RESUMO
In a prospective study of 42 patients with myelofibrosis with myeloid metaplasia (MMM), peripheral blood (PB) and bone marrow (BM) interphase cytogenetics and PB CD34 enumeration were performed concomitantly with BM karyotype analysis. Interphase cytogenetics was performed with a panel of fluorescence in situ hybridization (FISH) probes that were capable of detecting most of the known recurrent cytogenetic lesions in MMM. There was a close concordance in the results of interphase cytogenetics between PB and BM, regardless of the PB CD34 count. In general, FISH-detectable abnormalities were also detected by BM karyotype. Although complementary, interphase cytogenetics may not always provide the necessary karyotypic information in MMM.
Assuntos
Células da Medula Óssea/patologia , Aberrações Cromossômicas , Mielofibrose Primária/genética , Adulto , Idoso , Antígenos CD34/sangue , Antígenos de Neoplasias/sangue , Feminino , Humanos , Hibridização in Situ Fluorescente , Interfase/genética , Cariotipagem , Masculino , Pessoa de Meia-Idade , Mielofibrose Primária/complicações , Mielofibrose Primária/patologia , Estudos ProspectivosRESUMO
Mantle-cell lymphoma (MCL) has a poorer prognosis than other small B-cell lymphomas, thus a definitive diagnosis is essential. The t(11;14)(q13;q32) associated with MCL juxtaposes portions of CCND1 (11q13) and IGH (14q32), resulting in over-expression of cyclin D1. In this study, a highly sensitive two-colour fluorescence in situ hybridization (FISH) method was developed to detect t(11;14)(q13;q32) in nuclei isolated from paraffin-embedded tissue. Twenty-three MCLs, 13 normal controls and nine small B-cell lymphomas other than MCL were studied by FISH. Each MCL had been previously investigated to detect genomic IGH-CCND1 fusion by polymerase chain reaction (PCR) using DNA extracted from frozen tissue. The IGH-CCND1 fusion detection rate in the MCLs was 96% by FISH compared with 35% by PCR. By FISH, one MCL and three small B-cell lymphomas other than MCL harboured abnormalities involving only IGH. Less than 1% of cells showed false-positive IGH-CCND1 fusion in normal specimens by FISH. Thus, this highly sensitive FISH assay is very useful in confirming the diagnosis of MCL, has wide applicability as it may be performed on both paraffin-embedded and fresh tissue, and may also facilitate detection of translocations involving these loci in tumours other than MCL.
Assuntos
Cromossomos Humanos Par 11 , Cromossomos Humanos Par 14 , Linfoma de Célula do Manto/diagnóstico , Translocação Genética , Estudos de Casos e Controles , Humanos , Imunofenotipagem , Hibridização in Situ Fluorescente , Interfase , Leucemia Linfocítica Crônica de Células B/diagnóstico , Linfonodos , Tonsila Palatina , Projetos Piloto , Reação em Cadeia da Polimerase , Sensibilidade e EspecificidadeRESUMO
The goal of this work was to optimize dendritic cell (DC) preparations obtained from patients suffering from chronic myeloid leukemia (CML) and compare them with DC prepared from normal CD14+ mononuclear cells (MNC). We studied normal DC and bcr-abl+ leukemic DC (CML-DC) yields, expression of membrane molecules, differentiation status, and ability to stimulate T cells. We isolated DC precursors from PBMC by CD14-specific immunoadsorption and cultured them for 7 days in GM-CSF and IL-4, followed by a 3-day incubation to fully differentiate the cells. We evaluated cultures of CML-DC using RPMI 1640 medium supplemented with FBS and X-VIVO 15 medium containing human AB serum. In contrast to cells matured in RPMI 1640, virtually all cells incubated in X-VIVO 15 expressed CD83, a marker of mature DC. CML-DC and normal DC were indistinguishable in expression of CD83, resulting in the highest percentage reported so far. The yields of normal DC and CML-DC from CD14+ cells were indistinguishable. The percentage of bcr-abl+ cells in PBMC varied among patients between 65% and 97% and the final CML-DC preparations were >98% bcr-abl+ the highest purity of bcr-abl+ cells to date. Normal DC and CML-DC were equally effective in stimulating proliferation of allogeneic and autologous T cells. These techniques provide highly enriched, mature, functional CML-DC.
Assuntos
Células Dendríticas/citologia , Proteínas de Fusão bcr-abl/sangue , Receptores de Lipopolissacarídeos/sangue , Divisão Celular , Técnicas de Cocultura , Meios de Cultura , Meios de Cultura Livres de Soro , Citocinas , Células Dendríticas/efeitos dos fármacos , Humanos , Separação Imunomagnética , Hibridização in Situ Fluorescente , Leucemia Mielogênica Crônica BCR-ABL Positiva/sangue , Leucócitos Mononucleares , Manejo de Espécimes , Linfócitos T/citologiaRESUMO
D-FISH uses DNA probes with fluorescence in situ hybridization to detect two fusion signals in cells with a t(9;22)(q34;q11.2) from patients with chronic myeloid leukemia (CML). Using D-FISH, 147 patients with CML were studied and considerable macro genetic variation was observed among their Ph-chromosomes. Typical D-FISH signal patterns were observed for 81% of patients, but three different atypical patterns were seen in 19% of patients. Atypical patterns among Ph-chromosomes were consistent with loss of the 3' portion of BCR that is usually translocated to chromosome 9, or loss of the 5' segment of ABL that usually remains on chromosome 9, or loss of both the 3' translocated BCR and 5' residual ABL hybridization sites. Atypical patterns were associated with all forms of Ph-chromosomes including t(9;22)(q34;q1.2), complex translocations and masked. The normal range for 500 interphase nuclei for patients with typical patterns is < 1%. By comparison, the normal range for patients with either of two atypical patterns was < or = 1.8% and for patients with the other atypical pattern was < or = 23%. Thus, special scoring criteria are needed to detect and quantify nuclei with atypical patterns using D-FISH. The proportion of patients that responded to therapy with interferon alpha-2b or interferon alpha-2b and ara-C for 36 patients with typical patterns was similar to 7 patients with atypical patterns.
Assuntos
Genes abl/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Proteínas Oncogênicas/genética , Proteínas Tirosina Quinases , Proteínas Proto-Oncogênicas , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Cromossomos Humanos Par 22/genética , Cromossomos Humanos Par 9/genética , Citarabina/uso terapêutico , Humanos , Hibridização in Situ Fluorescente , Interferon alfa-2 , Interferon-alfa/uso terapêutico , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/terapia , Cromossomo Filadélfia , Valor Preditivo dos Testes , Proteínas Proto-Oncogênicas c-bcr , Proteínas Recombinantes , Sensibilidade e Especificidade , Translocação GenéticaRESUMO
Buccal smear analysis is a noninvasive, fast, and relatively inexpensive diagnostic method. It is used commonly where rapid gender identification is necessary or, more recently, for detection of aneusomy, microdeletion syndromes, and a variety of polymerase chain reaction-based molecular genetic tests. Previously we have shown that maternal cells can contaminate buccal smears taken from breast-fed infants, resulting in difficulty with test interpretation. The aim of this study was to determine optimal timing and technique for buccal smear collection in breast-fed infants in order to avoid diagnostic errors. We analyzed prospectively 50 breast-fed male infants for presence of cells with XX signal pattern from buccal mucosa scrapings at different times after breast feeding. The efficiency of mucosal cleaning on elimination of maternal cells was evaluated by comparing the proportion of XX cells before and after wiping of buccal mucosa with a cotton swab. Maternal cells were present in 23 of 48 (47.9%) samples collected within 5 min of feeding. The proportion of XX signal pattern was significantly (P = 0.001) reduced in samples collected at 30 min (8/48, P = 0.001) and > or =60 min (2/29, P = 0.0002) after feeding. Mucosal cleaning prior to smear collection significantly decreased the number of XX positive samples from 23 of 48 to 10 of 48 (P = 0.002). Buccal smears should not be obtained in nursing neonates until at least 60 min after breast feeding. In addition, prior to sample collection, buccal mucosa should be cleaned thoroughly with a cotton swab applicator. The same guidelines are applicable to older nursing infants.
Assuntos
Aleitamento Materno , Mucosa Bucal/citologia , Manejo de Espécimes , Feminino , Guias como Assunto , Humanos , Hibridização in Situ Fluorescente , Recém-Nascido , Masculino , Processos de Determinação SexualRESUMO
Using a highly sensitive fluorescence in situ hybridization method with probes for BCR and ABL1 (D-FISH), we studied 37 paired sets of bone marrow and blood specimens, collected within 24 to 96 hours of each other, from 10 patients before and during treatment for chronic myeloid leukemia (CML). The normal range for 500 interphase nuclei was =4 (=0.8%) nuclei based on 10 bone marrow and 10 blood specimens from normal individuals. The percentage of neoplastic nuclei was usually lower in blood than bone marrow. However, changes in the percentage of neoplastic nuclei in blood and bone marrow tracked closely over the course of therapy and with the results of quantitative cytogenetic studies on bone marrow. This result indicates that D-FISH is useful to test blood from patients with CML to monitor therapy. Moreover, by analysis of 6,000 nuclei with D-FISH, residual disease was identified in bone marrow and blood for patients in complete cytogenetic remission. Consequently, D-FISH analyses of interphase nuclei from blood could substitute for Q-cytogenetic studies on bone marrow. Thus, it may not be necessary to collect bone marrow samples so frequently to monitor therapy in CML.
Assuntos
Biomarcadores Tumorais/genética , DNA de Neoplasias/análise , Proteínas de Fusão bcr-abl/genética , Fatores Imunológicos/uso terapêutico , Hibridização in Situ Fluorescente/métodos , Interferon-alfa/uso terapêutico , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Antimetabólitos Antineoplásicos/uso terapêutico , Biomarcadores Tumorais/análise , Células Sanguíneas/química , Células Sanguíneas/ultraestrutura , Medula Óssea/química , Medula Óssea/ultraestrutura , Núcleo Celular/química , Núcleo Celular/ultraestrutura , Terapia Combinada , Citarabina/uso terapêutico , DNA de Neoplasias/genética , Proteínas de Fusão bcr-abl/análise , Genes abl , Humanos , Interferon alfa-2 , Leucemia Mielogênica Crônica BCR-ABL Positiva/sangue , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/terapia , Especificidade de Órgãos , Proteínas Recombinantes , Indução de Remissão , Resultado do TratamentoRESUMO
We investigated a new method using fluorescence in situ hybridization and DNA probes that span the common breakpoints of t(9;22)(q34;q11.2) and that detect double BCR/ABL fusion (D-FISH) in bone marrow cells with this translocation, one on the abnormal chromosome 9 and one on the Philadelphia chromosome (Ph chromosome). D-FISH patterns were abnormal in 30 of 30 specimens with classic, simple, complex, and masked Ph chromosomes. Based on 200 nuclei from each of 30 normal specimens, the mean percentage of false-positive cells was 0.25 +/- 0.39. Thirty-seven specimens from 10 patients were studied before treatment and two or more times at 4-month intervals after treatment with interferon-alpha2b (IFN-alpha2b) with or without ara-C. Based on 200 nuclei, the results of D-FISH in these specimens correlated closely with quantitative cytogenetics and accurately quantified disease within a few percent. We studied 6, 000 nuclei for each of six specimens, three normal and three from patients with chronic myeloid leukemia (CML) in cytogenetic remission. The normal cutoff for 6,000 nuclei was 0.079% and patients in cytogenetic remission had residual disease ranging from 7 (0.117%) to 53 (0.883%) Ph-positive nuclei. We conclude that D-FISH can detect the Ph chromosome and its variant translocations and accurately quantify disease in CML at diagnosis and at all times after treatment, including cytogenetic remission.
Assuntos
Genes abl , Hibridização in Situ Fluorescente/métodos , Leucemia Mielogênica Crônica BCR-ABL Positiva/diagnóstico , Cromossomo Filadélfia , Cromossomos Humanos Par 22 , Cromossomos Humanos Par 9 , Humanos , Interfase , Leucemia Mielogênica Crônica BCR-ABL Positiva/terapia , Metáfase , Neoplasia Residual/diagnóstico , Translocação GenéticaRESUMO
Conventional X- and Y-chromatin and fluorescent in situ hybridization (FISH) analysis based on X- and Y-chromosome specific probes were conducted from buccal smear, on 15 normal males, 15 normal females, and 9 cases suspected of sex chromosome anomalies. The proportion of X- and Y-chromatin in normal females and males was 12% +/- 3% and 51.5% +/- 4.9%, respectively, by the conventional X- and Y-chromatin procedure. The CEP-X/Y analysis by FISH for the same specimens provided a proportion of 98.8% +/- 0.7% cells with XX signals in the normal females and 99.8% +/- 0.4% cells with XY signals in the normal males. The FISH method was superior to the conventional procedure in nine cases suspected of sex chromosome anomalies, including one case of mosaicism. The results of CEP-X/Y will sometimes be false; it will not detect structural anomalies of sex chromosomes, and it is not intended to detect low level mosaicism. However, the test is useful for rapid screening of sex chromosome aneuploidy at a fraction of the cost for chromosome analysis. The FISH test is also appropriate to detect tissue specific sex chromosome mosaicism, especially if it is relatively high. This FISH test is best used as an adjunct to chromosome analysis whenever possible.