responded to induction and 10 (36%) achieved MRD-. Two-year progression-free survival (PFS) was 71.4% (CI95, 56.5-90.3%). There was no significant difference in median PFS between the high-risk and the standard-risk groups. Ofatumumab consolidation didn't convert MRD + to MRD-. In the MRD + group, we saw selective loss of CD20 antigens during therapy. In conclusion, risk-adapted CIT is feasible in treatment-naïve CLL. Ofatumumab consolidation didn't improve depth of response in MRD + patients. Loss of targetable CD20 likely reduces efficacy of consolidation therapy.
Assuntos
Leucemia Linfocítica Crônica de Células B , Anticorpos Monoclonais Humanizados/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Humanos , Imunoterapia , Leucemia Linfocítica Crônica de Células B/diagnóstico , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Resultado do TratamentoRESUMO
Chronic lymphocytic lymphoma (CLL) has one of the highest familial risks among cancers. Monoclonal B-cell lymphocytosis (MBL), the precursor to CLL, has a higher prevalence (13%-18%) in families with 2 or more members with CLL compared with the general population (5%-12%). Although, the rate of progression to CLL for high-count MBLs (clonal B-cell count ≥500/µL) is â¼1% to 5%/y, no low-count MBLs have been reported to progress to date. We report the incidence and natural history of MBL in relatives from CLL families. In 310 CLL families, we screened 1045 relatives for MBL using highly sensitive flow cytometry and prospectively followed 449 of them. MBL incidence was directly age- and sex-adjusted to the 2010 US population. CLL cumulative incidence was estimated using Kaplan-Meier survival curves. At baseline, the prevalence of MBL was 22% (235/1045 relatives). After a median follow-up of 8.1 years among 449 relatives, 12 individuals progressed to CLL with a 5-year cumulative incidence of 1.8%. When considering just the 139 relatives with low-count MBL, the 5-year cumulative incidence increased to 5.7%. Finally, 264 had no MBL at baseline, of whom 60 individuals subsequently developed MBL (2 high-count and 58 low-count MBLs) with an age- and sex-adjusted incidence of 3.5% after a median of 6 years of follow-up. In a screening cohort of relatives from CLL families, we reported progression from normal-count to low-count MBL to high-count MBL to CLL, demonstrating that low-count MBL precedes progression to CLL. We estimated a 1.1% annual rate of progression from low-count MBL, which is in excess of that in the general population.
Assuntos
Linfócitos B/patologia , Leucemia Linfocítica Crônica de Células B/etiologia , Linfocitose/complicações , Adulto , Idoso , Idoso de 80 Anos ou mais , Progressão da Doença , Feminino , Humanos , Incidência , Estimativa de Kaplan-Meier , Linfocitose/diagnóstico , Linfocitose/etiologia , Linfocitose/patologia , Masculino , Pessoa de Meia-Idade , LinhagemRESUMO
Vaccinations are effective in preventing infections; however, it is unknown if patients with chronic lymphocytic leukemia (CLL) who are treatment naïve (TN) or receiving Bruton tyrosine kinase inhibitors (BTKi's) respond to novel adjuvanted vaccines. Understanding the effect of BTKi's on humoral immunity is timely because BTKi's are widely used and vaccination against coronavirus disease 2019 is urgently needed. In 2 open-label, single-arm clinical trials, we measured the effect of BTKi's on de novo immune response against recombinant hepatitis B vaccine (HepB-CpG) and recall response against recombinant zoster vaccine (RZV) in CLL patients who were TN or on BTKi. The primary end point was serologic response to HepB-CpG (anti-hepatitis B surface antibodies ≥10 mIU/mL) and RZV (≥fourfold increase in anti-glycoprotein E). The response rate to HepB-CpG was lower in patients on BTKi (3.8%; 95% confidence interval [CI], 0.7-18.9) than patients who were TN (28.1%; 95% CI, 15.6-45.4; P = .017). In contrast, the response rate to RZV did not differ significantly between the BTKi (41.5%; 95% CI, 27.8-56.6) and TN cohorts (59.1%; 95% CI, 38.7-76.7; P = .2). BTKi's were associated with a decreased de novo immune response following HepB-CpG, whereas recall immune response following RZV was not significantly affected by BTKi therapy. These trials were registered at www.clinicaltrials.gov as #NCT03685708 (Hep-CpG) and #NCT03702231 (RZV).
Assuntos
Vacinas contra Hepatite B/imunologia , Vacina contra Herpes Zoster/imunologia , Imunidade , Leucemia Linfocítica Crônica de Células B/imunologia , Inibidores de Proteínas Quinases/efeitos adversos , Vacinas Sintéticas/imunologia , Adjuvantes Imunológicos , Tirosina Quinase da Agamaglobulinemia/antagonistas & inibidores , Idoso , Feminino , Humanos , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Masculino , Pessoa de Meia-Idade , Avaliação de Resultados da Assistência ao Paciente , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , VacinaçãoRESUMO
Previously an increased risk for monoclonal gammopathy of undetermined significance (MGUS), a precursor of multiple myeloma (MM), was reported among Vietnam veterans exposed to Agent Orange and its contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Dysregulated expression of certain microRNAs (miRNAs) was demonstrated in MGUS and MM. Given the important role of miRNAs in cellular homeostasis, the aim of this study was to determine if there was an association between serum levels of selected miRNAs and TCDD in 47 MGUS cases identified in our previous investigation using serum specimens and exposure data archived by the Air Force Health Study (AFHS). A total of 13 miRNA levels (let-7a, let-7i, miR-16, miR-20a, miR-21, miR-34a, miR-106b, miR-146a, miR-181a, miR-192, miR-205, miR-335, and miR-361) was measured in serum stored during the 2002 AFHS follow-up and the relationship to lipid-adjusted serum TCDD levels in 1987 was determined. miR-34a showed the strongest relationship with TCDD; after age-adjustment, this positive association was more pronounced. In contrast, the other 12 miRNAs displayed absolute values of age adjusted coefficient estimates below 1.16 and non-significant p-values. The observed strong positive association between high body burdens of TCDD and miR-34a, a tumor suppressor regulated by p53, in this MGUS population warrants clarification of the TCDD-miR-34a relationship and its role in the pathogenesis of MGUS and risk for MM.
Assuntos
Herbicidas/efeitos adversos , MicroRNAs/sangue , Gamopatia Monoclonal de Significância Indeterminada/sangue , Dibenzodioxinas Policloradas/efeitos adversos , Veteranos/estatística & dados numéricos , Idoso , Idoso de 80 Anos ou mais , Humanos , Masculino , Pessoa de Meia-Idade , Gamopatia Monoclonal de Significância Indeterminada/etiologia , Estudos Prospectivos , Estados UnidosRESUMO
The safety and efficacy of ibrutinib (420 mg) in chronic lymphocytic leukemia (CLL) were evaluated in a phase 2 study; 51 patients had TP53 aberration (TP53 cohort) and 35 were enrolled because of age 65 years or older (elderly cohort). Both cohorts included patients with treatment-naive (TN) and relapsed/refractory (RR) CLL. With the median follow-up of 4.8 years, 49 (57.0%) of 86 patients remain on study. Treatment was discontinued for progressive disease in 20 (23.3%) patients and for adverse events in 5 (5.8%). Atrial fibrillation occurred in 18 (20.9%) patients for a rate of 6.4 per 100 patient-years. No serious bleeding occurred. The overall response rate at 6 months, the primary study endpoint, was 95.8% for the TP53 cohort (95% confidence interval, 85.7%-99.5%) and 93.9% for the elderly cohort (95% confidence interval, 79.8%-99.3%). Depth of response improved with time: at best response, 14 (29.2%) of 48 patients in the TP53 cohort and 9 (27.3%) of 33 in the elderly cohort achieved a complete response. Median minimal residual disease (MRD) in peripheral blood was 3.8 × 10-2 at 4 years, with MRD-negative (<10-4) remissions in 5 (10.2%) patients. In the TP53 cohort, the estimated 5-year progression-free survival (PFS) was 74.4% in TN-CLL compared with 19.4% in RR-CLL (P = .0002), and overall survival (OS) was 85.3% vs 53.7%, respectively (P = .023). In the elderly cohort, the estimated 5-year PFS and OS in RR-CLL were 64.8% and 71.6%, respectively, and no event occurred in TN-CLL. Long-term administration of ibrutinib was well tolerated and provided durable disease control for most patients. This trial was registered at www.clinicaltrials.gov as #NCT01500733.
Assuntos
Antineoplásicos/uso terapêutico , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Inibidores de Proteínas Quinases/uso terapêutico , Pirazóis/uso terapêutico , Pirimidinas/uso terapêutico , Adenina/análogos & derivados , Adulto , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos/administração & dosagem , Antineoplásicos/efeitos adversos , Medula Óssea/metabolismo , Feminino , Seguimentos , Humanos , Estimativa de Kaplan-Meier , Leucemia Linfocítica Crônica de Células B/diagnóstico , Leucemia Linfocítica Crônica de Células B/metabolismo , Leucemia Linfocítica Crônica de Células B/mortalidade , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Neoplasia Residual , Piperidinas , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/efeitos adversos , Pirazóis/administração & dosagem , Pirazóis/efeitos adversos , Pirimidinas/administração & dosagem , Pirimidinas/efeitos adversos , Resultado do TratamentoRESUMO
Chronic lymphocytic leukemia (CLL) is a progressive malignancy of mature B-cells that involves the peripheral blood (PB), lymph nodes (LNs) and bone marrow (BM). Although the majority of CLL cells are in a resting state, small populations of proliferating cells exist; however, the anatomical site of active cell proliferation remains to be definitively determined. Based on findings that CLL cells in LNs have increased expression of B-cell activation genes, we tested the hypothesis that the fraction of 'newly born' cells would be highest in the LNs. Using a deuterium oxide (2H) in vivo labeling method in which patients consumed deuterated (heavy) water (2H2O), we determined CLL cell kinetics in concurrently obtained samples from LN, PB and BM. The LN was identified as the anatomical site harboring the largest fraction of newly born cells, compared to PB and BM. In fact, the calculated birth rate in the LN reached as high a 3.3% of the clone per day. Subdivision of the bulk CLL population by flow cytometry identified the subpopulation with the CXCR4dimCD5bright phenotype as containing the highest proportion of newly born cells within each compartment, including the LN, identifying this subclonal population as an important target for novel treatment approaches.
Assuntos
Linfócitos B/patologia , Medula Óssea/patologia , Proliferação de Células , Leucemia Linfocítica Crônica de Células B/patologia , Leucócitos Mononucleares/patologia , Linfonodos/patologia , Idoso , Apoptose , Linfócitos B/imunologia , Linfócitos B/metabolismo , Medula Óssea/imunologia , Medula Óssea/metabolismo , Feminino , Citometria de Fluxo , Humanos , Imunofenotipagem , Leucemia Linfocítica Crônica de Células B/imunologia , Leucemia Linfocítica Crônica de Células B/metabolismo , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Linfonodos/imunologia , Linfonodos/metabolismo , Ativação Linfocitária , Masculino , Pessoa de Meia-Idade , Células Tumorais CultivadasRESUMO
Gaucher disease (GD) is an autosomal recessive disorder caused by mutations in the gene encoding acid-ß-glucosidase, resulting in functional disruptions in degradation of glycosphingolipids and lysosomal accumulation of the substrates. The most frequent clinical presentations of GD are thrombocytopenia, splenomegaly and bone pain. Prior to advent of enzyme replacement therapy, splenectomy was performed for complications of hypersplenism such as severe thrombocytopenia and transfusion dependency. Though there is evidence about worsening bone disease after splenectomy, there is no systematic study to assess its effects on the immune system in GD patients. In order to investigate the long-term immunological effects of splenectomy, we used flow cytometry to compare the immunophenotypes of GD patients who had undergone splenectomy (SGD) to those with intact spleen. The results show that SGD patients have significantly fewer CD27(+)/IgM(+) B-cells but more CD4(+)/CD45RO(+) and CD8(+)/CD45RO(+) T-cells. The most surprising finding was an almost complete absence of circulating dendritic cells in SGD patients. In addition, splenectomized subjects had comorbidities, the most common being monoclonal gammopathy of undetermined significance (MGUS). Taken together, these results highlight the persistence of multiple immune alterations and comorbidities coexisting in higher frequency in the SGD group and they are not affected by GD specific therapy.
Assuntos
Comorbidade , Doença de Gaucher/cirurgia , Sistema Imunitário , Esplenectomia/efeitos adversos , Adulto , Idoso , Linfócitos B/imunologia , Estudos de Casos e Controles , Células Dendríticas , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Gamopatia Monoclonal de Significância Indeterminada/etiologia , Linfócitos T/imunologiaAssuntos
Citometria de Fluxo/métodos , Mieloma Múltiplo/diagnóstico , Neoplasia Residual/diagnóstico , Plasmócitos/patologia , Antineoplásicos/uso terapêutico , Citometria de Fluxo/instrumentação , Humanos , Imunofenotipagem , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/mortalidade , Mieloma Múltiplo/patologia , Neoplasia Residual/tratamento farmacológico , Neoplasia Residual/mortalidade , Neoplasia Residual/patologia , Plasmócitos/efeitos dos fármacos , Prognóstico , Análise de SobrevidaRESUMO
PURPOSE: Clinical trials of ibrutinib combined with anti-CD20 monoclonal antibodies (mAb) for chronic lymphocytic leukemia (CLL) report encouraging results. Paradoxically, in preclinical studies, in vitro ibrutinib was reported to decrease CD20 expression and inhibit cellular effector mechanisms. We therefore set out to investigate effects of in vivo ibrutinib treatment that could explain this paradox. EXPERIMENTAL DESIGN: Patients received single-agent ibrutinib (420 mg daily) on an investigator-initiated phase II trial. Serial blood samples were collected pretreatment and during treatment for ex vivo functional assays to examine the effects on CLL cell susceptibility to anti-CD20 mAbs. RESULTS: We demonstrate that CD20 expression on ibrutinib was rapidly and persistently downregulated (median reduction 74%, day 28, P < 0.001) compared with baseline. Concomitantly, CD20 mRNA was decreased concurrent with reduced NF-κB signaling. An NF-κB binding site in the promoter of MS4A1 (encoding CD20) and downregulation of CD20 by NF-κB inhibitors support a direct transcriptional effect. Ex vivo, tumor cells from patients on ibrutinib were less susceptible to anti-CD20 mAb-mediated complement-dependent cytotoxicity than pretreatment cells (median reduction 75%, P < 0.001); however, opsonization by the complement protein C3d, which targets cells for phagocytosis, was relatively maintained. Expression of decay-accelerating factor (CD55) decreased on ibrutinib, providing a likely mechanism for the preserved C3d opsonization. In addition, ibrutinib significantly inhibited trogocytosis, a major contributor to antigen loss and tumor escape during mAb therapy. CONCLUSIONS: Our data indicate that ibrutinib promotes both positive and negative interactions with anti-CD20 mAbs, suggesting that successfully harnessing maximal antitumor effects of such combinations requires further investigation.
Assuntos
Antineoplásicos/uso terapêutico , Pirazóis/uso terapêutico , Pirimidinas/uso terapêutico , Rituximab/efeitos dos fármacos , Adenina/análogos & derivados , Antígenos CD20/genética , Antígenos CD20/metabolismo , Antineoplásicos/farmacologia , Biópsia , Medula Óssea/patologia , Antígenos CD55/genética , Antígenos CD55/metabolismo , Ensaios Clínicos Fase II como Assunto , Proteínas do Sistema Complemento/imunologia , Citotoxicidade Imunológica , Interações Medicamentosas , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Humanos , Leucemia Linfocítica Crônica de Células B/diagnóstico , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/metabolismo , NF-kappa B/metabolismo , Piperidinas , Pirazóis/farmacologia , Pirimidinas/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Rituximab/farmacologiaRESUMO
PURPOSE: Chronic lymphocytic leukemia (CLL) cells depend on microenvironmental interactions for proliferation and survival that are at least partially mediated through B-cell receptor (BCR) signaling. Ibrutinib, a Bruton tyrosine kinase inhibitor, disrupts BCR signaling and leads to the egress of tumor cells from the microenvironment. Although the on-target effects on CLL cells are well defined, the impact on the microenvironment is less well studied. We therefore sought to characterize the in vivo effects of ibrutinib on the tumor microenvironment. EXPERIMENTAL DESIGN: Patients received single-agent ibrutinib on an investigator-initiated phase II trial. Serial blood and tissue samples were collected pretreatment and during treatment. Changes in cytokine levels, cellular subsets, and microenvironmental interactions were assessed. RESULTS: Serum levels of key chemokines and inflammatory cytokines decreased significantly in patients on ibrutinib. Furthermore, ibrutinib treatment decreased circulating tumor cells and overall T-cell numbers. Most notably, a reduced frequency of the Th17 subset of CD4(+)T cells was observed concurrent with reduced expression of activation markers and PD-1 on T cells. Consistent with direct inhibition of T cells, ibrutinib inhibited Th17 differentiation of murine CD4(+)T cells in vitro Finally, in the bone marrow microenvironment, we found that ibrutinib disaggregated the interactions of macrophages and CLL cells, inhibited secretion of CXCL13, and decreased the chemoattraction of CLL cells. CONCLUSIONS: In conjunction with inhibition of BCR signaling, these changes in the tumor microenvironment likely contribute to the antitumor activity of ibrutinib and may impact the efficacy of immunotherapeutic strategies in patients with CLL. See related commentary by Bachireddy and Wu, p. 1547.
Assuntos
Antineoplásicos/uso terapêutico , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/patologia , Inibidores de Proteínas Quinases/uso terapêutico , Pirazóis/uso terapêutico , Pirimidinas/uso terapêutico , Microambiente Tumoral/efeitos dos fármacos , Adenina/análogos & derivados , Idoso , Idoso de 80 Anos ou mais , Animais , Antineoplásicos/farmacologia , Medula Óssea/patologia , Comunicação Celular/efeitos dos fármacos , Comunicação Celular/imunologia , Diferenciação Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Movimento Celular/imunologia , Citocinas/sangue , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Humanos , Imunofenotipagem , Mediadores da Inflamação/sangue , Mediadores da Inflamação/metabolismo , Leucemia Linfocítica Crônica de Células B/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Masculino , Camundongos , Pessoa de Meia-Idade , Piperidinas , Inibidores de Proteínas Quinases/farmacologia , Pirazóis/farmacologia , Pirimidinas/farmacologia , Subpopulações de Linfócitos T/efeitos dos fármacos , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Células Th17/efeitos dos fármacos , Células Th17/imunologia , Células Th17/metabolismoRESUMO
The FDA has co-sponsored three workshops to address minimal residual disease (MRD) detection in acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), and acute myeloid leukemia (AML) as well as an FDA-NCI roundtable symposium on MRD detection and its use as a response biomarker in Multiple Myeloma (MM). As clinical outcomes in MM continue to improve with the introduction of new therapeutics, consideration of biomarkers and their development as validated surrogate endpoints that can be used in the place of traditional clinical trial endpoints of progression-free survival (PFS) will be fundamental to expeditious drug development. This article will describe the FDA drug approval process, the regulatory framework through which a biomarker can be used as a surrogate endpoint for drug approval, and how MRD detection in MM fits within this context. In parallel, this article will also describe the FDA current device clearance process with emphasis on the analytical development as it might apply to an in vitro diagnostic assay for the detection of MRD in MM. It is anticipated that this Special Issue may possibly represent how MRD might serve as a drug development tool in hematological malignancies.
Assuntos
Antígenos CD/análise , Antineoplásicos/uso terapêutico , Aprovação de Drogas/legislação & jurisprudência , Citometria de Fluxo/normas , Mieloma Múltiplo/diagnóstico , Neoplasia Residual/diagnóstico , Antígenos CD/genética , Antígenos CD/imunologia , Biomarcadores Farmacológicos/análise , Aprovação de Equipamentos/legislação & jurisprudência , Expressão Gênica , Humanos , Imunofenotipagem , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/imunologia , Mieloma Múltiplo/mortalidade , Neoplasia Residual/tratamento farmacológico , Neoplasia Residual/imunologia , Neoplasia Residual/mortalidade , Plasmócitos/efeitos dos fármacos , Plasmócitos/imunologia , Plasmócitos/patologia , Prognóstico , Indução de Remissão , Análise de Sobrevida , Estados Unidos , United States Food and Drug AdministrationRESUMO
Chronic lymphocytic leukemia (CLL) is characterized by immune dysregulation, often including hypogammaglobulinemia, which contributes to a high rate of infections and morbidity. Ibrutinib, a covalent inhibitor of Bruton tyrosine kinase (BTK), inhibits B-cell receptor signaling and is an effective, US Food and Drug Administration (FDA)-approved treatment of CLL. Inactivating germline mutations in BTK cause a severe B-cell defect and agammaglobulinemia. Therefore, we assessed the impact of ibrutinib on immunoglobulin levels, normal B cells, and infection rate in patients with CLL treated with single-agent ibrutinib on a phase 2 investigator-initiated trial. Consistent with previous reports, immunoglobulin G (IgG) levels remained stable during the first 6 months on treatment, but decreased thereafter. In contrast, there were a transient increase in IgM and a sustained increase in IgA (median increase 45% at 12 months, P < .0001). To distinguish the effects on clonal B cells from normal B cells, we measured serum free light chains (FLCs). In κ-clonal CLL cases, clonal (κ) FLCs were elevated at baseline and normalized by 6 months. Nonclonal (λ) FLCs, which were often depressed at baseline, increased, suggesting the recovery of normal B cells. Consistently, we observed normal B-cell precursors in the bone marrow and an increase in normal B-cell numbers in the peripheral blood. Patients with superior immune reconstitution, as defined by an increase in serum IgA of ≥50% from baseline to 12 months, had a significantly lower rate of infections (P = .03). These data indicate that ibrutinib allows for a clinically meaningful recovery of humoral immune function in patients with CLL. This trial was registered at www.clinicaltrials.gov as #NCT015007330.
Assuntos
Linfócitos B , Imunidade Humoral/efeitos dos fármacos , Imunoglobulinas , Infecções , Leucemia Linfocítica Crônica de Células B , Pirazóis/administração & dosagem , Pirimidinas/administração & dosagem , Recuperação de Função Fisiológica , Adenina/análogos & derivados , Tirosina Quinase da Agamaglobulinemia , Idoso , Idoso de 80 Anos ou mais , Linfócitos B/imunologia , Linfócitos B/metabolismo , Medula Óssea/imunologia , Medula Óssea/metabolismo , Feminino , Seguimentos , Humanos , Imunoglobulinas/sangue , Imunoglobulinas/imunologia , Infecções/sangue , Infecções/tratamento farmacológico , Infecções/imunologia , Leucemia Linfocítica Crônica de Células B/sangue , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/imunologia , Masculino , Piperidinas , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/imunologia , Proteínas Tirosina Quinases/metabolismo , Recuperação de Função Fisiológica/efeitos dos fármacos , Recuperação de Função Fisiológica/imunologia , Fatores de TempoRESUMO
A surface-labeled lyophilized lymphocyte (sLL) preparation has been developed using human peripheral blood mononuclear cells prelabeled with a fluorescein isothiocyanate conjugated anti-CD4 monoclonal antibody. The sLL preparation is intended to be used as a reference material for CD4+ cell counting including the development of higher order reference measurement procedures and has been evaluated in the pilot study CCQM-P102. This study was conducted across 16 laboratories from eight countries to assess the ability of participants to quantify the CD4+ cell count of this reference material and to document cross-laboratory variability plus associated measurement uncertainties. Twelve different flow cytometer platforms were evaluated using a standard protocol that included calibration beads used to obtain quantitative measurements of CD4+ T cell counts. There was good overall cross-platform and counting method agreement with a grand mean of the laboratory calculated means of (301.7 ± 4.9) µL(-1) CD4+ cells. Excluding outliers, greater than 90% of participant data agreed within ±15%. A major contribution to variation of sLL CD4+ cell counts was tube to tube variation of the calibration beads, amounting to an uncertainty of 3.6%. Variation due to preparative steps equated to an uncertainty of 2.6%. There was no reduction in variability when data files were centrally reanalyzed. Remaining variation was attributed to instrument specific differences. CD4+ cell counts obtained in CCQM-P102 are in excellent agreement and show the robustness of both the measurements and the data analysis and hence the suitability of sLL as a reference material for interlaboratory comparisons and external quality assessment.
Assuntos
Linfócitos T CD4-Positivos , Fluoresceína-5-Isotiocianato , Leucócitos Mononucleares , Fenótipo , Anticorpos/análise , Contagem de Linfócito CD4/métodos , Contagem de Linfócito CD4/normas , Linfócitos T CD4-Positivos/química , Fluoresceína-5-Isotiocianato/análise , Liofilização/métodos , Humanos , Leucócitos Mononucleares/química , Projetos PilotoAssuntos
Biomarcadores Tumorais/análise , Citometria de Fluxo/estatística & dados numéricos , Mieloma Múltiplo/diagnóstico , Neoplasia Residual/diagnóstico , Biomarcadores Tumorais/genética , Congressos como Assunto , Humanos , Mieloma Múltiplo/genética , Mieloma Múltiplo/mortalidade , Mieloma Múltiplo/patologia , Neoplasia Residual/genética , Neoplasia Residual/mortalidade , Neoplasia Residual/patologia , Análise de SobrevidaRESUMO
Circulating monoclonal B cells may be detected in healthy adults, a condition called monoclonal B-cell lymphocytosis (MBL). MBL has also been identified in donated blood, but no systematic study of blood donors has been reported. Using sensitive and specific laboratory methods, we detected MBL in 149 (7.1%; 95% confidence interval, 6.0% to 8.3%) of 2098 unique donors ages 45 years or older in a Midwestern US regional blood center between 2010 and 2011. Most of the 149 donors had low-count MBL, including 99 chronic lymphocytic leukemia-like (66.4%), 22 atypical (14.8%), and 19 CD5(-) (12.8%) immunophenotypes. However, 5 donors (3.4%) had B-cell clonal counts above 500 cells per µL, including 3 with 1693 to 2887 cells per µL; the clone accounted for nearly all their circulating B cells. Four donors (2.7%) had 2 distinct MBL clones. Of 51 MBL samples in which immunoglobulin heavy chain (IGH)V-D-J genotypes could be determined, 71% and 29% used IGHV3- and IGHV4-family genes, respectively. Sequencing revealed 82% with somatic hypermutation, whereas 18% had >98% germ-line identity, including 5 with entirely germ-line sequences. In conclusion, MBL prevalence is much higher in blood donors than previously reported, and although uncommon, the presence of high-count MBL warrants further investigations to define the biological fate of the transfused cells in recipients.
Assuntos
Linfócitos B/patologia , Doadores de Sangue/estatística & dados numéricos , Linfocitose/epidemiologia , Idoso , Idoso de 80 Anos ou mais , Anticorpos Monoclonais , Linfócitos B/imunologia , Feminino , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Imunofenotipagem , Leucemia Linfocítica Crônica de Células B/sangue , Leucemia Linfocítica Crônica de Células B/epidemiologia , Contagem de Linfócitos , Linfocitose/sangue , Linfocitose/genética , Masculino , Pessoa de Meia-Idade , PrevalênciaRESUMO
BACKGROUND: Deletion 13q14.3 is the most common cytogenetic abnormality in chronic lymphocytic leukemia (CLL). Previously it was reported that miR-15/16 is the target of 13q14 deletions and plays a tumor suppressor role by suppressing Bcl-2. Therefore, Bcl-2 expression was examined more closely to determine whether it would predict 13q14 deletion status. METHODS: A multi-color flow panel consisting of anti-Bcl-2/anti-lambda/anti-kappa/CD19/CD5/CD3/CD20 was performed. The ability of Bcl-2 to predict 13q14 deletion was tested using the conventional Bcl-2 index (c-index): mean fluorescence intensity (MFI) of CLL clone/MFI of residual T cells. Fifty-four untreated CLL/MBL patients were studied. Bimodal Bcl-2 expression was evaluated to test the ability of Bcl-2 to detect intraclonal heterogeneity. Other CLL prognostic markers including CD38, CD49d, CD26, and CD69 were evaluated. FISH was performed on selected sorted populations. RESULTS: The Bcl-2 c-index strongly predicts del13q14 P < 0.0001. A statistically significant association was observed between the percentage of cells carrying the deletion and the level of Bcl-2 expression P < 0.05. Cells sorted based on Bcl-2 expression showed enrichment of both hemizygous and homozygous del 13q14 cells. Also, we observed that an alteration in Bcl-2 level over time predicts changes in 13q14 deletion status. And a statistically significant correlation between the bimodal pattern of CD69 expression and the presence of 13q14 deletion was found P < 0.0001. CONCLUSION: Bcl-2 expression using the c-index strongly predicts 13q14 deletion and can be used to distinguish homozygous, heterozygous, and diploid CLL clonal cells. Further systematic studies of this biomarker are needed for confirmation and expansion of these findings.
Assuntos
Citometria de Fluxo/métodos , Genes bcl-2/genética , Leucemia Linfocítica Crônica de Células B/genética , Deleção de Sequência , Idoso , Animais , Aberrações Cromossômicas , Cromossomos Humanos Par 13 , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Leucemia Linfocítica Crônica de Células B/diagnóstico , Leucemia Linfocítica Crônica de Células B/patologia , Masculino , Pessoa de Meia-Idade , PrognósticoRESUMO
Overexpression of zeta-chain-associated protein 70 (ZAP-70) was recently recognized as an independent prognostic marker for the aggressive form of chronic lymphocytic leukemia (CLL). The objective of this study was to demonstrate the feasibility and implementation of quantitative detection of ZAP-70 protein in B cells to clearly distinguish patients with CLL with the aggressive form of the disease. B cells were isolated from patient blood and lysed. Released ZAP-70 protein was detected using an immunomagnetic fluorescence assay. The assay protocol was developed using Jurkat cells and recombinant ZAP-70 (rZAP-70). The limit of detection was determined to be lower than 125 Jurkat cells and 39 pg of rZAP-70 protein. The signal response was linear over a wide dynamic range, from 125 to 40 000 Jurkat cells per test (R(2) = 0.9987) and from 0 to 40 000 pg rZAP-70 protein per test (R(2) = 0.9928). The results from 20 patients with CLL correlated strongly with flow cytometry analysis. Concordance between the two methods for positive and negative results was 100% (7/7) and 92% (12/13), respectively, while the overall concordance between the two methods was 95%. The assay reported here is a simple, reliable and reproducible method for quantitative detection of ZAP-70 in patient leukemic cells, without the need for cell fixation or permeabilization. The ZAP-70 signal was linear over a wide dynamic range, which we believe enables quantitative assessment of small changes in ZAP-70 expression over the course of the disease and in response to therapeutic intervention.
Assuntos
Linfócitos B/metabolismo , Leucemia Linfocítica Crônica de Células B/metabolismo , Proteína-Tirosina Quinase ZAP-70/análise , Citometria de Fluxo , Imunofluorescência/métodos , Humanos , Separação Imunomagnética/métodos , Células Jurkat , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/patologia , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína-Tirosina Quinase ZAP-70/genéticaRESUMO
To transform the linear fluorescence intensity scale obtained with fluorescent microspheres to an antibody bound per cell (ABC) scale, a biological cell reference material is needed. Optimally, this material should have a reproducible and tight ABC value for the expression of a known clinical reference biomarker. In this study, we characterized commercially available cryopreserved peripheral blood mononuclear cells (PBMCs) and two lyophilized PBMC preparations, Cyto-Trol and PBMC-National Institute for Biological Standard and Control (NIBSC) relative to freshly prepared PBMC and whole blood samples. It was found that the ABC values for CD4 expression on cryopreserved PBMC were consistent with those of freshly obtained PBMC and whole blood samples. By comparison, the ABC value for CD4 expression on Cyto-Trol is lower and the value on PBMC-NIBSC is much lower than those of freshly prepared cell samples using both conventional flow cytometry and CyTOF™ mass cytometry. By performing simultaneous surface and intracellular staining measurements on these two cell samples, we found that both cell membranes are mostly intact. Moreover, CD4(+) cell diameters from both lyophilized cell preparations are smaller than those of PBMC and whole blood. This could result in steric interference in antibody binding to the lyophilized cells. Further investigation of the fixation effect on the detected CD4 expression suggests that the very low ABC value obtained for CD4(+) cells from lyophilized PBMC-NIBSC is largely due to paraformaldehyde fixation; this significantly decreases available antibody binding sites. This study provides confirmation that the results obtained from the newly developed mass cytometry are directly comparable to the results from conventional flow cytometry when both methods are standardized using the same ABC approach.