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1.
Blood ; 129(11): 1469-1479, 2017 03 16.
Artigo em Inglês | MEDLINE | ID: mdl-28049639

RESUMO

Disease progression in patients with chronic lymphocytic leukemia (CLL) treated with ibrutinib has been attributed to histologic transformation or acquired mutations in BTK and PLCG2. The rate of resistance and clonal composition of PD are incompletely characterized. We report on CLL patients treated with single-agent ibrutinib on an investigator-initiated phase 2 trial. With median follow-up of 34 months, 15 of 84 evaluable patients (17.9%) progressed. Relapsed/refractory disease at study entry, TP53 aberration, advanced Rai stage, and high ß-2 microglobulin were independently associated with inferior progression-free survival (P < .05 for all tests). Histologic transformation occurred in 5 patients (6.0%) and was limited to the first 15 months on ibrutinib. In contrast, progression due to CLL in 10 patients (11.9%) occurred later, diagnosed at a median 38 months on study. At progression, mutations in BTK (Cys481) and/or PLCG2 (within the autoinhibitory domain) were found in 9 patients (10.7%), in 8 of 10 patients with progressive CLL, and in 1 patient with prolymphocytic transformation. Applying high-sensitivity testing (detection limit ∼1 in 1000 cells) to stored samples, we detected mutations up to 15 months before manifestation of clinical progression (range, 2.9-15.4 months). In 5 patients (6.0%), multiple subclones carrying different mutations arose independently, leading to subclonal heterogeneity of resistant disease. For a seamless transition to alternative targeted agents, patients progressing with CLL were continued on ibrutinib for up to 3 months, with 19.8 months median survival from the time of progression. This trial was registered at www.clinicaltrials.gov as #NCT01500733.


Assuntos
Evolução Clonal , Resistencia a Medicamentos Antineoplásicos , Leucemia Linfocítica Crônica de Células B/patologia , Pirazóis/uso terapêutico , Pirimidinas/uso terapêutico , Adenina/análogos & derivados , Tirosina Quinase da Agamaglobulinemia , Idoso , Transformação Celular Neoplásica , Progressão da Doença , Intervalo Livre de Doença , Seguimentos , Humanos , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/mortalidade , Leucemia Linfocítica Crônica de Células B/fisiopatologia , Fosfolipase C gama/genética , Piperidinas , Proteínas Tirosina Quinases/genética
2.
Leukemia ; 31(6): 1340-1347, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28074063

RESUMO

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 Cultivadas
3.
JAMA Oncol ; 1(6): 746-54, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26181891

RESUMO

IMPORTANCE: Carfilzomib-lenalidomide-dexamethasone therapy yields deep responses in patients with newly diagnosed multiple myeloma (NDMM). It is important to gain an understanding of this combination's tolerability and impact on minimal residual disease (MRD) negativity because this end point has been associated with improved survival. OBJECTIVE: To assess the safety and efficacy of carfilzomib-lenalidomide-dexamethasone therapy in NDMM and high-risk smoldering multiple myeloma (SMM). DESIGN, SETTING, AND PARTICIPANTS: Clinical and correlative pilot study at the National Institutes of Health Clinical Center. Patients with NDMM or high-risk SMM were enrolled between July 11, 2011, and October 9, 2013. Median follow-up was 17.3 (NDMM) and 15.9 months (SMM). INTERVENTIONS: Eight 28-day cycles were composed of carfilzomib 20/36 mg/m2 on days 1, 2, 8, 9, 15, and 16; lenalidomide 25 mg on days 1 through 21; and dexamethasone 20/10 mg (cycles 1-4/5-8) on days 1, 2, 8, 9, 15, 16, 22, and 23. Patients who achieved at least stable disease subsequently received 24 cycles of lenalidomide extended dosing. MAIN OUTCOMES AND MEASURES: Primary end points were neuropathy of grade 3 or greater (NDMM) and at least very good partial response rates (SMM). Minimal residual disease was also assessed. RESULTS: Of 45 patients with NDMM, none had neuropathy of grade 3 or greater. Of 12 patients with high-risk SMM, the most common of any-grade adverse events were lymphopenia (12 [100%]) and gastrointestinal disorders (11 [92%]). All patients with SMM achieved at least a very good partial response during the study period. Among the 28 patients with NDMM and the 12 with SMM achieving at least a near-complete response, MRD negativity was found in 28 of 28 (100% [95% CI, 88%-100%]), 11 of 12 (92% [95% CI, 62%-100%]) (multiparametric flow cytometry), 14 of 21 (67% [95% CI, 43%-85%]), and 9 of 12 (75% [95% CI, 43%-94%]) (next-generation sequencing), respectively. In patients with NDMM, 12-month progression-free survival for MRD-negative vs MRD-positive status by flow cytometry and next-generation sequencing was 100% vs 79% (95% CI, 47%-94%; P < .001) and 100% vs 95% (95% CI, 75%-99%; P = .02), respectively. CONCLUSIONS AND RELEVANCE: Carfilzomib-lenalidomide-dexamethasone therapy is tolerable and demonstrates high rates of MRD negativity in NDMM, translating into longer progression-free survival in patients achieving MRD negativity. Carfilzomib-lenalidomide-dexamethasone therapy also demonstrates efficacy in high-risk SMM.


Assuntos
Inibidores da Angiogênese/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Dexametasona/administração & dosagem , Mieloma Múltiplo/tratamento farmacológico , Oligopeptídeos/administração & dosagem , Inibidores de Proteassoma/administração & dosagem , Talidomida/análogos & derivados , Adulto , Idoso , Idoso de 80 Anos ou mais , Inibidores da Angiogênese/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Biomarcadores Tumorais/genética , Dexametasona/efeitos adversos , Intervalo Livre de Doença , Esquema de Medicação , Feminino , Humanos , Lenalidomida , Masculino , Maryland , Pessoa de Meia-Idade , Mieloma Múltiplo/diagnóstico , Mieloma Múltiplo/genética , Mieloma Múltiplo/mortalidade , National Institutes of Health (U.S.) , Neoplasia Residual , Oligopeptídeos/efeitos adversos , Projetos Piloto , Inibidores de Proteassoma/efeitos adversos , Fatores de Risco , Talidomida/administração & dosagem , Talidomida/efeitos adversos , Fatores de Tempo , Resultado do Tratamento , Estados Unidos
5.
Lancet Oncol ; 16(2): 169-76, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25555420

RESUMO

BACKGROUND: Patients with chronic lymphocytic leukaemia (CLL) with TP53 aberrations respond poorly to first-line chemoimmunotherapy, resulting in early relapse and short survival. We investigated the safety and activity of ibrutinib in previously untreated and relapsed or refractory CLL with TP53 aberrations. METHODS: In this investigator-initiated, single-arm phase 2 study, we enrolled eligible adult patients with active CLL with TP53 aberrations at the National Institutes of Health Clinical Center (Bethesda, MD, USA). Patients received 28-day cycles of ibrutinib 420 mg orally once daily until disease progression or the occurrence of limiting toxicities. The primary endpoint was overall response to treatment at 24 weeks in all evaluable patients. This study is registered with ClinicalTrials.gov, number NCT01500733, and is fully enrolled. FINDINGS: Between Dec 22, 2011, and Jan 2, 2014, we enrolled 51 patients; 47 had CLL with deletion 17p13.1 and four carried a TP53 mutation in the absence of deletion 17p13.1. All patients had active disease requiring therapy. 35 enrolled patients had previously untreated CLL and 16 had relapsed or refractory disease. Median follow-up was 24 months (IQR 12.9-27.0). 33 previously untreated patients and 15 patients with relapsed or refractory CLL were evaluable for response at 24 weeks. 32 (97%; 95% CI 86-100) of 33 previously untreated patients achieved an objective response, including partial response in 18 patients (55%) and partial response with lymphocytosis in 14 (42%). One patient had progressive disease at 0.4 months. 12 (80%; 95% CI 52-96) of the 15 patients with relapsed or refractory CLL had an objective response: six (40%) achieved a partial response and six (40%) a partial response with lymphocytosis; the remaining three (20%) patients had stable disease. Grade 3 or worse treatment-related adverse events were neutropenia in 12 (24%) patients (grade 4 in one [2%] patient), anaemia in seven (14%) patients, and thrombocytopenia in five (10%) patients (grade 4 in one [2%] patient). Grade 3 pneumonia occurred in three (6%) patients, and grade 3 rash in one (2%) patient. INTERPRETATION: The activity and safety profile of single-agent ibrutinib in CLL with TP53 aberrations is encouraging and supports its consideration as a novel treatment option for patients with this high-risk disease in both first-line and second-line settings. FUNDING: Intramural Research Program of the National Heart, Lung, and Blood Institute and the National Cancer Institute, Danish Cancer Society, Novo Nordisk Foundation, National Institutes of Health Medical Research Scholars Program, and Pharmacyclics Inc.


Assuntos
Resistencia a Medicamentos Antineoplásicos/genética , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Mutação/genética , Recidiva Local de Neoplasia/tratamento farmacológico , Pirazóis/uso terapêutico , Pirimidinas/uso terapêutico , Proteína Supressora de Tumor p53/genética , Adenina/análogos & derivados , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Seguimentos , Humanos , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/mortalidade , Leucemia Linfocítica Crônica de Células B/patologia , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/mortalidade , Recidiva Local de Neoplasia/patologia , Estadiamento de Neoplasias , Piperidinas , Prognóstico , Método Simples-Cego , Taxa de Sobrevida
6.
Blood ; 125(1): 56-70, 2015 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-25359990

RESUMO

Germ-line GATA2 gene mutations, leading to haploinsufficiency, have been identified in patients with familial myelodysplastic syndrome/acute myeloid leukemia, monocytopenia and mycobacterial infections, Emberger syndrome, and dendritic cell, monocyte, B-, and NK-cell deficiency. GATA2 mutations have also been reported in a minority of patients with congenital neutropenia and aplastic anemia (AA). The bone marrow (BM) from patients with GATA2 deficiency is typically hypocellular, with varying degrees of dysplasia. Distinguishing GATA2 patients from those with AA is critical for selecting appropriate therapy. We compared the BM flow cytometric, morphologic, and cytogenetic features of 28 GATA2 patients with those of 32 patients being evaluated for idiopathic AA. The marrow of GATA2 patients had severely reduced monocytes, B cells, and NK cells; absent hematogones; and inverted CD4:CD8 ratios. Atypical megakaryocytes and abnormal cytogenetics were more common in GATA2 marrows. CD34(+) cells were comparably reduced in GATA2 and AA. Using these criteria, we prospectively identified 4 of 32 patients with suspected AA who had features suspicious for GATA2 mutations, later confirmed by DNA sequencing. Our results show that routine BM flow cytometry, morphology, and cytogenetics in patients who present with cytopenia(s) can identify patients for whom GATA2 sequencing is indicated.


Assuntos
Anemia Aplástica/diagnóstico , Doenças da Medula Óssea/diagnóstico , Fator de Transcrição GATA2/deficiência , Adulto , Anemia Aplástica/genética , Anemia Aplástica/metabolismo , Antígenos CD34/metabolismo , Medula Óssea/patologia , Doenças da Medula Óssea/genética , Estudos de Coortes , Citogenética , Feminino , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Mutação , Análise de Sequência de DNA , Adulto Jovem
7.
Blood ; 123(6): 809-21, 2014 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-24227816

RESUMO

Haploinsufficiency of the hematopoietic transcription factor GATA2 underlies monocytopenia and mycobacterial infections; dendritic cell, monocyte, B, and natural killer (NK) lymphoid deficiency; familial myelodysplastic syndromes (MDS)/acute myeloid leukemia (AML); and Emberger syndrome (primary lymphedema with MDS). A comprehensive examination of the clinical features of GATA2 deficiency is currently lacking. We reviewed the medical records of 57 patients with GATA2 deficiency evaluated at the National Institutes of Health from January 1, 1992, to March 1, 2013, and categorized mutations as missense, null, or regulatory to identify genotype-phenotype associations. We identified a broad spectrum of disease: hematologic (MDS 84%, AML 14%, chronic myelomonocytic leukemia 8%), infectious (severe viral 70%, disseminated mycobacterial 53%, and invasive fungal infections 16%), pulmonary (diffusion 79% and ventilatory defects 63%, pulmonary alveolar proteinosis 18%, pulmonary arterial hypertension 9%), dermatologic (warts 53%, panniculitis 30%), neoplastic (human papillomavirus+ tumors 35%, Epstein-Barr virus+ tumors 4%), vascular/lymphatic (venous thrombosis 25%, lymphedema 11%), sensorineural hearing loss 76%, miscarriage 33%, and hypothyroidism 14%. Viral infections and lymphedema were more common in individuals with null mutations (P = .038 and P = .006, respectively). Monocytopenia, B, NK, and CD4 lymphocytopenia correlated with the presence of disease (P < .001). GATA2 deficiency unites susceptibility to MDS/AML, immunodeficiency, pulmonary disease, and vascular/lymphatic dysfunction. Early genetic diagnosis is critical to direct clinical management, preventive care, and family screening.


Assuntos
Fator de Transcrição GATA2/genética , Síndromes de Imunodeficiência/mortalidade , Leucemia Mieloide Aguda/mortalidade , Síndromes Mielodisplásicas/mortalidade , Adolescente , Adulto , Idoso , Criança , Feminino , Fator de Transcrição GATA2/deficiência , Estudos de Associação Genética , Haploinsuficiência , Hematopoese , Humanos , Síndromes de Imunodeficiência/genética , Síndromes de Imunodeficiência/patologia , Lactente , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Sistema Linfático , Masculino , Pessoa de Meia-Idade , Síndromes Mielodisplásicas/genética , Síndromes Mielodisplásicas/patologia , Prognóstico , Estudos Prospectivos , Taxa de Sobrevida , Adulto Jovem
8.
Cytometry B Clin Cytom ; 84(4): 237-47, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23674504

RESUMO

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 , Prognóstico
9.
Exp Hematol ; 41(9): 772-8, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23689118

RESUMO

Eradication of minimal residual disease (MRD) after allotransplantation in persons with chronic lymphocytic leukemia (CLL) is associated with lower rates of relapse. Rapid engraftment of donor lymphocyte elements can contribute to MRD control, but it remains unclear whether this strategy will benefit patients. In this study, we report the incidence of MRD eradication and graft-versus-host disease (GvHD) in persons with rapid versus later donor T lymphocyte engraftment after lymphodepleting chemotherapy and reduced intensity conditioning (RIC) allotransplantation. Twenty-seven subjects received lymphodepleting chemotherapy to facilitate donor engraftment followed by fludarabine and cyclophosphamide RIC and a blood cell allograft. MRD was monitored by multicolor flow cytometry after transplantation. Complete donor T lymphoid chimerism (TLC) and myeloid chimerism (MC) were achieved in 25 subjects at a median of 28 days (range, 14-60 days) and 21 days (range, 14-180 days), respectively. Achieving complete donor TLC by day 14 versus day 28 or later correlated with occurrence of grade 2 or higher acute GvHD (90% [95% confidence interval {CI}, 78-100%] versus 35% [95% CI, 16-54%]; p = 0.014) and better control of MRD in the bone marrow at day 100, median 0% (range, 0-0.1%) versus 8.5% (range, 0-92%; p = 0.016). Among 11 persons with early donor TLC, none had progressive disease, and seven died of treatment -related mortality (TRM). In persons with later development of TLC, 8 of 16 had progressive disease and 2 died of TRM. Time to donor myeloid chimerism had no effect on outcomes. Rapid establishment of donor TLC resulted in more complete eradication of early MRD, but greater incidence of acute GvHD and TRM in persons with CLL undergoing RIC allotransplantation.


Assuntos
Sobrevivência de Enxerto , Efeito Enxerto vs Leucemia , Leucemia Linfocítica Crônica de Células B/terapia , Transplante de Células-Tronco de Sangue Periférico , Quimeras de Transplante , Condicionamento Pré-Transplante , Doença Aguda , Adulto , Idoso , Feminino , Seguimentos , Doença Enxerto-Hospedeiro/etiologia , Doença Enxerto-Hospedeiro/mortalidade , Doença Enxerto-Hospedeiro/terapia , Humanos , Leucemia Linfocítica Crônica de Células B/mortalidade , Masculino , Pessoa de Meia-Idade , Neoplasia Residual , Estudos Retrospectivos , Linfócitos T , Fatores de Tempo , Transplante Homólogo
11.
Clin Cancer Res ; 18(7): 1979-91, 2012 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-22351695

RESUMO

PURPOSE: Chronic lymphocytic leukemia (CLL), a malignancy of mature B cells, is incurable with chemotherapy. Signals from the microenvironment support leukemic cell survival and proliferation and may confer chemotherapy resistance. ON 01910.Na (Rigosertib), a multikinase phosphoinositide 3-kinase (PI3K) inhibitor, is entering phase III trials for myelodysplastic syndrome. Our aim was to analyze the efficacy of ON 01910.Na against CLL cells in vitro and investigate the molecular effects of this drug on tumor biology. EXPERIMENTAL DESIGN: Cytotoxicity of ON 01910.Na against CLL cells from 34 patients was determined in vitro with flow cytometry of cells stained with Annexin V and CD19. Global gene expression profiling on Affymetrix microarrays, flow cytometry, Western blotting, and cocultures with stroma cells were used to delineate ON 01910.Na mechanism of action. RESULTS: ON 01910.Na induced apoptosis in CLL B cells without significant toxicity against T cells or normal B cells. ON 01910.Na was equally active against leukemic cells associated with a more aggressive disease course [immunoglobulin heavy-chain variable region unmutated, adverse cytogenetics] than against cells without these features. Gene expression profiling revealed two main mechanisms of action: PI3K/AKT inhibition and induction of ROS that resulted in an oxidative stress response through activating protein 1 (AP-1), c-jun-NH(2)-terminal kinase, and ATF3 culminating in the upregulation of NOXA. ROS scavengers and shRNA mediated knockdown of ATF3- and NOXA-protected cells from drug-induced apoptosis. ON 01910.Na also abrogated the prosurvival effect of follicular dendritic cells on CLL cells and reduced SDF-1-induced migration of leukemic cells. CONCLUSIONS: These data support the clinical development of ON 01910.Na in CLL.


Assuntos
Glicina/análogos & derivados , Estresse Oxidativo/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Sulfonas/farmacologia , Fator 3 Ativador da Transcrição/genética , Fator 3 Ativador da Transcrição/metabolismo , Apoptose/efeitos dos fármacos , Western Blotting , Linhagem Celular Tumoral , Feminino , Perfilação da Expressão Gênica , Glicina/farmacologia , Células HEK293 , Células HL-60 , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Região Variável de Imunoglobulina/genética , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/metabolismo , Leucemia Linfocítica Crônica de Células B/patologia , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mutação , Análise de Sequência com Séries de Oligonucleotídeos , Fosfatidilinositol 3-Quinases/genética , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Células Tumorais Cultivadas
12.
Cytometry B Clin Cytom ; 82(2): 67-77, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22031337

RESUMO

INTRODUCTION: Zeta-chain-associated protein kinase 70 (ZAP-70) has been identified as an independent prognostic marker in chronic lymphocytic leukemia (CLL). Based on our previous studies, we have developed a combined one-tube technology with multiple internal controls to optimize ZAP-70 assessment. METHODS: Forty-eight untreated CLL cases were examined for ZAP-70 expression using a modified 7-color one-tube assay. Normal donor (ND) whole blood is stained with CD3 APC-Cy7 and CD19 APC. In a second tube, patient whole blood is stained with CD5 PE-Cy7, CD19 PerCP-Cy5.5, and CD20 eFluor450. After surface staining and fixation, these two tubes are combined. After saponin permeabilization, the cells were stained with two anti-ZAP-70 clones (1E7.2/AF488 and SBZAP/PE). The results obtained from this modified tube were compared with those obtained concurrently using the non-mixed single sample tubes. Five different methods of ZAP-70 expression analysis were evaluated: percentage positive cells using ND T-cells as a reference; the internal patient T-cell/clone ratio; ND T-cell/clone ratio; clone/ND B-cell ratio; and modified Z-index. RESULT: Overall, the combined patient and ND mix tube performed better than the non-mixed single sample tube. The strongest correlations between ZAP-70 expression and immunoglobulin heavy chain variable (IGHV) mutational status were seen with percentage positive ND T-cell, ND T-cell/clone ratio, and clone/ND B-cell ratio for both 1E7.2 and SBZAP clone (P < 0.0001). CONCLUSION: The modified one tube method combining the ND and patient sample provides highly reliable results that correlate with the IGHV mutational status. This method should be considered as part of the next step in standardization of the ZAP-70 assay in CLL.


Assuntos
Citometria de Fluxo/métodos , Leucemia Linfocítica Crônica de Células B/diagnóstico , Proteína-Tirosina Quinase ZAP-70/análise , Proteína-Tirosina Quinase ZAP-70/sangue , Idoso , Idoso de 80 Anos ou mais , Anticorpos Monoclonais , Antígenos CD19/imunologia , Antígenos CD20/imunologia , Linfócitos B/citologia , Biomarcadores Tumorais/análise , Biomarcadores Tumorais/biossíntese , Complexo CD3/imunologia , Antígenos CD5/imunologia , Feminino , Humanos , Cadeias Pesadas de Imunoglobulinas/análise , Cadeias Pesadas de Imunoglobulinas/genética , Masculino , Pessoa de Meia-Idade , Coloração e Rotulagem/métodos , Linfócitos T/citologia
14.
Haematologica ; 96(8): 1221-5, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21508125

RESUMO

A novel, genetic immunodeficiency syndrome has been recently described, herein termed "MonoMAC". It is characterized by severe circulating monocytopenia, NK- and B-lymphocytopenia, severe infections with M. avium complex (MAC), and risk of progression to myelodysplasia/acute myelogenous leukemia. Detailed bone marrow analyses performed on 18 patients further define this disorder. The majority of patients had hypocellular marrows with reticulin fibrosis and multilineage dysplasia affecting the myeloid (72%), erythroid (83%) and megakaryocytic (100%) lineages. Cytogenetic abnormalities were present in 10 of 17 (59%). Despite B-lymphocytopenia, plasma cells were present but were abnormal (e.g. CD56(+)) in nearly half of cases. Increased T-cell large granular lymphocyte populations were present in 28% of patients. Chromosomal breakage studies, cell cycle checkpoint functions, and sequencing of TERT and K-RAS genes revealed no abnormalities. MonoMAC appears to be a unique, inherited syndrome of bone marrow failure. We describe distinctive bone marrow features to help in its recognition and diagnosis. (Clinicaltrials.gov identifiers: NCT00018044, NCT00923364, NCT01212055).


Assuntos
Síndromes de Imunodeficiência/diagnóstico , Leucopenia/patologia , Monócitos/patologia , Síndromes Mielodisplásicas/diagnóstico , Síndromes Mielodisplásicas/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Análise Citogenética , Progressão da Doença , Feminino , Instabilidade Genômica/genética , Humanos , Síndromes de Imunodeficiência/complicações , Imunofenotipagem , Leucemia Mieloide Aguda/patologia , Linfócitos/patologia , Masculino , Pessoa de Meia-Idade , Mutação/genética , Síndromes Mielodisplásicas/complicações , Mielofibrose Primária/patologia , Risco , Adulto Jovem
15.
Cytometry B Clin Cytom ; 80(5): 300-8, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21472849

RESUMO

BACKGROUND: ZAP-70 expression is a stage independent prognostic marker in CLL. However, interlaboratory variation is large, and there is neither a consensus nor a regulatory approved methodology. METHODS: Two anti-ZAP70 clones (1E7.2 and SBZAP) were compared in 45 untreated CLL patients. Nine different methods for ZAP-70 expression analysis were evaluated: M1, isotype control to determine negative; M2, internal residual T-cell to determine positive; M3, normal donor (ND) T-cell to determine positive; M4, internal T-cell/clone ratio; M5, ND residual T-cell/clone ratio; M6, clone/normal remaining B-cell ratio; M7, clone/ND B- cell ratio; M8, CLL-Z score; M9, modified CLL-Z score. A scoring system was designed integrating both 1E7.2 and SBZAP clones to assign ZAP-70 expression. RESULTS: The correlation coefficients for the four selected highest statistically significant methods were as follows (M1 = 0.71, M3 = 0.72, M7 = 0.67, and M9 = 0.64). These four methods were used to generate a combined score. The two reagents showed agreement using the designed scoring system for 37/45 samples (82%), and 8/45 (18%) showed equivocal result with one of the two clones. Seven of the eight equivocal samples were resolved using the scoring system. CONCLUSIONS: Four of the nine methods of analysis were compared for each reagent. The use of two independent ZAP-70 reagents increases analytical certitude and the scoring method aids in the resolution of equivocal results. The combined use of two reagents, four methods of analysis, and a scoring method allowed for assignment of ZAP-70 expression in 44/45 samples (98%) tested and improved performance of this important prognostic assay.


Assuntos
Anticorpos Monoclonais , Citometria de Fluxo/métodos , Leucemia Linfocítica Crônica de Células B , Proteína-Tirosina Quinase ZAP-70/imunologia , Idoso , Idoso de 80 Anos ou mais , Anticorpos Monoclonais/sangue , Especificidade de Anticorpos , Antígenos de Superfície/sangue , Antígenos de Superfície/imunologia , Feminino , Expressão Gênica/imunologia , Humanos , Leucemia Linfocítica Crônica de Células B/sangue , Leucemia Linfocítica Crônica de Células B/diagnóstico , Leucemia Linfocítica Crônica de Células B/imunologia , Masculino , Pessoa de Meia-Idade , Proteína-Tirosina Quinase ZAP-70/sangue
16.
Cytometry B Clin Cytom ; 80(5): 309-17, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21472850

RESUMO

INTRODUCTION: In a companion methodological study, we compared two anti-ZAP-70 clones (1E7.2 AF 488 and SBZAP PE) and four selected methods of analysis. Clinical correlations are required for validation. METHODS: Multicolor flow-cytometric evaluation of ZAP-70, CD38, CD69, CD26, CD49d, and CD27 was tested in 45 untreated-CLL patients. Four methods of ZAP-70 expression analysis and a scoring system were designed. A correlation analysis between ZAP-70 score, immunoglobulin heavy chain variable (IGHV) mutational status, fluorescence in situ hybridization, and these biomarkers was undertaken. RESULTS: There is a strong correlation between ZAP-70 expression and IGHV mutational status. The scoring system for a single reagent (P = 0.0006 or 0.0002) favors the use of multiple methods of analysis. The combined score was substantially equivalent (P = 0.0003). There was also a correlation with del 13q14 (P = 0.017) and trisomy12 (P = 0.011). A correlation for CD38 and ZAP-70 score was seen using both 1E7.2 AF488 and SBZAP PE when ≥20% or ≥7% cutoff was used. A positive correlation was seen for CD49d expression using both reagents. CD26 showed a correlation with ZAP-70 expression, but it was dependent upon the method of analysis. CD69 and CD27 showed no statistically significant correlation. CONCLUSION: In our study population, ZAP-70 expression is the better predictor of the IGHV mutational status. The correlation analysis confirms that the use of four methods of analysis with a single reagent or both reagents is superior to the use of a single method of analysis. The routine use of CD38, CD49d, and CD26 will require standardization.


Assuntos
Citometria de Fluxo/métodos , Imunoensaio/normas , Cadeias Pesadas de Imunoglobulinas , Leucemia Linfocítica Crônica de Células B , Proteína-Tirosina Quinase ZAP-70 , Idoso , Idoso de 80 Anos ou mais , Anticorpos Monoclonais/sangue , Antígenos de Superfície/sangue , Antígenos de Superfície/imunologia , Biomarcadores Tumorais/sangue , Feminino , Expressão Gênica/imunologia , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/imunologia , Hibridização in Situ Fluorescente , Leucemia Linfocítica Crônica de Células B/sangue , Leucemia Linfocítica Crônica de Células B/diagnóstico , Leucemia Linfocítica Crônica de Células B/imunologia , Masculino , Pessoa de Meia-Idade , Mutação , Proteína-Tirosina Quinase ZAP-70/sangue , Proteína-Tirosina Quinase ZAP-70/imunologia
17.
Blood ; 117(2): 563-74, 2011 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-20940416

RESUMO

Chronic lymphocytic leukemia (CLL), an incurable malignancy of mature B lymphocytes, involves blood, bone marrow, and secondary lymphoid organs such as the lymph nodes (LN). A role of the tissue microenvironment in the pathogenesis of CLL is hypothesized based on in vitro observations, but its contribution in vivo remains ill-defined. To elucidate the effects of tumor-host interactions in vivo, we purified tumor cells from 24 treatment-naive patients. Samples were obtained concurrently from blood, bone marrow, and/or LN and analyzed by gene expression profiling. We identified the LN as a key site in CLL pathogenesis. CLL cells in the LN showed up-regulation of gene signatures, indicating B-cell receptor (BCR) and nuclear factor-κB activation. Consistent with antigen-dependent BCR signaling and canonical nuclear factor-κB activation, we detected phosphorylation of SYK and IκBα, respectively. Expression of BCR target genes was stronger in clinically more aggressive CLL, indicating more effective BCR signaling in this subtype in vivo. Tumor proliferation, quantified by the expression of the E2F and c-MYC target genes and verified with Ki67 staining by flow cytometry, was highest in the LN and was correlated with clinical disease progression. These data identify the disruption of tumor microenvironment interactions and the inhibition of BCR signaling as promising therapeutic strategies in CLL. This study is registered at http://clinicaltrials.gov as NCT00019370.


Assuntos
Leucemia Linfocítica Crônica de Células B/patologia , Linfonodos/patologia , NF-kappa B/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo , Transdução de Sinais/fisiologia , Microambiente Tumoral/fisiologia , Adulto , Proliferação de Células , Separação Celular , Feminino , Citometria de Fluxo , Perfilação da Expressão Gênica , Humanos , Hibridização in Situ Fluorescente , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/metabolismo , Linfonodos/metabolismo , Masculino , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , Receptores de Antígenos de Linfócitos B/genética
18.
Pediatr Blood Cancer ; 54(1): 173-5, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19785027

RESUMO

The association of neurofibromatosis 1 (NF1), juvenile xanthogranulomas (JXG), and juvenile myelomonocytic leukemia (JMML) has been previously reported. We describe herein this triad in a Caucasian male infant with a pathogenic mutation in the NF1 gene (neurofibromin). The clinical course from initial presentation to final diagnosis is detailed; the physical features and hematologic characteristics are discussed. The patient underwent bone marrow transplantation and is currently in remission. Children with concurrent cutaneous café-au-lait and JXG lesions should be evaluated and monitored closely for the possible development of JMML.


Assuntos
Leucemia Mielomonocítica Juvenil/diagnóstico , Neurofibromatose 1/diagnóstico , Xantogranuloma Juvenil/diagnóstico , Transplante de Medula Óssea , Humanos , Lactente , Leucemia Mielomonocítica Juvenil/complicações , Leucemia Mielomonocítica Juvenil/terapia , Masculino , Mutação/genética , Neurofibromatose 1/complicações , Neurofibromatose 1/terapia , Neurofibromina 1/genética , Xantogranuloma Juvenil/complicações , Xantogranuloma Juvenil/terapia
19.
J Pediatr Hematol Oncol ; 30(8): 612-7, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18799940

RESUMO

Hypocalcemia is a rare complication of osteosarcoma, having been described in only 4 reports. We present the case of a 16-year-old male with metastatic osteosarcoma of the right humerus who was found to have severe asymptomatic hypocalcemia. Cytogenetic analysis of peripheral blood revealed a microdeletion in band 22q11.2. Following amputation of the tumor-bearing extremity, the patient's calcium levels increased, but did not normalize. These findings suggested that the etiology of his hypocalcemia was osteoblastic utilization of calcium by the tumor, exacerbated by 22q11.2 deletion syndrome.


Assuntos
Neoplasias Ósseas/complicações , Síndrome de DiGeorge/complicações , Hipocalcemia/etiologia , Osteossarcoma/complicações , Absorciometria de Fóton , Adolescente , Amputação Cirúrgica , Neoplasias Ósseas/patologia , Neoplasias Ósseas/fisiopatologia , Síndrome de DiGeorge/patologia , Síndrome de DiGeorge/fisiopatologia , Humanos , Úmero/patologia , Úmero/cirurgia , Hipocalcemia/patologia , Hipocalcemia/fisiopatologia , Hibridização In Situ , Masculino , Osteossarcoma/patologia , Osteossarcoma/fisiopatologia
20.
Int J Oncol ; 33(2): 239-44, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18636143

RESUMO

The Cancer and Leukemia Group B has performed central review of karyotypes submitted by institutional cytogenetics laboratories from patients with acute myeloid (AML) and acute lymphoblastic (ALL) leukemia since 1986. We assessed the role of central karyotype review in maintaining accurate, high quality cytogenetic data for clinical and translational studies using two criteria: the proportion of karyotypes rejected (i.e. inadequate), and, among accepted (i.e. adequate) cases, the proportion of karyotypes whose interpretation was changed on central karyotype review. We compared the first four years during which central karyotype review was performed with a recent 4-year period and found that the proportion of rejected samples decreased significantly for both AML and ALL. However, during the latter period, central karyotype reviews still found 8% of AML and 16% of ALL karyotypes inadequate. Among adequate cases, the karyotype was revised in 26% of both AML and ALL samples. Some revisions resulted in changing the patients' assignment to particular World Health Organization diagnostic categories and/or moving patients from one prognostic group to another. Overall, when both data on rejection rates and data on karyotype revisions made in accepted cases were considered together, 32% of AML and 38% of ALL samples submitted were either rejected or revised on central karyotype review during the recent 4-year period. These data underscore the necessity of continued central karyotype review in multi-institutional cooperative group studies.


Assuntos
Citogenética/normas , Cariotipagem , Leucemia Mieloide Aguda/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Adulto , Humanos , Leucemia Mieloide Aguda/diagnóstico , Leucemia-Linfoma Linfoblástico de Células Precursoras/diagnóstico
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