白血病MLL基因重排及其融合基因检测的临床意义

目的:研究白血病MLL(mixed lineage leukemia)基因重排及其融合基因的检测及其临床意义。方法:采用荧光原位杂交(FISH)检测70例白血病患者MLL基因重排,对于MLL基因重排的患者,用巢式RT-PCR方法检测常见6种MLL融合基因类型。结果:9例白血病有MLL基因重排,发生率为12.86,5例B细胞系急性淋巴细胞白血病(B-ALL),其中融合基因2例为MLL/ENL,1例MLL/AF4,2例未扩出融合基因产物;3例为急性髓细胞白血病M5(AML-M5),其中2例融合基因均为MLL/AF9,1例未扩出融合基因产物;1例为幼年型粒单核细胞白血病(JMML),其融合基因为MLL/ENL。结论:巢式RT-PCR是检测MLL基因重排及其融合基因类型简便有效的方法,MLL基因重排见于B-ALL、AML-M5、JMML,预后差。     

Rearrangements of the MLL gene in therapy-related acute myeloid leukemia in patients previously treated with agents targeting DNA- topoisomerase II

Chromosome band 11q23 is frequently involved in acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) de novo, as well as in myelodysplastic syndromes (MDS) and lymphoma. Five percent to 15% of patients treated with chemotherapy for a primary neoplasm develop therapy-related AML (t-AML) that may show rearrangements, usually translocations involving band 11q23 or, less often, 21q22. These leukemias develop after a relatively short latent period and often follow the use of drugs that inhibit the activity of DNA-topoisomerase II (topo II). We previously identified a gene, MLL (myeloid-lymphoid leukemia or mixed-lineage leukemia), at 11q23 that is involved in the de novo leukemias. We have studied 17 patients with t-MDS/t-AML, 12 of whom had cytogenetically detectable 11q23 rearrangements. Ten of the 12 t-AML patients had received topo II inhibitors and 9 of these, all with balanced translocations of 11q23, had MLL rearrangements on Southern blot analysis. None of the patients who had not received topo II inhibitors showed an MLL rearrangement. Of the 5 patients lacking 11q23 rearrangements, some of whom had monoblastic features, none had an MLL rearrangement, although 4 had received topo II inhibitors. Our study indicates that the MLL gene rearrangements are similar both in AML that develops de novo and in t-AML. The association of exposure to topo II- reactive chemotherapy with 11q23 rearrangements involving the MLL gene in t-AML suggests that topo II may play a role in the aberrant recombination events that occur in this region both in AML de novo and in t-AML.     

Therapy-related MDS/leukemia carrying dup(11) (q21q23) with MLL gene tandem duplication

A 42-year-old woman had been given a diagnosis of malignant lymphoma, follicular, small cleaved cell. She had undergone chemotherapy including etoposide (1,500 mg/total) and was in her second complete remission. Four years and 4 months later, refractory anemia with excess of blasts (RAEB) with dup(11) (q21q23) x 2 developed in the patient and progressed to acute myeloid leukemia (AML-M5b). Despite regression of the AML to RAEB, a clone with the additional abnormality of del(20) (q11q13.1) appeared and transformed the RAEB into AML-M6. Rearrangement of the MLL gene was observed, and FISH analysis demonstrated that the signal sequences from the gene's 5' and 3'-terminal regions had detached. RT-PCR techniques detected a tandem duplication of MLL gene exons 2 through 8. This was considered to be one of the mechanisms behind the formation of the 11q23 abnormality observed in this patient.     

Gene rearrangements in bone marrow cells of patients with acute myelogenous leukemia

At diagnosis, clonal gene rearrangement probes [retinoic acid receptor (RAR)-alpha, major breakpoint cluster region (M-bcr), immunoglobulin (Ig)-JH, T cell receptor (TcR)-beta, myeloid lymphoid leukemia (MLL) or cytokine genes (GM-CSF, G-CSF, IL-3)] were detected in bone marrow samples from 71 of 153 patients with acute myelogenous leukemia (AML) (46%): in 41 patients with primary AML (pAML) (58%) and in 30 patients with secondary AML (42%). In all cases with promyelocytic leukemia (AML-M3) RAR-alpha gene rearrangements were detected (n = 9). Gene rearrangements in the Ig-JH or the TcR-beta or GM-CSF or IL-3 or MLL gene were detected in 12, 10, 16 and 12% of the cases, respectively, whereas only few cases showed gene rearrangements in the M-bcr (6%) or G-CSF gene (3%). Survival of pAML patients with TcR-beta gene rearrangements was longer and survival of pAML patients with IL-3 or GM-CSF gene rearrangement was shorter than in patients without those rearrangements. No worse survival outcome was seen in patients with rearrangements in the MLL, Ig-JH or M-bcr gene. In remission of AML (CR), clonal gene rearrangements were detected in 23 of 48 cases (48%) if samples were taken once in CR, in 23 of 26 cases (88%) if samples were taken twice in CR and in 23 of 23 cases (100%) if samples were studied three times in CR. All cases with gene rearrangements at diagnosis showed the same kind of rearrangement at relapse of the disease (n = 12). Our data show that (1) populations with clonal gene rearrangements can be regularly detected at diagnosis, in CR and at relapse of AML. (2) Certain gene rearrangements that are detectable at diagnosis have a prognostic significance for the patients' outcome. Our results point out the significance of gene rearrangement analyses at diagnosis of AML in order to identify 'poor risk' patients - independently of the karyotype. Moreover, the persistence of clonal cells in the further course of AML can be studied by gene rearrangement analysis.     

Therapy-related acute myelogenous leukemia (AML-M6) with add(11) (q23) and del(20) (q11.2) developing via myelodysplastic syndrome after chemotherapy for malignant lymphoma

A 62-year-old woman was diagnosed as having malignant lymphoma, diffuse large B-cell type. She underwent chemotherapy with the standard dose of CHOP and MINE regimens, resulting in complete remission. Four months later, the myelodysplastic syndrome of RA (refractory anemia) with pancytopenia developed and rapidly progressed to acute myelogenous leukemia (AML-M6) in 4 months. Cytogenetic analysis for the bone marrow specimens of both periods of MDS and AML-M6 revealed complex karyotypic abnormalities involving chromosome 5, 7, 11q23 and 20q11.2. Neither rearrangement of the MLL gene by Southern blot analysis nor tandem duplication of MLL gene by RT-PCR technique was detected. The patient was died from progression of leukemia and pneumonia. The autopsy showed no residual disease of lymphoma-related disease.     

急性白血病11q23/MLL基因易位重排及其临床意义

目的：研究11q23/MLL基因易位重排在急性白血病（AL）中的发生率、产生融合基因的常见类型及其临床意义.方法：用荧光原位杂交技术，MLL双色断裂分离重排探针检测50例AL患者（49例初治，1例难治）的11q23/MLL基因，用流式细胞仪检测免疫表型，对于11q23/MLL基因易位重排阳性的患者，用巢式RTPCR方法检测11q23/MLL基因易位重排形成的6种常见融合基因类型.结果：6例AL有11q23/MLL基因易位重排，发生率为12%，2例为AML-M5，4例为ALL且均为B-ALL.2例11q23/MLL基因易位重排阳性的AML M5患者融合基因均为MLL/AF9，其中1例为初治，发病时左侧小腿有白血病细胞浸润，本例患者化疗1疗程获CR;1例为难治性AL患者，于第3个疗程化疗后才达CR.4例11q23/MLL基因易位重排阳性的B-ALL患者中有2例于诊断后3周内死于全身衰竭和感染，化疗未获CR，其中1例患者的融合基因为MLL/ENL，1例未扩出融合基因产物;1例于诊断后第2天因DIC脑出血死亡，未进行化疗，其融合基因为MLL/AF9;1例发病时胸椎有白血病细胞浸润，1疗程化疗后获CR，其融合基因产物未扩出.结论：荧光原位杂交技术是检测AL11q23/MLL基因易位重排快速、灵敏的方法，巢式RT-PCR是检测11q23/MLL基因易位重排所产生的融合基因类型简便可行的方法;有11q23/MLL基因易位重排的AL患者临床症状凶险，预后差。     

Secondary acute myeloid leukemia with translocation (4;11) and MLL/AF4 rearrangement in a 15-year-old boy treated for common acute lymphoblastic leukemia 11 years earlier

Summary Secondary acute myeloid leukemia occurring in a 15-year-old boy 11 years after initial treatment of a common lymphoblastic leukemia (c-ALL) is described. Initial complete remission was terminated after 4 years by an isolated testicular relapse, followed by first bone marrow relapse within 18 months. After he achieved remission again, an allogeneic bone marrow transplantation from his HLA-identical brother was performed. Five years and 9 months later, the patient developed thrombocytopenia, leukopenia, and anemia, but bone marrow biopsies at this time demonstrated only myelofibrosis, with no blast cell population present. A polymerase chain reaction assay of a peripheral blood sample recognized the mRNA fusion region for the MLL/AF4 rearrangement, i.e., the molecular equivalent of the translocation (4;11)(q21,q23). Four weeks later, a blast cell population with AML-M1 morphology according to the FAB classification appeared in the bone marrow, and translocation (4;11) was detected by cytogenetics. Thus, secondary leukemias with chromosomal 11q23 rearrangement can develop after a long latency period and can be diagnosed earlier with the PCR technique.     

Human homologue of the rat chondroitin sulfate proteoglycan, NG2, detected by monoclonal antibody 7.1, identifies childhood acute lymphoblastic leukemias with t(4;11)(q21;q23) or t(11;19)(q23;p13) and MLL gene rearrangements

Monoclonal antibody 7.1, which recognizes the chondroitin sulfate proteoglycan molecule NG2, was used to screen prospectively blast cells from 104 consecutive children at initial presentation with acute lymphoblastic leukemia (ALL). Reactivity with this antibody was found in 9 cases (8.6%), of whom 5 had a t(4;11)(q21;q23) and 4 had a t(11;19)(p13;q23). None of the NG2- cases had either translocation. Southern blot analysis disclosed MLL gene rearrangement in only the 9 cases with 7.1 reactivity plus the t(4;11)(q21;q23) or t(11;19)(q23;p13) translocation. MLL gene rearrangements were not detected in 89 patient leukemic samples that did not express NG2, including 7 patients with del(11)(q23) or inv(11)(p13q23). As expected from the association with t(4;11) and t(11;19), NG2+ cases were significantly more likely to be infants, to have hyperleukocytosis and central nervous system involvement, to be CD10-, and to express myeloid- associated antigens CD15 and CD65. Despite short follow-up duration, 3 of the NG2+ cases have relapsed while the other 101 patients remain in remission. Thus, blast cell surface expression of NG2 is useful for identifying patients with ALL having t(4;11) or t(11;19) translocations that are associated with poor prognosis, especially in the infant age group.     

Rearrangement of the MLL gene confers a poor prognosis in childhood acute lymphoblastic leukemia, regardless of presenting age

MLL gene rearrangements are associated with an extremely poor prognosis in infants with acute lymphoblastic leukemia (ALL), but little is known about their clinical significance in older children. Therefore, we studied 45 cases of childhood ALL with abnormalities of chromosome 11q23 for rearrangement of the MLL gene to determine if this feature confers a uniformly poor prognosis. MLL gene rearrangements were detected in all 18 cases with the common t(4;11), t(9;11) or t(11;19) translocations, whereas only 5 of 12 patients with either unbalanced or uncommon balanced translocations demonstrated a rearrangement. Abnormalities of the MLL gene were not detected in any of the 15 cases with a deletion or inversion of the chromosomes 11q23 region. The presence of an MLL rearrangement was significantly associated with age less than 1 year (P < .001), leukocyte count >50 x 10(9)/L (P = .003), and the absence of leukemic cell CD10 expression (P < .001). In a stratified statistical analysis adjusted for age and treatment protocol, MLL gene rearrangement was correlated with an inferior treatment outcome (P = .028). The 4-year event-free survival estimate (+/- SE) was 10% +/- 6.5% for cases with a rearranged MLL gene and 64% +/- 19.2% for other cases. When infants were excluded from the analysis, MLL rearrangement was still significantly associated with a poor outcome (P = .02), and remained so with the exclusion of t(4;11)- positive cases (P = .05). Thus, regardless of presenting age, MLL gene rearrangement identifies a high-risk subgroup of patients who are not likely to be cured with conventional treatment.     

Acute myelomonocytic leukemia (AML-M4) and translocation t(6;9)(p23);q34): two additional patients with prominent myelodysplasia

Two patients with acute myelomonocytic leukemia (AML-M4) and a specific chromosomal translocation t(6;9)(p23;q34) are reported and compared to 21 AML patients with the same translocation collected from the literature. Our observation suggest that AML with t(6;9)(p23;q34) is characterized by myelodysplasia, basophilia, and a variety of blast cell morphologies (M1, M2, M4) with a greater proportion of the cases than previously appreciated being examples of acute myelomonocytic leukemia (AML-M4). The consistent association of myelodysplasia provokes the proposal that this subtype of de novo AML is a result of an acute stem cell disorder. The poor outcome with standard AML chemotherapy experienced in this group of relatively young patients necessitates consideration of alternative therapeutic strategies such as early bone marrow transplantation.     

MLL Gene Rearrangement in Acute Myelogenous Leukemia After Exposure to Tegafur/Uracil

We report a case of acute myelogenous leukemia (AML) with MLL (myeloid-lymphoid leukemia or mixed-lineage leukemia) gene rearrangement after exposure to tegafur/uracil. Cytogenetic and clinical findings in this patient: t(11;17) (q23;q25), AML-M4 morphology, development of AML within a short latent period after first exposure to tegafur/uracil, and good response to remission induction chemotherapy but short remission duration, have been considered typical features of therapy-related acute myelogenous leukemia (t-AML) after exposure to topoisomerase II-targeting agents. This case report suggests that t-AML may develop after exposure to tegafur/uracil and that MLL gene rearrangement may not necessarily be specific to t-AML after exposure to topoisomerase II-targeting agents.     

Successful Outcome of Mismatched Hematopoietic Stem Cell Transplantation from a Related Donor in an Infant with Acute Lymphoblastic Leukemia and 9;11 Translocation: Case Report and Review of the Literature

Although infants with acute lymphoblastic leukemia (ALL) and MLL gene rearrangements have a poor prognosis, those with acute myeloid leukemia (AML) have been shown to have a superior outcome with intensive chemotherapy alone despite the presence of MLL gene rearrangements. We report the case of an ALL infant with t(9;11), a common cytogenetic abnormality in infant AML, who after relapse underwent successful hematopoietic stem cell transplantation (HSCT) from her HLA 2-loci-mismatched mother. Analysis of the outcome among ALL infants with MLL gene rearrangements registered in the Japan Infant Leukemia Study between 1996 and 1999 showed the event-free survival of patients with t(9;11) was not different from that of those with other 11q23 translocations. Most of the patients with t(9;11) described in the reviewed literature also experienced either induction failure or early relapse after achievement of complete remission, but some of them were rescued with subsequent HSCT. These findings suggest that infant ALL with t(9;11) has features distinct from those of infant AML with the same karyotype and that the prognosis among these patients can be improved only with the combination of intensive chemotherapy and HSCT An appropriate strategy for the treatment of ALL infants with different 11q23 translocations must be clarified.     

Morphological and cytogenetic changes in therapy-related leukemia developed in a t(8;21)-acute myeloid leukemia (M2) patient: sequential cytogenetic and molecular analyses

A patient with acute myeloid leukemia (AML)-M2 with t(8;21)(q22;q22) achieved complete remission with remission-induction chemotherapy followed by consolidation and intensification chemotherapies. T(8;21)(q22;q22) disappeared, but chimeric AML1/MTG8 was continuously detected in bone marrow cells. Following the development of therapy-related leukemia after 1 year, evolution of therapy-related AML-M4 with t(11;17)(q23;q25) and the rearrangement of the MLL gene were observed, while AML/MTG8 disappeared. After reinduction and following intermittent chemotherapies, a subsequent alternative transformation to AML-M2 occurred after detection of t(3;21)(q21;q22), with a break in the AML1 gene shown by interphase fluorescence in situ hybridization analysis. This leukemia transformed to AML-M4 after t(9;22)(q34;q11), with a minor BCR/ABL rearrangement, and then finally to AML-M2. This therapy-related leukemia was resistant to chemotherapy. These findings indicate that alterations in cytogenetic and molecular events caused by chemotherapeutic agents contribute to the sequential evolution of new leukemic clones with different morphology.     

Two new translocations involving the 11q23 region map outside the MLL locus in myeloid leukemias

BACKGROUND AND OBJECTIVES: In acute leukemias, chromosomal translocations involving the 11q23 band are frequently, but not invariably, associated with MLL gene rearrangement and their finding is associated with a poor prognosis. We observed two new translocations with a breakpoint in the 11q23 region at standard cytogenetic analysis: a previously undescribed t(3;11)(q21;q23) in a 70-year old woman with a fulminating form of AML-M1 and a new translocation t(6;11)(q15;q23) in a 61-year old man with an atypical chronic myelogenous leukemia. In these two patients, involvement of the MLL gene was analyzed by molecular cytogenetic techniques which also allowed a more precise mapping of the breakpoints. DESIGN AND METHODS: The MLL gene was analyzed by Southern blot and by fluorescent in situ hybridization (FISH) with a double-color MLL probe. A panel of 11q, 3q and 6q cosmid/YAC probes mapping around the breakpoints was used for breakpoint mapping. RESULTS: In both patients, FISH analysis and Southern blot showed that the MLL gene was not rearranged; in patient 1, MLL was retained on the 11q+ derivative, whereas in patient 2 it moved to the 6q- chromosome. In the t(3;11) we localized the chromosome 11 breakpoint at 11q23.3, in a region flanked by CP-939H3 and cos1p3, distal to the MLL locus; in the t(6;11) the break occurred at 11q21, in a region flanked by CP-819A5 and CP-829A6, proximal to the MLL locus. INTERPRETATION AND CONCLUSIONS: Our cases add two new translocations to the list of chromosomal anomalies involving the long arm of chromosome 11, and show that apparent translocation t(11q23) may involve loci and genes other than MLL. Characterizing the molecular heterogeneity of 11q23 translocations may identify some prognostic significance.     

MLL-rearranged leukemias: insights from gene expression profiling

Gene expression analysis of human leukemias has provided insight into disease classification and mechanisms of oncogenesis. Its success is particularly evident for acute leukemias with rearrangement of the mixed lineage leukemia (MLL) gene on chromosome 11q23. Unlike most other recurrent translocations, MLL rearrangements are found in leukemias classified as acute myelogenous leukemia (AML) or acute lymphoblastic leukemia (ALL). In addition, MLL-rearranged leukemias often express both myeloid- and lymphoid-associated genes. These unusual characteristics have generated much interest in the cell of origin and the mechanism of transformation by MLL rearrangements. Here we review insights gained from characterization of MLL-rearranged human leukemias by genome-wide expression profiling and compare these to data from model systems.     

Superior outcome of infant acute myeloid leukemia with intensive chemotherapy: results of the Japan Infant Leukemia Study Group.

This study analyzed data on 35 infants with acute myeloid leukemia (AML) who were treated with intensive chemotherapy between 1995 and 1998 in Japan. The incidence of boys, younger age (< 6 months old), and hyperleukocytosis at onset was high in patients with the M4/M5 subtype (n = 23) in the French-American-British classification, compared with the non-M4/M5 subtype (n = 12). Thirteen (56%) and 16 (70%) patients with the M4/M5 subtype also showed 11q23 translocations and MLL gene rearrangements, respectively, whereas only one patient with the non-M4/M5 subtype had this rearrangement. All 35 patients were treated with the ANLL91 protocol consisting of etoposide, high-dose cytarabine, and anthracyclines. Overall survival and the event-free survival (EFS) rates at 3 years of all patients were 76% (95% confidence interval [CI], 61.3%-90.7%) and 72% (95% CI, 56.4%-87.9%), respectively. EFS showed no significant difference between 2 subgroups divided by age, gender, presence of the MLL gene rearrangements, and white blood cell count at onset; EFS in patients with the M4/M5 subtype tended to be better than those with the non-M4/M5 subtype. Although all 6 patients who underwent allogeneic stem cell transplantation (SCT) have been in complete remission, no benefit of SCT was confirmed. These findings suggest that the intensive chemotherapy with the ANLL91 protocol might have been responsible for the observed good outcome of infant AML, even without SCT. The presence of the MLL gene rearrangements or the age at onset had no impact on the outcome of infant AML.