RESUMEN
The partial tandem duplication (PTD) of ALL1 (MLL) is one of the more common molecular abnormalities in adult de novo acute myeloid leukemia (AML) and carries a poor prognosis. The PTD of ALL1 is identified in leukemic blasts at the genomic level by ALL1 rearrangement upon Southern analysis and by genomic fusion following DNA PCR. The genomic defect encodes for a unique fusion transcript that is readily detected by reverse transcription-PCR (RT-PCR). To determine if the ALL1 fusion transcript is specific for leukemic blasts or instead can be found with any frequency in normal cells, we analyzed 52 bone marrow and 8 peripheral blood samples from 60 normal donors by nested RT-PCR. Ten of 60 samples (16%; 7 bone marrow and 3 peripheral blood) contained a unique transcript showing a fusion of two ALL1 exons that was consistent with the PTD of ALL1. However, a corresponding genomic rearrangement or a unique genomic fusion of ALL1 could not be demonstrated by Southern analysis or DNA PCR, respectively. Marked differences were observed in the size and sequence of the ALL1 fusion transcripts detected in normal donors, compared to those detected in leukemic patients with a PTD of ALL1. Moreover, although the ALL1 fusion transcripts seen in AML always maintain the open reading frame, the open reading frame was preserved in only 5 of 10 fusion transcripts from normal donors. Finally, in contrast to leukemic blasts with the PTD of ALL1, the fusion transcripts in normal cells could not be detected in the poly(A)+ RNA fraction by RT-PCR. In summary, the origin and the composition of the ALL1 fusion transcripts found in normal cells appear to be distinct from those found in the leukemic cells. The data accumulated thus far suggest that the ALL1 fusion product detected in normal tissue results from the process of differential mRNA splicing rather than true ALL1 gene rearrangement. These findings also suggest caution in the use of RT-PCR for detection of minimal residual disease in AML patients with the PTD of ALL1.
Asunto(s)
Médula Ósea/química , Proteínas de Unión al ADN/sangre , Leucemia Mieloide/metabolismo , Familia de Multigenes , Proto-Oncogenes , Factores de Transcripción , Enfermedad Aguda , Antígenos CD34 , Secuencia de Bases , Proteínas de Unión al ADN/análisis , Células Madre Hematopoyéticas/química , N-Metiltransferasa de Histona-Lisina , Humanos , Leucemia Mieloide/genética , Proteína de la Leucemia Mieloide-Linfoide , ARN/análisis , Mapeo RestrictivoRESUMEN
Rearrangements of the ALL-1 gene by reciprocal translocations involving chromosome band 11q23 are frequently associated with human acute leukemia. We have previously reported the detection of ALL-1 gene rearrangements in adult patients with acute myeloid leukemia lacking cytogenetic evidence of 11q23 translocations. These included 2 of 19 patients with normal karyotypes as well as 3 of 4 patients with trisomy 11 as a sole cytogenetic abnormality. Rearrangement of the ALL-1 genes in two of the patients with trisomy 11 was shown to result from a direct tandem duplication of a portion of the gene spanning exons 2-6. Here we report the characterization of the ALL-1 gene rearrangement in one of the previously reported acute myeloid leukemia patients with a normal karyotype. ALL-1 rearrangement in this patient results from a direct tandem duplication of a portion of the gene spanning exons 2-8. RNA polymerase chain reaction and DNA sequence analysis show that the partially duplicated ALL-1 gene is transcribed into mRNA capable of encoding a partially duplicated protein. Sequence analysis of the genomic fusion region provides evidence for Alu-mediated homologous recombination as a mechanism for partial duplication of the ALL-1 gene.
Asunto(s)
Cromosomas Humanos Par 11 , Exones/genética , Reordenamiento Génico/genética , Leucemia Mieloide/genética , Proto-Oncogenes/genética , Secuencias Repetitivas de Ácidos Nucleicos/genética , Enfermedad Aguda , Secuencia de Bases , Southern Blotting , Humanos , Datos de Secuencia Molecular , Reacción en Cadena de la PolimerasaRESUMEN
A partial nontandem duplication (PNTD) of mixed lineage leukemia (MLL) gene is described in B-cell acute lymphoid leukemia without structural cytogenetic abnormalities at 11q23 and 9p22. A duplicated portion of MLL is interrupted by the insertion of a region of 9p22 that includes the 3'-end of the AF9 gene. The PNTD encodes: (a) a PNTD transcript; (b) a partial tandem duplication of MLL; and (c) a chimeric transcript fusing MLL to the 3'-end of AF9, mimicking the t(9;11)(p22;q23) and expressed 1024-fold higher than the other two. The MLL PNTD, therefore, contributes toward leukemogenesis through simultaneous production of fusion transcripts that are otherwise encoded by three distinct genetic defects.
Asunto(s)
Linfoma de Burkitt/genética , Proteínas de Unión al ADN/genética , Reordenamiento Génico , Proto-Oncogenes , ARN Mensajero/genética , Factores de Transcripción , Empalme Alternativo/genética , Southern Blotting , Rotura Cromosómica , Cromosomas Humanos Par 11/genética , Cromosomas Humanos Par 9/genética , Exones , N-Metiltransferasa de Histona-Lisina , Humanos , Proteína de la Leucemia Mieloide-Linfoide , Proteínas Nucleares/genética , Proteínas Recombinantes de Fusión/genética , Secuencias Repetitivas de Ácidos Nucleicos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Translocación Genética , Células Tumorales CultivadasRESUMEN
Translocations which involve chromosome band 11q23 are frequently found in infants and adults with acute myeloid leukemia (AML) or acute lymphoblastic leukemia. We previously cloned a gene called ALL-1 which spans the 11q23 breakpoint and is rearranged in most cases of leukemia with 11q23 abnormalities. In the present report, we have investigated the occurrence of ALL-1 rearrangement in cases of AML without cytogenetic evidence of 11q23 abnormalities. We detected molecular rearrangements of the ALL-1 gene in 3 of 4 patients with de novo AML and trisomy 11 as a sole chromosomal abnormality. Furthermore, we found DNA rearrangements of ALL-1 in 2 of 19 patients with de novo AML and normal cytogenetics. We conclude that molecular rearrangement of ALL-1 often can be detected in de novo AML, despite the absence of cytogenetic abnormalities involving 11q23.
Asunto(s)
Cromosomas Humanos Par 11 , Reordenamiento Génico/genética , Leucemia Mieloide/genética , Translocación Genética/genética , Enfermedad Aguda , Adulto , Anciano , Femenino , Humanos , Cariotipificación , Masculino , Persona de Mediana EdadRESUMEN
Gains of a single chromosome are frequent cytogenic findings in human cancer, but no molecular rearrangement has been consistently associated with any trisomy. In acute myeloid leukemia (AML), trisomy 11 (+11) occurring as a sole abnormality is the third most common trisomy. We have shown that the ALL1 gene, located at 11q23, can be rearranged as a result of a partial tandem duplication in two such cases of AML. To test the hypothesis that the partial tandem duplication of ALL1 is the recurrent molecular defect in cases of AML presenting with +11 as a sole cytogenic abnormality, we performed Southern analysis and PCR for defects of ALL1 in 17 cases of AML and one case of myelodysplastic syndrome with +11 or +11q but without cytogenic evidence of a structural abnormality involving 11q23. Twelve cases (67%) had rearrangement of ALL1, including 10 of 11 patients (91%) with +11 as a sole abnormality and 2 of 7 cases (29%) with +11 and other aberrations; all were classified as FAB M1 or M2. In 10 of the 12 cases, material was available for additional characterization; a partial tandem duplication of ALL1 was detected in each of these 10 cases (100%). Four cases demonstrated previously unreported duplications, two of which were detectable only by reverse transcription-PCR. Four patients with the ALL1 duplication also displayed a loss of material from 7q, suggesting an association between these two findings. We conclude that the partial tandem duplication of ALL1 is present in most, if not all, cases of AML with +11 as a sole abnormality, and can be found in cases of AML with +11 or +11q accompanied by other cytogenic abnormalities. The duplication is more prevalent in AML than was recognized previously in part because its size and location vary considerably, requiring a variety of molecular probes for detection. Our finding of the ALL1 duplication as a consistent defect in patients with +11 represents the first identification of a specific gene rearrangement associated with recurrent trisomy in human cancer.
Asunto(s)
Cromosomas Humanos Par 11/genética , Exones/genética , Reordenamiento Génico/genética , Leucemia Mieloide/genética , Síndromes Mielodisplásicos/genética , Secuencias Repetitivas de Ácidos Nucleicos/genética , Trisomía , Enfermedad Aguda , Adulto , Anciano , Aberraciones Cromosómicas/genética , Trastornos de los Cromosomas , Femenino , Humanos , Cariotipificación , Leucemia Mieloide/complicaciones , Masculino , Persona de Mediana Edad , Reacción en Cadena de la PolimerasaRESUMEN
Approximately 45% of adults with acute myeloid leukemia (AML) have normal cytogenetics and therefore lack structural abnormalities that can assist in the localization and characterization of molecular defects. The partial tandem duplication of the ALL1 (MLL) gene has been found in several such cases of AML, yet its frequency and clinical significance are unclear. We performed Southern analysis of the ALL1 gene in pretreatment samples from 98 AML patients with normal cytogenetics. Eleven of 98 such patients (11%; 95% confidence interval, 6-19%) showed rearrangement of ALL1 at diagnosis. The partial tandem duplication of ALL1 was responsible for ALL1 rearrangement in all such cases examined, making it a frequent molecular defect in adult AML patients with normal cytogenetics. Furthermore, patients with ALL1 rearrangement had a significantly shorter duration of complete remission when compared to patients without ALL1 rearrangement (P = 0.01; median, 7.1 versus 23.2 months). This defect defines for the first time a subset of AML patients with normal cytogenetics who have short durations of complete remission and thus require new therapeutic approaches.
Asunto(s)
Reordenamiento Génico , Genes Supresores de Tumor/genética , Leucemia Mieloide/genética , Enfermedad Aguda , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Southern Blotting , Femenino , Humanos , Masculino , Persona de Mediana EdadRESUMEN
There is substantial evidence to suggest that aberrant DNA methylation in the regulatory regions of expressed genes may play a role in hematologic malignancy. In the current report, the Restriction Landmark Genomic Scanning (RLGS) method was used to detect aberrant DNA methylation (M) in acute myeloid leukemia (AML). RLGS-M profiles were initially performed using DNA from diagnostic, remission, and relapse samples from a patient with AML. Rp18, one of the eight spots found that was absent in the relapse sample, was cloned. Sequence analysis showed that the spot represented a portion of the WIT-1 gene on human chromosome 11p13. Rp18 was missing in the relapse sample due to a distinct DNA methylation pattern of the WIT-1 gene. Twenty-seven AML patients that entered CR after therapy (i.e., chemosensitive) were studied and only 10 (37%) of the diagnostic bone marrow (BM) samples showed methylation of WIT-1. However, seven of eight (87.5%) diagnostic BM samples from primary refractory AML (chemosensitive) showed methylation of WIT-1. The incidence of WIT-1 methylation in primary refractory AML was significantly higher than that noted in chemosensitive AML (P=0.018). Together, these results indicate that RLGS-M can be used to find novel epigenetic alterations in human cancer that are undetectable by standard methods. In addition, these results underline the potential importance of WIT-1 methylation in chemoresistant AML.
Asunto(s)
Metilación de ADN , Leucemia Mieloide/genética , Enfermedad Aguda , Southern Blotting , Células de la Médula Ósea/patología , ADN de Neoplasias/genética , ADN de Neoplasias/metabolismo , Humanos , Leucemia Mieloide/patología , RecurrenciaRESUMEN
The AML1/ETO fusion transcript can be detected by reverse transcription polymerase chain reaction (RT-PCR) in patients with t(8;21)-associated acute myeloid leukemia (AML) in long-term complete remission (CR). Quantitation of the amount of the fusion transcript during CR may therefore be more predictive of cure or relapse than a simple qualitative assessment. Real Time PCR, a fluorometric-based technique, allows simple and rapid quantitation of a target sequence during the extension phase of PCR amplification, in contrast to end-point quantitative methods. Six patients with t(8;21)(q22;q22) AML, who achieved CR were studied by Real Time RT-PCR at different time intervals following diagnosis and high-dose cytarabine and anthracycline-based induction therapy. Five patients had a diagnostic bone marrow (BM) sample available for molecular analysis. Each patient showed > or = 10(3) copies of the AML1/ETO fusion transcript at diagnosis, and each showed a 2- to 4-log decrease in copy number following successful induction chemotherapy. This is comparable to the log-fold reduction in leukemic blasts that is thought to occur in patients successfully cytoreduced into CR by induction chemotherapy. The sixth patient showed a relatively high copy number immediately following successful remission induction chemotherapy, which continued to increase during early CR and was later followed by relapse. Real Time RT-PCR appears to offer advantages over previously used quantitative RT-PCR methods by providing absolute quantitation of the target sequence, expanding the dynamic range of quantitation to over six orders of magnitude, eliminating the post-PCR processing, and reducing labor and carryover contamination. These features make this an attractive method to prospectively evaluate the prognostic value of AML1/ETO fusion transcript quantitation in a larger patient population with t(8;21)(q22;q22) AML in CR.
Asunto(s)
Leucemia Mieloide Aguda/diagnóstico , Proteínas de Fusión Oncogénica , Reacción en Cadena de la Polimerasa/métodos , Factores de Transcripción/análisis , Enfermedad Aguda , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Cromosomas Humanos Par 21/genética , Cromosomas Humanos Par 8/genética , Subunidad alfa 2 del Factor de Unión al Sitio Principal , Estudios de Factibilidad , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Neoplasia Residual , Proteína 1 Compañera de Translocación de RUNX1 , Proteínas Recombinantes de Fusión/análisis , Inducción de Remisión , Translocación Genética/genéticaRESUMEN
11q23 translocations (t(11q23)) are recurring cytogenetic abnormalities in both acute myeloid leukemia (AML) and acute lymphoblastic leukemia, involving the same gene, ALL1 (or MLL). Mixed lineage antigen expression has been reported in these leukemias, but its frequency and clinical significance are unknown. We immunophenotyped leukemia cells from 19 adult de novo AML patients with t(11q23) by multiparameter flow cytometry. Translocations included t(6;11)(q27;q23), t(9;11)(p22;q23), t(9;11;19)(p22;q23;q13.3), t(2;11)(11;17)(q37;q11q23;q11), t(11;17)(q23;q25), t(11;19)(q23;p13.1), t(11;19)(q23;p13.3) and t(11;22)(q23;q11). FAB types were M4 and M5. The committed stem cell and myeloid antigens HLADr, CD4dim, CD11b, CD13, CD15, CD32, CD33, CD38 and CD64 were each expressed in 80-100% of cases, and the early stem cell and lymphoid antigens CD34, CD56, CD3, CD2 and CD7 in 42, 39, 16, 5 and 5%, respectively. Antigen expression frequencies did not differ from those in 443 adequately karyotyped M4 and M5 cases without t(11q23). Fifteen patients (79%) attained complete remission (CR); median CR duration and survival were 10.0 and 15.1 months. CR duration and survival did not correlate with antigen expression. In particular, patients with t(9;11) survived longer than those with other t(11q23) (median not reached vs 7.6 months; P = 0.048), but antigen expression did not differ in the two groups. Thus frequencies of lymphoid antigen expression are similar in AML with t(11q23) and in other FAB M4 and M5 cases, treatment outcome does not differ in t(11q23) cases with and without lymphoid antigen expression, and better outcome of patients with t(9;11) compared to other t(11q23) does not correlate with differences in antigen expression. Mixed lineage antigen expression is not a distinctive feature of AML with t(11q23).
Asunto(s)
Cromosomas Humanos Par 11 , Leucemia Mielomonocítica Aguda/genética , Leucemia Mielomonocítica Aguda/inmunología , Proto-Oncogenes , Factores de Transcripción , Translocación Genética , Adolescente , Adulto , Antígenos CD/análisis , Médula Ósea/patología , Mapeo Cromosómico , Proteínas de Unión al ADN/genética , Supervivencia sin Enfermedad , Femenino , Citometría de Flujo/métodos , Reordenamiento Génico , Antígenos HLA-DR/análisis , Células Madre Hematopoyéticas/inmunología , Células Madre Hematopoyéticas/patología , N-Metiltransferasa de Histona-Lisina , Humanos , Inmunofenotipificación , Cariotipificación , Leucemia Mielomonocítica Aguda/mortalidad , Leucemia Mielomonocítica Aguda/terapia , Masculino , Persona de Mediana Edad , Proteína de la Leucemia Mieloide-Linfoide , Recurrencia , Análisis de Supervivencia , Dedos de ZincRESUMEN
Clonal chromosome translocations, deletions, and inversions have been repeatedly observed for decades in approximately two thirds of all cases of acute myeloid leukemia (AML). With the dramatic advances in molecular biology that have occurred during the past two decades, these structural cytogenetic abnormalities have now provided invaluable clues as to the location of genes known or suspected of inducing leukemia. In most instances, leukemogenesis in AML results from gene fusion, when segments from two different genes are fused together to give rise to a chimeric structure consisting of the 5' end of one gene and the 3' end of another. Exceptions to this, however, do exist. In cases of AML that lack cytogenetic abnormalities, investigators are now also beginning to elucidate the genes involved in malignant transformation. Together, these observations support the notion that AML is heterogeneous at the molecular level, and suggest that clinicians will need to continue to take cytogenetic and molecular characteristics into consideration to optimize patient therapy.
Asunto(s)
Aberraciones Cromosómicas/genética , Cromosomas Humanos/genética , Leucemia Mieloide/genética , Translocación Genética/genética , Enfermedad Aguda , Inversión Cromosómica , Cromosomas Humanos Par 11/genética , Humanos , Biología MolecularRESUMEN
The significance of full donor hematopoietic engraftment or hematopoietic chimerism (HC) following allogeneic bone marrow transplantation (BMT) remains unresolved. To study this phenomenon properly, informative genetic loci must be prospectively identified and followed for several years after allogeneic BMT in large numbers of consecutive patients treated with uniform conditioning and immunosuppressive regimens. In addition, sensitive methods that can be performed on small numbers of cells are required in order to extend the analysis for HC to individual hematopoietic lineages and specific anatomic compartments (eg bone marrow, peripheral blood, and lymph nodes). Although polymerase chain reaction (PCR) amplification of polymorphic minisatellite and microsatellite DNA loci has improved the sensitivity of detection of HC, a single rapid, sensitive, and highly informative test that can consistently distinguish donor from recipient elements for the majority of allogenic BMT patients has not been described. In the present report, we describe a single PCR that simultaneously amplifies four microsatellites and was able to identify an informative locus in 48 of 50 (96%) consecutive recipient/donor pairs. A HC of as little as 0.5% could be detected, and HC of 10% could be detected in as few as 100 cells. This technique should allow the detection of an informative microsatellite locus in most patients undergoing allogeneic BMT, using minimal amounts of DNA and requiring 16 h for completion.
Asunto(s)
Trasplante de Médula Ósea , Hematopoyesis , Secuencia de Bases , Southern Blotting , Quimera , Humanos , Repeticiones de Microsatélite , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Trasplante HomólogoRESUMEN
Recent advances in cytogenetics and molecular biology have led to the realization that cancer is a genetic disorder. This finding is especially evident in acute leukemia where the identification of nonrandom cytogenetic abnormalities has paved the way for the discovery and characterization of associated oncogenes and novel mechanisms of leukemogenesis. New insights into the function of these genes and their products are beginning to provide information about their role in normal cell function and leukemogenesis. This article briefly reviews the most recent advances in this exciting field.
Asunto(s)
Aberraciones Cromosómicas , Leucemia/genética , Oncogenes , Enfermedad Aguda , Aneuploidia , Regulación Leucémica de la Expresión Génica , Humanos , Cariotipificación , Leucemia/clasificación , Leucemia/patología , Proteínas de Fusión Oncogénica/genética , Translocación GenéticaRESUMEN
Chromosome abnormalities resulting in gene fusions are commonly associated with acute myeloid leukemia (AML), however, the molecular mechanism(s) responsible for these defects are not well understood. The partial tandem duplication of the ALL1 (MLL) gene is found in patients with AML and trisomy 11 as a sole cytogenetic abnormality and in 11% of patients with AML and normal cytogenetics. This defect results from the genomic fusion of ALL1 intron 6 or intron 8 to ALL1 intron 1. Here, we examined the DNA sequence at the genomic fusion in nine cases of AML with a tandem duplication of ALL1 spanning exons 2-6. Each breakpoint occurred within intron 6 of the ALL1 breakpoint cluster region and within a discrete 3.8-kb region near the 3' end of intron 1. In seven cases, a distinct point of fusion of intron 6 with intron 1 could not be identified. Instead, the sequence gradually diverged from an Alu element in intron 6 to an Alu element in intron 1 through a heteroduplex fusion. Thus, these rearrangements appear to be the result of a recombination event between homologous Alu sequences in introns 6 and 1. In two cases, the genomic junction was distinct and involved the fusion of a portion of an Alu element in intron 6 with non-Alu sequence in intron 1. These data support the hypothesis that a recombination event between homologous Alu sequences is responsible for the partial tandem duplication of ALL1 in the majority of AML cases with this genetic defect. Although Alu element-mediated homologous recombination events in germline cells are thought to be responsible for partial gene duplications or deletions in many inherited diseases, this appears to be the first demonstration identifying Alu element-mediated recombination as a consistent mechanism for gene rearrangement in somatic tissue.
Asunto(s)
Aberraciones Cromosómicas , Trastornos de los Cromosomas , Proteínas de Unión al ADN/genética , Leucemia Mieloide/genética , Familia de Multigenes , Proto-Oncogenes , Recombinación Genética , Factores de Transcripción , Enfermedad Aguda , Secuencia de Bases , Clonación Molecular , Reparación del ADN , Replicación del ADN , Proteínas de Unión al ADN/biosíntesis , Exones , N-Metiltransferasa de Histona-Lisina , Humanos , Intrones , Modelos Genéticos , Datos de Secuencia Molecular , Proteína de la Leucemia Mieloide-Linfoide , Conformación de Ácido Nucleico , Proteínas Recombinantes/biosíntesis , Alineación de Secuencia , Homología de Secuencia de Ácido Nucleico , Dedos de ZincRESUMEN
Presence of specific chromosomal abnormalities is one of the most important prognostic factors in acute myeloid leukemia (AML). Recent advances in molecular biology have allowed structural and functional characterization of many of these genomic rearrangements. In many instances, AML is associated with gene fusion, whereby segments from two different genes fuse to give rise to a chimeric structure consisting of the 5' end of one gene and the 3' end of another. Recombinant DNA technology has also permitted the creation of animal models for in vivo studies of the leukemogenic role of several of these chimeric proteins. This review presents current information on the molecular biology and the clinical significance of three of the most common molecular subtypes of AML. In the first two, t(8;21)(q22;q22) and inv(16)(p13q22), disruption of genes encoding for subunits of core-binding factor (CBF), a transcriptional regulator of normal hematopoiesis, occurs, suggesting a common leukemogenic pathway. The third subgroup is characterized by disruption of MLL, a gene that is located at chromosome band 11q23 and encodes a putative transcriptional regulator. This gene is commonly involved in reciprocal translocations of chromosome 11q23 with other gene partners, but, in some instances, MLL disruption occurs by a mechanism of partial tandem duplication, in the absence of any other partner gene. Current data suggest that identification of specific genetic abnormalities in newly diagnosed AML patients is important to predict clinical outcome and, perhaps, to select different therapeutic strategies. The predictive value of detecting these molecular markers to predict cure or relapse in AML patients in complete remission following definitive treatment is still uncertain.
Asunto(s)
Proteínas de Unión al ADN/genética , Leucemia Mieloide/genética , Proteínas de Neoplasias , Proto-Oncogenes , Factores de Transcripción/genética , Enfermedad Aguda , Factores de Unión al Sitio Principal , Reordenamiento Génico , N-Metiltransferasa de Histona-Lisina , Humanos , Proteína de la Leucemia Mieloide-LinfoideRESUMEN
The molecular defects responsible for tumorigenesis in adult de novo acute myeloid leukemia (AML) with a normal karyotype or an additional copy of one chromosome (i.e., trisomy) remain largely unknown. We recently discovered that approximately 90% of adult patients with de novo AML and trisomy 11 (+11) as a sole abnormality and 11% of adult patients with de novo AML and normal cytogenetics carry a molecular rearrangement of the ALL1 (MLL, HRX, or HTRX) gene. The rearranged ALL1 gene has been shown to result from the direct tandem duplication of a portion of ALL1 itself. To better understand the underlying mechanisms of leukemogenesis, we asked whether in cytogenetically normal cases one or both chromosomes carry the mutated allele and whether in trisomic cases the mutation is present in one, two, or three chromosomes. Herein we show that in cytogenetically normal cases of AML and in cases with +11 as a sole cytogenetic abnormality, only one chromosome contains the mutated ALL1 allele. Thus a single mutated ALL1 allele with the partial tandem duplication is sufficient for ALL1-associated leukemogenesis, irrespective of the number of normal genes present. The frequently occurring specific association of +11 and ALL1 gene mutation in the leukemic clone remains unexplained.
Asunto(s)
Cromosomas Humanos Par 11 , Proteínas de Unión al ADN/genética , Leucemia Mieloide Aguda/genética , Proto-Oncogenes , Factores de Transcripción , Adulto , Alelos , Amplificación de Genes , N-Metiltransferasa de Histona-Lisina , Humanos , Proteína de la Leucemia Mieloide-Linfoide , TrisomíaRESUMEN
Gene rearrangements involving MLL (also known as ALL1, HRX, or Htrx) are among the most common molecular abnormalities associated with acute leukemia. These leukemias generally have one allele involved in a rearrangement, while the remaining allele is uninvolved and demonstrates a germline MLL configuration. In this study, we describe a leukemic cell line that does not have a germline MLL allele and thus cannot produce a normal MLL gene product. We show that the ML-1 cell line, derived from a patient with acute myeloid leukemia, has one allele involved in a t(6;11)(q27;q23), while the remaining MLL allele is deleted. Cloning of the genomic breakpoints on the derivative(6) and der(11) chromosomes demonstrated a balanced translocation between MLL on chromosome band 11q23 and AF6 on chromosome band 6q27. Sequence analysis of the derivative chromosomes revealed that a 186-bp segment of MLL intron 6, downstream of the breakpoint, had been duplicated, inverted, and inserted between MLL and AF6 on the der(11) chromosome. In light of the fact that ML-1 cells can be induced to differentiate along the granulocyte and macrophage lineages, the finding that ML-1 lacks a germline MLL allele demonstrates that a normal MLL gene is not required for survival, proliferation, or differentiation of this cell line.
Asunto(s)
Proteínas de Unión al ADN/genética , Eliminación de Gen , Leucemia Mieloide/genética , Proto-Oncogenes , Factores de Transcripción , Adulto , Secuencia de Bases , Línea Celular , Bandeo Cromosómico , N-Metiltransferasa de Histona-Lisina , Humanos , Hibridación Fluorescente in Situ , Cariotipificación , Masculino , Datos de Secuencia Molecular , Proteína de la Leucemia Mieloide-Linfoide , Mapeo Restrictivo , Células Tumorales CultivadasRESUMEN
The ALL-1 gene, located on chromosome band 11q23, is fused to a variety of other genes by reciprocal chromosomal translocations present in 5-10% of human acute leukemias. We have recently reported the detection by Southern blot of ALL-1 gene rearrangements in adult patients with acute myeloid leukemia lacking cytogenetic evidence of 11q23 translocations. These include 2 of 19 patients with normal karyotypes as well as 3 of 4 patients with trisomy 11. To characterize the abnormal ALL-1 genes, we cloned the ALL-1 rearrangements from two patients with trisomy 11. Characterization of the clones, together with Southern blot analysis, indicates that the ALL-1 rearrangement in both patients is the result of a direct tandem duplication of a portion of the ALL-1 gene spanning exons 2-6. The partial ALL-1 duplication is also detected by Southern blot analysis in a patient with a normal karyotype. RNA PCR and DNA sequence analysis show that the partially duplicated ALL-1 gene is transcribed into mRNA capable of encoding a partially duplicated protein. Partial duplication of ALL-1, in which a portion of a putative protooncogene is fused with itself, represents an additional genetic mechanism for leukemogenesis. Our findings suggest that the presence of trisomy in malignancy may sometimes indicate the partial duplication of a cellular protooncogene.
Asunto(s)
Cromosomas Humanos Par 11 , Leucemia Mieloide/genética , Familia de Multigenes , Enfermedad Aguda , Adulto , Secuencia de Bases , Southern Blotting , Mapeo Cromosómico , Cartilla de ADN , Reordenamiento Génico , Humanos , Intrones , Cariotipificación , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Mapeo Restrictivo , TrisomíaRESUMEN
We have identified a gene at 11q23, telomeric to MLL, that encodes a guanine nucleotide exchange factor (GEF). This gene is transcribed into a 9.5-kb mRNA containing a 4.6-kb ORF. By Northern analysis, it was found to be expressed in all human tissues examined including peripheral blood leukocytes, spleen, prostate, testis, ovary, small intestine, colon, and minimally in thymus. Analysis of the predicted protein sequence indicates that it has strong homology to several members of the family of Rho GEFs that includes such oncogenes as Dbl, Vav, Tiam, and Bcr. A patient with primary acute myeloid leukemia (AML) and a karyotype of 51,XY,+8,+19,+3mar was found to have the 5' end of MLL at exon 6 fused in-frame with the 3' end of almost the entire ORF of this gene, which we named LARG for leukemia-associated Rho GEF. Transcriptional orientation of both genes at 11q23 is from centromere to telomere, consistent with other data that suggest the MLL-LARG fusion resulted from an interstitial deletion rather than a balanced translocation. LARG does not appear to have any homology with other MLL partner genes reported thus far. Thus, LARG represents an additional member of the GEF family and a novel MLL fusion partner in acute myeloid leukemia.
Asunto(s)
Cromosomas Humanos Par 11 , Proteínas de Unión al ADN/genética , Factores de Intercambio de Guanina Nucleótido/genética , Leucemia Mielomonocítica Aguda/genética , Proto-Oncogenes , Factores de Transcripción , Adulto , Secuencia de Aminoácidos , Fusión Artificial Génica , Secuencia de Bases , Mapeo Cromosómico , ADN Complementario , N-Metiltransferasa de Histona-Lisina , Humanos , Masculino , Datos de Secuencia Molecular , Proteína de la Leucemia Mieloide-Linfoide , Factores de Intercambio de Guanina Nucleótido RhoRESUMEN
The allogeneic mixed lymphocyte reaction (MLR) is a complex in vitro assay of T-cell recognition and responsiveness in which interleukin-2 (IL-2) plays a central role. We have previously demonstrated that c-kit ligand (KL) can enhance IL-2-induced proliferation in a subset of human natural killer cells expressing the c-kit tyrosine kinase receptor. In the present study, we asked whether KL could enhance IL-2-mediated T-cell proliferation in the allogeneic MLR. We demonstrate that the vast majority of activated human T-cell clones express the c-kit mRNA transcript. Binding studies performed on activated T cells with radioiodinated KL were consistent with the expression of a single class of c-kit receptors. The addition of exogenous KL to the MLR led to an increase in tritiated thymidine (3[H]-TdR) incorporation in the absence of other exogenous cytokines, and did so in a dose-dependent fashion. A reproducible increase in 3[H]-TdR incorporation was noted at concentrations of KL, which approximate those normally found in vivo. Antibody blocking of KL binding to c-kit, T-cell depletion and sorting experiments suggest that the action of KL is mediated at least in part by a direct effect on both CD4+ and CD8+ T-cells. KL's enhancement of the MLR also requires the binding of IL-2 to its high-affinity IL-2 receptor. Given the abundance of KL normally found in human serum, these data suggest that this cytokine may have a role during T-cell activation in vivo.
Asunto(s)
Prueba de Cultivo Mixto de Linfocitos , Factor de Células Madre/farmacología , Subgrupos de Linfocitos T/inmunología , Animales , Replicación del ADN , Histocompatibilidad , Humanos , Inmunidad Celular , Interleucina-2/fisiología , Isoantígenos/inmunología , Activación de Linfocitos , Ratones , Proteínas Proto-Oncogénicas c-kit/biosíntesis , Proteínas Proto-Oncogénicas c-kit/clasificación , ARN Mensajero/biosíntesis , Receptores de Interleucina-2/efectos de los fármacos , Receptores de Interleucina-2/fisiología , Factor de Células Madre/biosíntesis , Factor de Células Madre/genética , Subgrupos de Linfocitos T/metabolismoRESUMEN
Following reports of childhood acute myeloid leukemia (AML) showing that patients with t(9; 11)(p22; q23) have a better prognosis than those with translocations between 11q23 and other chromosomes, we compared response to therapy and survival of 24 adult de novo AML patients with t(9; 11) with those of 23 patients with other 11q23 translocations [t(11q23)]. Apart from a higher proportion of French-American-British (FAB) M5 subtype in the t(9; 11) group (83% v 43%, P = .006), the patients with t(9; 11) did not differ significantly from patients with t(11q23) in terms of their presenting clinical or hematologic features. Patients with t(9; 11) more frequently had an extra chromosome(s) 8 or 8q as secondary abnormalities (46% v 9%, P = .008). All patients received standard cytarabine and daunorubicin induction therapy, and most of them also received cytarabine-based intensification treatment. Two patients, both with t(9; 11), underwent bone marrow transplantation (BMT) in first complete remission (CR). Nineteen patients (79%) with t(9; 11) and 13 (57%) with t(11q23) achieved a CR (P = .13). The clinical outcome of patients with t(9; 11) was significantly better: the median CR duration was 10.7 versus 8.9 months (P = .02), median event-free survival was 6.2 versus 2.2 months (P = .009), and median survival was 13.2 versus 7.7 months (P = .009). All patients with t(11q23) have died, whereas seven (29%) patients with t(9; 11) remain alive in first CR. Seven of eight patients with t(9; 11) who received postremission regimens with cytarabine at a dose of 100 (four patients) or 400 mg/m2 (2 patients) or who did not receive postremission therapy (2 patients) have relapsed. In contrast, 7 (64%) of 11 patients who received intensive postremission chemotherapy with high-dose cytarabine (at a dose 3 g/m2) (5 patients), or underwent BMT (2 patients) remain in continuous CR. We conclude that the outcome of adults with de novo AML and t(9; 11) is more favorable than that of adults with other 11q23 translocations; this is especially true for t(9; 11) patients who receive intensive postremission therapy.