RESUMO
KIT D816V is present in a majority of patients with systemic mastocytosis (SM). We determined the KIT D816V allele burden by quantitative real-time PCR in bone marrow and peripheral blood of 105 patients with mastocytosis. KIT D816V was detected in 92/105 patients (88%). Significant differences in the median allele burden were observed between disease subgroups: cutaneous mastocytosis (0.042%), indolent SM (0.285%), smoldering SM (5.991%), aggressive SM (9.346%), and SM with associated hematologic non-mast cell lineage disease (3.761%) (P < 0.001). The KIT D816V burden also correlated with serum tryptase (R = 0.5, P < 0.005) but not with mast cell infiltration in bone marrow or mediator symptoms. Moreover, the allele burden was of prognostic significance regarding survival (P < 0.01). Patients responding to cytoreductive therapy showed a significant decrease in KIT D816V (P < 0.05). To conclude, the KIT D816V burden correlates with the variant of mastocytosis, predicts survival, and is a valuable follow-up parameter in SM.
Assuntos
Alelos , Mastocitose/genética , Mastocitose/mortalidade , Mutação , Proteínas Proto-Oncogênicas c-kit/genética , Substituição de Aminoácidos , Humanos , Mastocitose/diagnóstico , Mastocitose/terapia , PrognósticoRESUMO
BACKGROUND: In chronic myeloid leukaemia (CML), clonal evolution with resistance to tyrosine kinase inhibitors (TKIs) is often triggered by BCR/ABL1 mutations. However, in the context of the complex pro-oncogenic signalling networks which ultimately lead to clonal expansion and disease progression, the exact contribution of BCR/ABL1 mutants remains uncertain. Recent data indicate that detection of BCR/ABL1 mutant subclones does not permit prediction of their expansion dynamics and their potential to become drivers of resistant disease. METHODS: To determine the patterns of clonal evolution and the distinct proliferation kinetics of individual BCR/ABL1 mutants during treatment, we employed ligase-dependent polymerase chain reaction (LD-PCR) analysis for quantitative surveillance of CML subclones with various tyrosine kinase domain (TKD) mutations including M244V, L248V, G250E, E255K, T315I, F317L-A/G, M351T and F359V. FINDINGS: Inadequate treatment responses were observed in 27 of 100 patients investigated and 16 were found to bear one or more BCR/ABL1 TKD mutations in separate subclones. Rapid subclone expansion upon onset or switch of TKI treatment was common and sometimes preceded corresponding changes in BCR/ABL1 transcript levels. Mutant subclones were found to respond differentially and sometimes unexpectedly to various treatment modalities. Decline and persistent depletion of specific mutation-bearing subclones in response to treatment could be documented by LD-PCR surveillance. INTERPRETATION: The observations show that quantitative monitoring of mutant BCR/ABL1 subclones by LD-PCR is a powerful tool for detection of clonal evolution, subclone-expansion and subclone-depletion and can contribute to optimised management of patients with CML.
Assuntos
Evolução Clonal/genética , Análise Mutacional de DNA/métodos , Proteínas de Fusão bcr-abl/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Mutação Puntual , Estudos de Coortes , Humanos , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Karyotype is an important prognostic factor in patients with newly diagnosed acute myeloblastic leukaemia (AML). The prognostic value of cytogenetics on the outcome of patients with AML in relapse has not yet been well defined. We analysed the clinical outcome of 152 patients with de novo, chemotherapy-treated AML in first relapse according to the cytogenetic classification of the United Kingdom Medical Research Council. The rate of second complete remission (CR) (88, 64 and 36%) and the probability of survival at 3 years (43, 18 and 0%) were significantly different between the favourable, intermediate and adverse cytogenetic risk groups, respectively. Compared to the favourable group, the relative risk (RR) of death (multivariate analyses) was 2.6 (confidence interval (CI): 1.5-4.4, P<0.001) for the intermediate and 3.7 (CI: 1.7-7.9, P=0.001) for the adverse group. The prognostic value of the duration of first CR was confirmed (RR of death: 2.0 (CI: 1.0-4.0) for each additional year in first CR), whereas the FLT3 mutation obtained at diagnosis did not markedly influence the outcome of patients with AML in relapse. In conclusion, our results indicate that both karyotype and the duration of first CR are independent prognostic factors for patients with de novo AML in first relapse.
Assuntos
Análise Citogenética , Leucemia Mieloide/diagnóstico , Leucemia Mieloide/genética , Doença Aguda , Classificação , Feminino , Seguimentos , Humanos , Cariotipagem , Leucemia Mieloide/mortalidade , Masculino , Pessoa de Meia-Idade , Prognóstico , Proteínas Proto-Oncogênicas/genética , Receptores Proteína Tirosina Quinases/genética , Recidiva , Indução de Remissão , Análise de Sobrevida , Resultado do Tratamento , Tirosina Quinase 3 Semelhante a fmsRESUMO
BACKGROUND: B-cell CLL (B-CLL) is accompanied by a progressive decrease in cellular immune functions, and treatment-related immunosuppression can further aggravate T-cell immunodeficiency. To reduce the risks of T-cell depletion, it seems feasible to collect autologous CD4+ cells at an early disease stage and subsequently reinfuse them during periods of profound T-cell depletion. METHOD: We describe a two-step cell-selection method to obtain highly enriched CD4+ T-cells from leukapheresis compounds of patients with CD23+ B-CLL. The double selection procedure was performed using the CellPro Ceperate device, and consisted of CD4+ selection followed by CD23 purging to further remove contaminating CD23+ B-cells from the CD4+ cell fraction. The results of eight runs performed with leukapheresis material obtained from eight patients with CD23+ B-CLL at different disease stages are presented. RESULTS: The CD4/CD23 double cell-selection procedure resulted in the purification of > 90% CD4+ cells. Median recovery of CD4+ T lymphocytes after selection was 46%, and was negatively affected by the initial tumor cell load. The final T-cell fraction still contained lymphocytes of the B-CLL clone, as determined by FACS and PCR. The cell-processing procedure had no impact on T-cell function, as assessed by the in vitro production of the cytokine interferon-gamma. Moreover, the selected CD4+ cells retained their capacity to co-stimulate mitogen-induced B-cell IgG production in vitro. CONCLUSION: The described CD4 selection/CD23 depletion strategy is a suitable approach to obtaining high numbers of functional active autologous CD4+ T cells for adoptive T-cell transfer in patients with CD23+ BCLL.
Assuntos
Linfócitos T CD4-Positivos/metabolismo , Separação Celular/métodos , Leucemia Linfocítica Crônica de Células B/terapia , Idoso , Linfócitos T CD4-Positivos/transplante , Humanos , Imunoglobulina G/metabolismo , Interferon gama/metabolismo , Leucemia Linfocítica Crônica de Células B/imunologia , Pessoa de Meia-Idade , Projetos Piloto , Receptores de IgE/imunologia , Receptores de IgE/metabolismo , Transplante AutólogoRESUMO
We studied the incidence of leukemia cutis (LC) in 381 consecutive patients with acute myeloid leukemia (AML) in a single institution and compared the demographic, hematological, and cytogenetic findings in AML patients with and without LC. We also examined the response to intensive chemotherapy, overall survival, and duration of remission in this patient population with regard to the presence of LC. The prevalence of LC was 3.7% in clinically diagnosed patients and 2.9% in biopsy-proven cases, respectively. Patients with and without LC did not differ with regard to age, sex, white blood cell counts, hemoglobin, fibrinogen, and platelet counts at diagnosis, but lactate dehydrogenase (LDH) was significantly higher in patients with LC. Various karyotype abnormalities were found, but in patients with LC numerical abnormalities of chromosome 8 were significantly more common ( P<0.0001). Patients with LC did not differ from patients without LC with regard to remission rate, but there was a trend towards shorter remission duration in patients with LC. We conclude that patients with LC have some features different from patients without this symptom. The increased frequency of numerical aberrations of chromosome 8 in patients with LC was the most interesting observation of our study. The pathophysiological significance of this finding remains to be determined.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Aberrações Cromossômicas , Cromossomos Humanos Par 8 , Leucemia Mieloide , Leucemia , Adulto , Fatores Etários , Idoso , Feminino , Humanos , Incidência , Leucemia/tratamento farmacológico , Leucemia/epidemiologia , Leucemia/genética , Leucemia Mieloide/tratamento farmacológico , Leucemia Mieloide/epidemiologia , Leucemia Mieloide/genética , Masculino , Pessoa de Meia-Idade , Indução de Remissão , Fatores Sexuais , Análise de SobrevidaRESUMO
T(14;18) chromosomal translocation is assumed to result from illegitimate rearrangement between the BCL2 proto-oncogene and the IGH locus during the D(H) to J(H) joining phase of V(D)J recombination in early B cells. Analysis of the breakpoint junctions suggests that translocation derives from the fusion between normal V(D)J recombination intermediates at the IGH locus and non-V(D)J-mediated broken-ends at the BCL2 locus. So far, BCL2 broken-ends have only been observed fused to coding-ends, raising questions concerning the molecular constraints of the illegitimate joining process. Using a combination of genome walking and long-range PCR assays, we describe in this report that in 4.5% (2/44) of the t(14;18), one of the BCL2 broken-ends is fused to a signal-end. The formation of these J(H)RSS/BCL2 junctions provides direct evidence that BCL2 broken-ends are capable of joining to both products of V(D)J recombination, suggesting their presence in the RAG-mediated post-cleavage complex. In addition, junctions generated by this alternative end-joining do not involve deletion of the chromosome 14 intervening sequences generally lost in the standard translocation, providing a unique opportunity to investigate the rearrangement status of this region in the translocated IGH allele. In both cases, a DJ(H) rearrangement could be detected 5' of the J(H)-RSS/BCL2 junction. These findings, together with the previously reported bias towards the most external D(H) and J(H) segments in standard breakpoints, strongly suggest that t(14;18) preferentially occurs during an attempted secondary D(H) to J(H) rearrangement. This unusual and restricted window of differentiation opens intriguing questions concerning the etiology of the translocation.
Assuntos
Quebra Cromossômica , Cromossomos Humanos Par 14/ultraestrutura , Cromossomos Humanos Par 18/ultraestrutura , Genes de Imunoglobulinas , Genes bcl-2 , Cadeias Pesadas de Imunoglobulinas/genética , Translocação Genética/genética , Sequência de Bases , Passeio de Cromossomo , Cromossomos Humanos Par 14/genética , Cromossomos Humanos Par 18/genética , DNA Nucleotidiltransferases/metabolismo , Rearranjo Gênico de Cadeia Pesada de Linfócito B , Humanos , Região de Junção de Imunoglobulinas/genética , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Proto-Oncogene Mas , VDJ RecombinasesRESUMO
Minimal residual disease in patients with acute myeloid leukemia (AML) with inversion(16) can be monitored by CBFbeta/MYH11 RT-PCR. While the association between molecular remission (MR) in bone marrow (BM) and peripheral blood (PB) and long-term clinical remission (CR) seems to be established, there are insufficient data on the kinetics of CBFbeta/MYH11. We have performed a prospective study in order to generate a reasonable and sufficient schedule for PCR-monitoring. 11 patients with AML and inversion (16) in complete hematological remission have been prospectively monitored by CBFbeta/MYH11 RT-PCR in their BM and PB during an observation period of 7 to 67 months (median 32 months). Patients were followed during consolidation chemotherapy with repetitive cycles of high-dose Ara-C and after autologous or allogeneic stem cell transplantation in 2nd CR or refractory AML. MR never coincided with achievement of CR but occurred between 2 and 8 months after hematological remission. All patients in continuous CR were PCR-negative after 1-8 (median 4) months. Two patients relapsed despite MR for 10 to 15 months. Molecular relapse preceded hematological relapse by 3 to 5 months. Three out of four patients who were not in MR after 8 months relapsed. Allogeneic stem cell transplantation was able to eradicate minimal residual disease in 4/4 patients. In 2 patients a temporary reconversion to PCR-positivity was reversed by reduction of immunosuppression. 1 patient did not become PCR-negative until compete withdrawal of immunosuppression. We suggest that BM and PB should be examined after the last consolidation treatment. In case of MR, PB should be examined every 1 to 2 months and BM examination should be done only in case of PCR-positivity in PB in order to confirm the molecular relapse and to identify an impending cytogenetic and/or hematological relapse. CBFbeta/MYH11 RT-PCR monitoring is able to predict relapse 3 to 5 months prior to overt hematological relapse, offers a window of opportunity for preemptive therapy of molecular relapse and confers implications for immunotherapy in the setting of allografting.
Assuntos
Leucemia Mieloide/diagnóstico , Proteínas de Fusão Oncogênica/genética , Doença Aguda , Adolescente , Adulto , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Sangue/metabolismo , Medula Óssea/metabolismo , Inversão Cromossômica , Cromossomos Humanos Par 16 , Feminino , Seguimentos , Transplante de Células-Tronco Hematopoéticas , Humanos , Leucemia Mieloide/genética , Leucemia Mieloide/terapia , Masculino , Pessoa de Meia-Idade , Neoplasia Residual/diagnóstico , Neoplasia Residual/genética , Guias de Prática Clínica como Assunto , Estudos Prospectivos , Recidiva , Indução de Remissão , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Mastocytosis is a term used for a group of disorders characterized by abnormal growth and accumulation of tissue mast cells (MC) in one or more organ systems. In patients with systemic mastocytosis (SM) the clinical course may be indolent or aggressive or even complicated by leukemic progression or an associated clonal hematologic non mast cell lineage disease (AHNMD). However, at first presentation (diagnosis) it may be difficult to define the category of disease and the prognosis. We report on a 48-year-old female patient with SM with urticaria pigmentosa-like skin lesions and mediator-related symptoms. She was found to have splenomegaly, a high infiltration grade (MC) in bone marrow biopsies (>30%), mild anemia, and a high serum tryptase level (>500 ng/ml). In addition, she exhibited discrete histologic signs of myeloproliferation in the 'non-affected' marrow and monoclonal blood cells established by C-KIT 2468A-->T mutation (Asp-816-Val) -analysis and HUMARA assay. Despite these findings, however, the clinical course was stable over years and no AHNMD or organ impairment developed. Because of the 'intermediate' clinical signs and absence of progression to aggressive disease, we proposed the term 'smouldering mastocytosis'.
Assuntos
Substituição de Aminoácidos , Mastocitose/genética , Mutação de Sentido Incorreto , Proteínas Proto-Oncogênicas c-kit/genética , Adulto , Anemia/etiologia , Medula Óssea/patologia , Contagem de Células , Células Clonais/química , Células Clonais/patologia , Códon/genética , Análise Mutacional de DNA , Progressão da Doença , Mecanismo Genético de Compensação de Dose , Feminino , Humanos , Hipotensão/etiologia , Mastócitos/patologia , Mastocitose/complicações , Mastocitose/tratamento farmacológico , Mastocitose/patologia , Células Mieloides/química , Células Mieloides/patologia , Receptores Androgênicos/análise , Serina Endopeptidases/sangue , Choque/etiologia , Síncope/etiologia , Triptases , Urticaria Pigmentosa/complicações , Urticaria Pigmentosa/tratamento farmacológico , Urticaria Pigmentosa/genética , Urticaria Pigmentosa/patologiaRESUMO
From 1987 to 1999 35 patients with poor prognosis non-Hodgkin's lymphoma (NHL) underwent allogeneic stem cell transplantation (SCT) at the University Hospitals of Vienna and Graz. Initial biopsy specimens were reclassified according to the Revised European-American Classification of Lymphoid Neoplasms (REAL). All patients surviving 28 days engrafted. Twenty-eight of them (93%) attained clinical remission. At the last follow-up 14 patients were alive and disease-free at a median of 5.0 (range, 2.3-12.9) years after allogeneic SCT. The actuarial overall survival is 35%. Five patients relapsed 1.8 to 27.6 months after transplant, the probability of relapse is 23%. Of the 21 deaths following SCT, seven were due to relapse/refractory disease and 14 due to transplant-related causes. The probability of treatment-related mortality is 48%. After SCT, minimal residual disease (MRD) was monitored by polymerase chain reaction (PCR) in seven patients with a BCL-2/IgH translocation and in 13 with a clonal immunoglobulin heavy chain (IgH) rearrangement. All 20 patients attained clinical remission rapidly and converted to PCR negativity. In the follow-up nine of these patients are in long-term clinical and molecular remission, six PCR-negative patients died of transplant-related causes and five patients relapsed. In summary, allogeneic stem cell transplantation has a curative potential for patients with refractory and recurrent non-Hodgkin's lymphoma. In our series long-term disease-free survival was associated with molecular disease eradication after SCT. Treatment-related mortality rate was high, thus earlier referral of selected patients to allogeneic SCT should be considered.
Assuntos
Transplante de Células-Tronco Hematopoéticas , Linfoma não Hodgkin/terapia , Adulto , Feminino , Rearranjo Gênico , Genes de Imunoglobulinas , Genes bcl-2 , Doença Enxerto-Hospedeiro/etiologia , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Linfoma não Hodgkin/genética , Linfoma não Hodgkin/mortalidade , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Prognóstico , Transplante HomólogoRESUMO
The t(11;14)(q13;q32) between the BCL-1 and immunoglobulin heavy chain gene (IgH) loci in mantle cell lymphoma (MCL) are believed to be mediated by the mechanism of V(D)J recombination similar to the t(14; 18) in follicular lymphoma (FL). We have recently shown that the t(14;18) event creates staggered double-strand breaks in the BCL-2 locus, and that the t(14;18) junctions contain templated nucleotide insertions (T-nucleotides; U. Jäger et al., Blood, 95: 3520-3529, 2000). Reasoning that the earlier (pregerminal center) B-cell origin of MCL might be reflected in a different molecular structure of the chromosomal breakpoints, we PCR-amplified diagnostic samples from 93 patients. Thirty-six samples (39%) were positive for the direct (BCL-1/J(H)) and 23 for both direct and reciprocal (D(H)/BCL-1) junctions. The breaks on chromosome 14 exhibited features of V(D)J-mediated recombination as shown by D(H) and J(H) coding end processing. However, duplications of BCL-1 sequences in 39% of the 23 patients indicate staggered double-strand breaks in the major translocation cluster region (MTC). This is incompatible with V(D)J recombination and indicates a different mechanism of cleavage. The use of J(H)6 in the junctions (39%) was similar to that in the immunoglobulin genes of normal B cells and B-CLL, but considerably less than in FL. Only 2 of 36 samples contained a BCL-1/DJ(H) rearrangement, which was indicative of a previous DJ(H) rearrangement. Most importantly, 19% of the BCL-1/IgH junctions with inserts of > or =5 nucleotides contained error-prone copies (T-nucleotides) of 8-12 nucleotides originating from the surrounding BCL-1 or IgH regions, a lower rate than in FL. No correlation was found between the addition of T-nucleotides and the rate of somatic mutation in the immunoglobulin genes. We conclude that the t(11;14) and t(14;18) use the same basic mechanism of translocation including V(D)J-mediated recombination, double-strand staggered breaks, and template-dependent, error-prone DNA-synthesis. However, the distinct differences in the utilization of J(H) regions suggest that the t(11;14) occurs predominantly during an attempted primary D(H)-J(H) rearrangement in early B cells, whereas the t(14;18) mostly occurs during secondary rearrangement. This is in agreement with the pregerminal center B-cell origin of MCL.
Assuntos
Genes de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias J de Imunoglobulina/genética , Linfoma de Célula do Manto/genética , Translocação Genética/genética , Sequência de Bases , Quebra Cromossômica/genética , Cromossomos Humanos Par 11 , Cromossomos Humanos Par 14 , Elementos de DNA Transponíveis/genética , Rearranjo Gênico de Cadeia Pesada de Linfócito B/genética , Genes bcl-1/genética , Humanos , Idiótipos de Imunoglobulinas/genética , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Moldes GenéticosRESUMO
BACKGROUND: Autografting of normal stem cells mobilized after chemotherapy is increasingly used in chronic myeloid leukemia (CML). Thus, quantification of possible contamination of progenitor cell apheresis with breakpoint cluster region (bcr)/Abelson murine leukemia (abl)-positive cells is of great clinical interest. STUDY DESIGN AND METHODS: Two molecular methods were compared to quantify bcr/abl positivity in leukapheresis components obtained after mobilizing chemotherapy in six patients with CML. To document the efficacy of in vivo purging, the leukapheresis procedures were monitored with interphase fluorescence in situ hybridization (FISH) and quantitative competitive PCR (QC-PCR) as a ratio of bcr/abl:abl. RESULTS: From the first to the last leukapheresis, bcr/abl positivity in FISH increased from a median of 11 percent to 33 percent. For bcr/abl transcripts, a simultaneous increase in consecutive leukapheresis procedures was seen. The median percentage of bcr cells in a bcr/abl:abl ratio was 3.1 percent in the first apheresis. In the last apheresis after the mobilization with mRNA, the QC-PCR showed a median of 19.5 percent. FISH and QC-PCR showed a statistical significant increase of bcr/abl positivity from the first to the last apheresis. CONCLUSIONS: Both FISH and QC-PCR were reliable methods of quantifying bcr/abl positivity, and they allowed selection of the optimal apheresis component for autologous transplantation. In both methods, a significant increase in bcr/abl positivity was seen from the first to the last leukapheresis. With FISH, results can be obtained within 24 hours. This method may prevent additional contaminated leukapheresis in case of increasing percentages of bcr/abl-positive cells.
Assuntos
Células-Tronco Hematopoéticas/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Proteínas Oncogênicas/metabolismo , Proteínas Tirosina Quinases , Proteínas Proto-Oncogênicas c-abl/metabolismo , Proteínas Proto-Oncogênicas , Adulto , Núcleo Celular/metabolismo , Doença Crônica , Citarabina/farmacologia , Feminino , Fator Estimulador de Colônias de Granulócitos/farmacologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Humanos , Idarubicina/farmacologia , Hibridização in Situ Fluorescente , Interfase , Leucaférese , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase/métodos , Proteínas Proto-Oncogênicas c-bcrRESUMO
A unique variant Philadelphia translocation accompanied by the loss of the short arm of chromosome 9 in a 32-year-old female with common acute lymphoblastic leukemia (cALL) is described. Furthermore, supernumerary chromosome 8 material was found as an insertion into the long arm of chromosome 2 and/or as ring chromosomes in addition to two normal chromosomes 8. The chromosomal abnormalities were identified by combined conventional chromosome banding analysis and fluorescence in situ hybridization (FISH). The BCR-ABL rearrangement was confirmed by FISH and reverse transcriptase-polymerase chain reaction (RT-PCR) studies. Possible mechanisms leading to this variant intra Philadelphia translocation are discussed. The aberrations found have prognostic implications, because 9p anomalies confer an adverse effect to the already poor prognosis of Philadelphia-positive ALL.
Assuntos
Proteínas de Fusão bcr-abl/genética , Proteínas de Fusão Oncogênica/genética , Cromossomo Filadélfia , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Adulto , Bandeamento Cromossômico , Cromossomos Humanos Par 2/genética , Cromossomos Humanos Par 22/genética , Cromossomos Humanos Par 8/genética , Cromossomos Humanos Par 9/genética , Feminino , Humanos , Hibridização in Situ Fluorescente , Cariotipagem , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Translocação GenéticaRESUMO
Structural rearrangements of the long arm of chromosome 3 involving bands 3q21 and 3q26 and leading either to a paracentric inversion inv (3)(q21q26) or a translocation between both homologous chromosomes--t(3;3)(q21q26)-- have been reported in patients with acute myelogenous leukemia (AML), myelodysplastic syndromes, myeloproliferative disorders, and chronic myelogenous leukemia in blast crisis. We describe three patients with de novo AML with these structural abnormalities who received multiple courses of conventional chemotherapy followed by unrelated donor (n=2) and autologous (n = 1) bone marrow transplantation (BMT). All three patients had early relapse: patients 1 and 2 had relapse 69 days and 306 days after BMT, respectively, and patient 3 immediately after autologous BMT. Despite further chemotherapy, they died without achieving another remission. These findings, together with other recorded similar cases, show that AML with structural abnormalities of the long arm of chromosome 3 as described has an extremely poor prognosis even with the most potent anti-leukemic treatment modalities.
Assuntos
Inversão Cromossômica , Cromossomos Humanos Par 3 , Leucemia Mieloide Aguda/genética , Translocação Genética , Adulto , Transplante de Medula Óssea , Cromossomos Humanos Par 7 , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Monossomia/genéticaRESUMO
We studied 210 unselected patients with acute myeloid leukaemia (AML) for MLL abnormalities. Twenty-seven patients (13%) with rearranged MLL genes were identified by means of Southern blot analysis. An MLL-AF6 fusion transcript was detected in six patients by a reverse transcriptase polymerase chain reaction (RT-PCR) for the MLL-AF6 translocation. Sequence analysis showed fusion of MLL exon 7 as well as exon 6 (two patients) or MLL exon 6 as well as exon 5 (four patients) to AF6 exon 2. In only three patients could the t(6;11) also be identified by cytogenetic and/or fluorescence in situ hybridization (FISH) analysis. The MLL-AF6-positive patients were monitored by RT-PCR for a period of 6-33 months. Complete haematological remission (CR) was achieved in all six cases, but was short in 5/6 patients (range 2.6-8.3 months). In these five patients, the MLL-AF6 transcripts were detected in every sample tested after induction and consolidation chemotherapy. One patient received autologous bone marrow transplantation (BMT) which also did not lead to PCR negativity. Intensive salvage therapy was unable to induce a second remission in the relapsed patients. One of the six MLL-AF6-positive patients achieved a molecular CR. He is still in CR at 33 months after diagnosis. Survival analysis indicates a poor prognosis in MLL-AF6-positive patients. The median event-free survival was 6.8 months, the median overall survival 15 months. Persistent PCR positivity was consistently associated with relapse. Thus, RT-PCR provides a valuable and sensitive tool for the identification of t(6;11)-positive AML and the monitoring of response to treatment in these patients. The results of RT-PCR may be useful to evaluate therapeutic procedures and to make treatment decisions, which will enable molecular remissions to be achieved and improve the clinical outcome in this group of patients.
Assuntos
Cromossomos Humanos Par 11 , Cromossomos Humanos Par 6 , Leucemia Mieloide/genética , Neoplasia Residual/genética , Translocação Genética , Doença Aguda , Adulto , Idoso , Fusão Gênica Artificial , Feminino , Rearranjo Gênico , Humanos , Hibridização in Situ Fluorescente , Cariotipagem , Masculino , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Reverse transcription polymerase chain reaction (RT-PCR) analysis is increasingly becoming part of the diagnostic and prognostic evaluation for hematologic and oncologic disorders. Currently, different RNA isolation methods are used in the diagnostic laboratories. No data are available on their suitability for sensitive detection of breakpoint cluster region-abelson (BCR-ABL) gene transcripts. We have extracted RNA from mononuclear cell (MNC) fractions and from lysed blood samples of 4 patients (1 with leukocytosis, 1 with chronic myelogeneous leukemia (CML) under interferon treatment, and 2 CML patients after bone marrow transplantation) with 3 RNA isolation reagents (TRIzol, RNAzol, FastTube reagent). RNA yield was slightly higher with RNAzol than with TRIzol as indicated by agarose gel electrophoresis and spectrophotometric measurement at 260 nm. The FastTube reagent was unsuitable for RNA isolation from MNC, and was not evaluated for lysed blood. Quantitative competitive RT-PCR amplification of the ABL gene showed comparable results for RNA isolated with RNAzol and TRIzol. In RNA samples extracted from lysed whole blood, the presence of amplifiable RNA/cDNA was confirmed by amplification of 4 selected reference genes (porphobilinogen deaminase (PBGD), ABL, the gene spanning the BCR on chromosome 22 and retinoic acid receptor alpha (RARA)) in a multiplex PCR. High quality, DNA-free RNA was obtained with RNAzol, and 1 BCR-ABL-positive (specific for translocation t [9; 221) cell among 2x10(4) normal cells was successfully detectable by single step RT-PCR. In RNA isolated with TRIzol, major contaminations with genomic DNA were observed which significantly impaired the interpretation of the results of RT-PCR analysis.
Assuntos
RNA/genética , RNA/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Sequência de Bases , Primers do DNA/genética , Estudos de Avaliação como Assunto , Genes abl , Humanos , Indicadores e Reagentes , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucocitose/genética , Receptores do Ácido Retinoico/genética , Receptor alfa de Ácido RetinoicoRESUMO
The human t(14;18) chromosomal translocation is assumed to result from illegitimate rearrangement between BCL-2 and D(H)/J(H) gene segments during V(D)J recombination in early B cells. De novo nucleotides are found inserted in most breakpoints and have been thus far interpreted as nontemplated N region additions. In this report, we have analyzed both direct (BCL-2/J(H)) and reciprocal (D(H)/BCL-2) breakpoints derived from 40 patients with follicular lymphoma with t(14;18). Surprisingly, we found that more than 30% of the breakpoint junctions contain a novel type of templated nucleotide insertions, consisting of short copies of the surrounding BCL-2, D(H), and J(H) sequences. The features of these templated nucleotides, including multiplicity of copies for 1 template and the occurrence of mismatches in the copies, suggest the presence of a short-patch DNA synthesis, templated and error-prone. In addition, our analysis clearly shows that t(14;18) occurs during a very restricted window of B-cell differentiation and involves 2 distinct mechanisms: V(D)J recombination, mediating the breaks on chromosome 14 during an attempted secondary D(H) to J(H) rearrangement, and an additional unidentified mechanism creating the initial breaks on chromosome 18. Altogether, these data suggest that the t(14;18) translocation is a more complex process than previously thought, involving the interaction and/or subversion of V(D)J recombination with multiple enzymatic machineries.
Assuntos
Cromossomos Humanos Par 14 , Cromossomos Humanos Par 18 , Genes de Imunoglobulinas , Genes bcl-2/genética , Cadeias Pesadas de Imunoglobulinas/genética , Linfoma Folicular/genética , Translocação Genética , Sequência de Bases , Medula Óssea/patologia , Mapeamento Cromossômico , Primers do DNA , Elementos de DNA Transponíveis , Mutação em Linhagem Germinativa , Humanos , Linfonodos/patologia , Linfoma Folicular/patologia , Dados de Sequência Molecular , Recombinação Genética , Alinhamento de Sequência , Moldes GenéticosRESUMO
The immunophenotypes of 12 acute promyelocytic leukemias (APL-M3; eight hypergranular, four microgranular) with documented PML-RAR-alpha fusion gene are presented. Bone marrow mononuclear cells were immunophenotyped using a panel of 20 monoclonal antibodies. The hypergranular APLs exhibited a mature myeloid phenotype as it has been described to be typical for M3. No lineage infidelity was detectable in classic M3 cases. In contrast, among the four cases of M3 variant, all leukemias showed marked expression of CD34 and two of four cases expressed the HLA-DR antigen. The CD2 antigen was expressed in three of four cases. Furthermore, one case showed expression of the CD56 antigen, and one case was positive for the blood group H antigen. The data suggest that microgranular APL is a heterogeneous entity with regard to the immunologic phenotype.
Assuntos
Imunofenotipagem/normas , Leucemia Promielocítica Aguda/genética , Leucemia Promielocítica Aguda/imunologia , Translocação Genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Monoclonais , Células da Medula Óssea/imunologia , Humanos , Leucemia Promielocítica Aguda/diagnóstico , Pessoa de Meia-Idade , Receptores do Ácido Retinoico/genética , Proteínas Recombinantes de Fusão/genética , Indução de RemissãoRESUMO
The benefit of all-trans-retinoic acid (ATRA) in the front line therapy of acute promyelocytic leukemia (APL) is well established, but its role in postremission therapy and in the treatment of relapse is currently under investigation. Moreover, the impact of cytosine arabinoside (Ara-C) in the therapy of APL has been questioned in recent studies. We report a prolonged third molecular remission (MR) in a patient with hyperleukocytotic APL after induction with ATRA, consolidation chemotherapy (CT) with intermittent intermediate dose Ara-C and maintenance therapy with intermittent ATRA. While the first two remissions were relatively short (8 months and 11 months, resp.), the duration of the third continuous CR (49+ months) is more than twice as long as the length of the two previous remissions combined. In this case Ara-C followed by intermittent ATRA maintenance was a safe and effective therapy for relapsed disease. A third molecular remission of such duration and quality is unusual.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Leucemia Promielocítica Aguda/tratamento farmacológico , Leucemia Promielocítica Aguda/patologia , Citarabina/administração & dosagem , Feminino , Humanos , Leucemia Promielocítica Aguda/fisiopatologia , Pessoa de Meia-Idade , Indução de Remissão , Fatores de Tempo , Tretinoína/administração & dosagemRESUMO
Identification of the inversion 16 in patients with acute myeloid leukemia (AML) is of great practical value since these patients have a relatively favorable prognosis, especially when treated with high-dose cytarabine. We compared the results of cytogenetic analysis and reverse transcriptase-polymerase chain reaction (RT-PCR) for core binding factor (CBF) beta/myosin heavy chain (MYH11) in 241 unselected cases of AML. In contrast with other studies, we found a high concordance between these 2 methods. Eighteen of 241 patients showed a cytogenetic anomaly of the chromosome 16. We detected the fusion transcript by RT-PCR in all 18 cases and in 2 additional patients with AML without any cytogenetic anomaly of chromosome 16. One patient had a normal diploid karyotype, and the second patient showed a trisomy 22 in karyotype analysis, which often is associated with inv(16). Only 8 of 20 CBF beta/MYH11-positive patients had M4Eo morphologic features. The much higher discrepancy between cytogenetic analysis and RT-PCR in other studies, especially in AMLs other than M4Eo, possibly indicates the necessity for PCR screening regardless of the French-American-British classification.
Assuntos
Cariotipagem , Leucemia Mieloide/genética , Proteínas de Fusão Oncogênica/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Doença Aguda , Adolescente , Adulto , Idoso , Cromossomos Humanos Par 16/genética , Cromossomos Humanos Par 22/genética , Primers do DNA/química , Humanos , Leucemia Mieloide/patologia , Pessoa de Meia-Idade , RNA Neoplásico/análise , Sensibilidade e Especificidade , TrissomiaRESUMO
Recurrence of the disease is the major problem in the treatment of acute myeloid leukemia (AML). The majority of patients who achieve a second remission will ultimately relapse. In this retrospective single-center study, we have analyzed the outcome of patients with a second relapse and tried to define the prognostic factors in intensively treated patients. Of 534 patients with AML, 62 had a second relapse. Thirty-three received further intensive chemotherapy (CT). Eighteen patients (55%) achieved a third complete remission (CR). The early death (ED) rate was only 9%. The overall survival (OS) of treated vs untreated patients was 6.9 vs 1.3 months, respectively (P = 0.01). The major selection criteria for a third CT were a favourable (t(15;17),t(8;21),inv(16)) or normal karyotype, long (>11 months) second CR (P < or = 0.005) and no previous bone marrow transplantation (BMT)(P < 0.01). Favorable or normal karyotype, second CR >11 months, as well as no previous BMT (P < 0.01) were associated with the achievement of a third CR. Favorable (P < 0.005) or normal karyotype (P < 0.01), as well as a second CR >11 months (P < 0.005) were associated with prolonged survival after CT. The median OS for patients receiving CT with favorable or normal cytogenetics, a second CR > 11 months, but no previous BMT was 26.5 months. Five patients with favorable or normal karyotype achieved a fourth or fifth remission. We conclude that intensive CT is associated with a survival benefit and good quality of life if patients are properly selected.