Comparative analysis of MLL partial tandem duplication and FLT3 internal tandem duplication mutations in 956 adult patients with acute myeloid leukemia

Partial tandem duplication (PTD) of the MLL gene and internal tandem duplication (ITD) of the juxtamembrane region of the FLT3 receptor tyrosine kinase gene have been described in acute myeloid leukemia (AML) patients, preferentially in those with normal cytogenetics. These alterations have been associated with a poor prognosis. In our study, we analyzed the prevalence and the potential prognostic impact of these aberrations in a large unselected and well-defined cohort of 956 patients with AML. Results were correlated with cytogenetic data and clinical outcome. MLL PTD was detected by RT-PCR, subsequent nucleotide sequencing, and Southern blotting. The overall incidence was found to be 5.0% (48/956), whereas FLT3 ITD was detected in 19.2% (184/956). Sixteen cases were positive for both alterations. The rate of MLL PTD in FLT3 ITD positive patients was significantly higher than that in FLT3 ITD negative patients [16/184 (8.7%); 32/772 (4.1%); P = 0.025]. However, both aberrations were highly increased in patients with normal karyotype (MLL PTD 35/431, P = 0.004; FLT3 ITD 132/334, P < 0.001). When restricted to this subgroup, the rate of MLL PTD in patients with FLT3 mutations was not significantly increased. No statistically significant differences were detected between patients positive for MLL PTD and patients negative for MLL PTD in the rate of complete remissions or the overall survival, although we did see a significantly shorter disease-free survival in patients age 60 or younger. In conclusion, although there is an overlap in the mutational spectrum in AML with FLT3 ITD and MLL PTD mutations, our data do not support a common mechanistic basis. Although associated with inferior disease-free survival, the results of this study do not unequivocally support the notion that MLL PTD mutations represent an independent prognostic factor.     

Pediatric T-cell acute lymphoblastic leukemia with aberrations of both MLL loci

Translocations involving the MLL gene at 11q23 have been implicated in acute lymphoblastic leukemia (ALL), as well as acute myeloid leukemia (AML). Such translocations result in gain of function fusion proteins that drive cell proliferation. Except in cases of T-cell ALL, MLL rearrangement is typically associated with a poor prognosis. We report a case of T-cell ALL with a t(11;19)(q23;p13.3) and deletion of the other chromosome 11 homolog at band q23. Fluorescence in situ hybridization (FISH) analyses confirmed involvement of the MLL loci in both the translocation and deletion. This case is unique in that deletions of 11q23 reported in ALL generally do not involve MLL. We are unaware of a previous report showing rearrangement of the MLL loci on both chromosome 11 homologues.     

Overlapping deletion regions at 11q23 in myelodysplastic syndrome and chronic lymphocytic leukemia, characterized by a novel BAC probe set

Translocations or deletions involving the 11q23 region have been observed in acute lymphoblastic leukemia (ALL), acute myelocytic leukemia (AML), myelodysplastic syndrome (MDS), and chronic lymphocytic leukemia (CLL). BAC probes encompassing the D11S29 and D11S924 markers and flanking the MLL gene were used in dual color fluorescence in situ hybridization. Fifteen patients with hematologic malignancies and cytogenetic abnormalities of 11q23 were analyzed. The BAC and MLL probes demonstrated split signals in five of 7 ALL or AML cases with translocations of 11q23. Of the remaining 2 cases, one had normal signals for both probe sets and the other had a submicroscopic deletion of the MLL 3' region. In one case of AML with del(11)(q23), deletion of the MLL 3' region and the region telomeric to the MLL gene was seen. Three CLL cases with deletion of part or the entire 11q23 region showed deletion of one copy of MLL, but retention of the region telomeric to MLL. However, in four MDS cases with deletions involving the 11q23 region, deletions of both the MLL gene and the flanking regions of the MLL gene were observed. Hence, the deletions on 11q23 are different but overlapping for CLL and MDS, implicating different genes involved for these diseases.     

Amplification of the 11q23 region in acute myeloid leukemia.

Cytogenetic abnormalities involving the 11q23 region are found in both acute lymphoblastic leukemia (ALL) and myeloid leukemia (AML). Molecular consequences of 11q23 translocations are the formation of chimeric genes, all of them involving the MLL (mixed-lineage leukemia) gene. To evaluate the usefulness of fluorescence in situ hybridization (FISH) in detecting MLL rearrangements in AML, we analyzed 181 patients with an MLL-specific probe. Among them, we detected three patients with multiple FISH signals, reflecting genomic amplification of this chromosomal region. Extra copies of MLL have been reported previously in four patients, but did not correspond to a true gene amplification. For the first time, we describe genomic amplification of the 11q23 region (up to more than 50 copies) in AML patients. This genomic amplification could affect MLL, but other genes in the vicinity could also be the primary target. Genes Chromosomes Cancer 26:166-170, 1999.     

Analysis of t(9;11) chromosomal breakpoint sequences in childhood acute leukemia: almost identical MLL breakpoints in therapy-related AML after treatment without etoposides

The translocation t(9;11)(p22;q23) is a recurring chromosomal abnormality in acute myeloid leukemia (AML) fusing two genes designated as MLL and AF9. Within MLL, almost all rearrangements cluster in an 8.3-kb restricted region and fuse 5' portions of MLL to a variety of heterologous genes in various 11q23 translocations. AF9 is one of the most common fusion partners of MLL. It spans more than 100 kb, and two breakpoint cluster regions (BCRs) have been identified in a telomeric region of intron 4 (BCR1) and within introns 7 and 8 (BCR2). We investigated 11 children's bone marrow or peripheral blood samples (3 AML, 5 t-AML, 2 ALL, 1 ALL relapse) and two cell lines (THP-1 and Mono-Mac-6) with cytogenetically diagnosed translocations t(9;11). By use of an optimized multiplex nested long-range PCR assay, a breakpoint-spanning DNA fragment from each sample was amplified and directly sequenced. In four patients and two cell lines, the AF9 breakpoints were located within BCR1 and in two patients within BCR2, respectively. However, in five patients the AF9 breakpoints were found outside the previously described BCRs within the centromeric region of intron 4 and even within intron 3 in one case. All five patients with a secondary AML, who had not received etoposides during treatment of the primary malignant disease, revealed almost identical MLL breakpoints very close to a breakage hot spot inducible by topoisomerase II inhibitors or apoptotic triggers in vitro. Sequence patterns around the breakpoints indicated involvement of a "damage-repair mechanism" in the development of t(9;11) similar to t(4;11) in infants' acute leukemia.     

Mutations of the FLT3 gene in adult acute myeloid leukemia: determination of incidence and identification of a novel mutation in a Thai population

FLT3 mutations have been reported to be the most frequent mutation in acute myeloid leukemia (AML). No data currently exist regarding FLT3 mutations in Southeast Asian patients. In this study, the incidence and type of FLT3 mutation in a large series of Thai AML patients were determined. FLT3 internal tandem duplication (ITD) mutations were observed in 24.6%, FLT3 tyrosine kinase domain mutations in 3.1%, and dual mutations in 2.7% of 256 newly diagnosed Thai AML patients. ITD mutations were mostly restricted to the juxtamembrane domain, and the in-frame ITD length varied from 21 to 201 base pairs. Six types of point mutations were identified, including Asp835Tyr, Asp835His, Asp835Glu, Asp835Ala, Ile836, and a novel mutation, Asp835Del/Ile836Val, which resulted in the loss of aspartic acid and substitution of isoleucine by valine. A rare leukemia karyotype, trisomy 11, was found to coexist with this novel FLT3 mutation, whereas the majority of patients with FLT3 mutations had a normal karyotype. Overall, FLT3 mutation was associated with a significantly higher white blood cell count and older age than the wild-type FLT3. In conclusion, the incidence of FLT3 mutation in Thailand is as high as that of western countries. The clinical significance of the novel mutation requires further studies in a larger population.     

Low frequency and variability of FLT3 mutations in Korean patients with acute myeloid leukemia

FLT3 mutations are common genetic changes, and are reported to have prognostic significance in acute myeloid leukemia (AML). The FLT3 internal tandem duplication (ITD) and the D835 activating mutation in the tyrosine kinase domain (TKD) were analyzed by polymerase chain reaction (PCR) in the genomic DNA of Korean patients with AML at diagnosis and during follow-up. There were 226 patients with AML enrolled between March 1996 and August 2005. The incidence of ITD and TKD at diagnosis was 13% (29/226) and 3% (6/226). When compared to Western and other Asian patients with AML, Korean patients had a lower frequency by about two-thirds of ITD and TKD. Among the non-M3 cases (N=203), the patients with an ITD had a significantly shorter event-free survival when compared with those without an ITD (p=0.0079). Among 54 relapsed patients, 9 patients had the FLT3 ITD at diagnosis. Six patients demonstrated a reappearance of the ITD and 3 patients remained negative at relapse. One patient, among 45 patients who relapsed, had a negative baseline ITD but acquired a de novo ITD at relapse. There were 101 samples from 93 patients in remission; they were all negative for an ITD. Among 34 patients who failed to achieve a remission, five patients had a persistent ITD and one patient had a de novo ITD. These results support the concept of resistance of FLT3 ITD leukemic clones to chemotherapy. Therefore, effective therapy with FLT3 targeting agents may improve the prognosis of non-M3 AML patients with the FLT3 mutation.     

逆转录-多重巢式聚合酶链反应技术在急性单核系白血病M4/M5 MLL基因重排检测中的应用

目的 探讨逆转录-多重巢式聚合酶链反应（多重PCR）技术在初诊M4／M5患者MLL基因重排检测中的价值。方法 采用骨髓直接或短期培养法制备染色体，应用R显带技术进行核型分析。采用多重PCR技术，检测40例初诊M4／M5患者中5种急性髓系白血病常见的MLL融合基因以及MLL部分串联重复。结果 R显带揭示7有涉及11q23的易位，包括t（6；11）（q27；q23）、t（9；11）（p21；q23）、t（11；17）（q23；q21）、t（11；19）（q23；p13．1），14例有其他核型异常，19例为正常核型。多重PCR证实了7例核型分析显示11q23易位标本中的6例，例3核型分析揭示46，XX，t（6；11）（q27；q23），多重PCR检测MLL/AF6为阴性；19例显带分析为正常核型标本中检出2例MLL部分串联重复。结论 多重PCR是对初诊M4／M5患者进行各种MLL重排筛检的有效方法。     

Granulocytic sarcomas in body cavities in childhood acute myeloid leukemias with 11q23/MLL rearrangements

Three childhood acute monoblastic leukemias (AML M5) with granulocytic sarcomas (GSs) are described. All displayed 11q23/MLL abnormalities, t(9;11)(p22;q23) in two cases and t(11;17)(q23;q21) in one case, constituting around 20% of all 11q23-positive AML cytogenetically investigated in our department. Two of the patients had GS in multiple locations, and all three had abdominal GS. In two of them, t(9;11)-positive GS was diagnosed prior to the diagnosis of AML. Fourteen (1.9%) of 752 published AML cases with 11q23 aberrations have had GS, either as a presenting feature or during disease progression. The incidence of GS has varied significantly (P < 0.05) between children (3.8%) and adults (0.8%). The most common AML subtype has been AML M5 ( approximately 75%) and the most frequent GS sites have been the skin, abdomen, orbit, and thorax. Considering the possibility of underreporting of GS in published cases and the relatively high frequency in our own series, we believe that 11q23/MLL rearrangements may predispose to GS development. Although extramedullary infiltrates in the skin are known to be frequent in cases of AML M5, which is often associated with 11q23 aberrations, the present findings indicate that GS in the abdomen, orbit, and thorax may also be common, especially in pediatric AML. Thus, the possibility of 11q23/MLL-positive GS should be suspected when tumors of uncertain derivation occur in these sites. Finally, the identification of 11q23/MLL abnormalities in GSs in two patients without overt AML underscores the importance of using cytogenetic and molecular genetic investigations as a diagnostic approach in the evaluation of tumorous lesions of unknown origin. Genes Chromosomes Cancer 27:136-142, 2000.     

MLL translocations with concurrent 3' deletions: interpretation of FISH results

Rearrangements involving the MLL gene at 11q23 occur in a clinically relevant subgroup of patients with acute lymphoblastic leukemia (ALL) at all ages, and therefore their accurate identification at diagnosis is important. It has become commonplace to screen ALL patients for rearrangements of MLL using a dual-color fluorescence in situ hybridization (FISH) assay. We report on 12 ALL patients with an unusual FISH result consisting of the following signal pattern: one 5' green, no 3' red, and one/two fusion signals. This configuration is consistent with a MLL translocation and simultaneous deletion of 3' MLL-a well-established phenomenon-which has been interpreted as a positive result. G-banded and complementary metaphase FISH analyses confirmed an 11q23/MLL translocation in 8 of the 12 cases, whereas in one case, the identification of a del(11)(q23) was restricted to G-banded analysis only. In three cases, an MLL rearrangement was excluded by extensive FISH analysis and/or Southern blotting. In conclusion, the loss of the 3' MLL signal should not be assumed to be the result of a concurrent translocation and deletion event, and such aberrant FISH signal patterns should be investigated further by alternative methods for determining their MLL status.     

FLT3 internal tandem duplication during myelodysplastic syndrome follow-up: a marker of transformation to acute myeloid leukemia

Myelodysplastic syndrome (MDS) is a clonal hematopoietic stem cell disorder characterized by ineffective hematopoiesis and risk for evolving to acute leukemia. Some molecular abnormalities related to acute myeloid leukemia (AML) transformation have been reported, such as FLT3 (FMS-like tyrosine kinase 3) mutations. FLT3, a member of the class 3 receptor tyrosine kinase family, mediates stem cell proliferation and differentiation, and its mutations, internal tandem duplication (ITD) and Asp835, have been reported in rare MDS patients. We studied FLT3 ITD, prospectively, in 50 MDS patients at diagnosis, at 6 and 12 months follow-up, and at any other time-point if AML transformation was detected. FLT3 ITD was not observed at diagnosis, but during follow-up the mutation was present in 2 of 50 patients (4%). Of these, one case exhibited FLT3 ITD at the end of the 6 months of follow-up in 8% of bone marrow cells; this case evolved into AML at 8 months, at which time FLT3 ITD was present in 85% of bone marrow cells. The other case exhibited FLT3 ITD in 68% of bone marrow cells at 7 months, precisely at the time of AML transformation. Although rare in MDS, FLT3 ITD is associated with a high probability of evolution to AML.     

11q23 abnormalities in patients with acute myelogenous leukemia and myelodysplastic syndrome as detected by molecular and cytogenetic analyses

11q23 chromosomal abnormalities and rearrangement of the mixed lineage leukemia (MLL) gene are important prognostic factors in acute myelogenous leukemia (AML) and myelodysplastic syndrome (MDS). However, the presence of 11q23 abnormalities does not always correlate with that of MLL gene rearrangement. We retrospectively compared the occurrence of 11q23 abnormalities (measured by karyotyping) and MLL gene rearrangement (measured by Southern blotting) in bone marrow from 311 consecutive adult patients with AML or MDS. 11q23 abnormalities were found in 18 patients (5.8%), of whom 7 (39%) did not have the MLL gene rearrangement. MLL gene rearrangement was detected in 35 patients (11.2%). Of these 35 patients, only 11 (31%) had cytogenetic evidence of 11q23 abnormalities. None of the 21 patients with chronic myelomonocytic leukemia had 11q23 abnormalities or MLL gene rearrangement. 11q23 abnormalities were associated with shorter survival than was a diploid karyotype. Both cytogenetic and molecular studies should be performed to detect 11q23 abnormalities in patients with AML or MDS.     

Chromosomal aberration of the 11q23 locus in acute leukemia and frequency of MLL gene translocation: results in 378 adult patients

Structural abnormality of the 11q23 band (11q23+) bearing the MLL gene translocation (MLL+) is a recurrent chromosome change observed in 3% to 7% of acute lymphoblastic leukemias and in 3% to 4% of acute myeloblastic leukemias. The resolution of conventional cytogenetics (CC) in detecting 11q23 rearrangement is limited when the translocative partner has a telomeric location; furthermore, CC can barely discriminate between true 11q23+/MLL+ and rearrangements clustering within the 11q22 to approximately 25 region without MLL involvement (MLL-). We characterized a series of 378 consecutive patients with adult acute leukemia by using CC, fluorescence in situ hybridization (FISH), and multiplex karyotyping (M-FISH) analysis. Our aim was to define the frequency of cryptic MLL+ cases and the frequency of MLL+ within 11q22 to approximately 25+ cases. As expected, FISH was more sensitive than CC in detecting MLL+ cases, but rather unexpectedly, 9 (45%) of 20 patients with 11q22 to approximately 25+ were MLL-. A better characterization of 11q22 to approximately 25+/MLL- leukemias is relevant for the identification of new, recurrent translocations. Moreover, these cases should be readily distinguishable from 11q23+/MLL+ cases. We recommend that karyotypic analysis always be complemented by molecular or FISH methods to unravel MLL rearrangements.     

Molecular cytogenetic analysis of gene rearrangements in childhood acute lymphoblastic leukemia

The aims of this study were to estimate the incidences of BCR/ABL, MLL, TEL/AML1 rearrangements, and p16 deletions in childhood acute lymphoblastic leukemia (ALL), to identify new abnormalities, and to demonstrate the usefulness of interphase fluorescence in situ hybridization (FISH). We performed G-banding analysis and FISH using probes for BCR/ABL, MLL, TEL/AML1 rearrangements, and p16 deletions on 65 childhood ALL patients diagnosed and uniformly treated at a single hospital. Gene rearrangements were identified in 73.8% of the patients using the combination of G-banding and FISH, while the chromosomal abnormalities were identified in 49.2% using G-banding alone. Gene rearrangements were disclosed by FISH in 24 (72.7%) of 33 patients with normal karyotype or no mitotic cell in G-banding. Among the gene rearrangements detected by FISH, the most common gene rearrangement was p16 deletion (20.3%) and the incidences of others were 14.1% for TEL/AML1, 11.3% for MLL, and 1.8% for BCR/ABL translocations. Infrequent or new aberrations such as AML1 amplification, MLL deletion, ABL deletion, and TEL/AML1 fusion with AML1 deletion were also observed. We established the rough incidences of gene rearrangements in childhood ALL, found new abnormalities and demonstrated the diagnostic capability of interphase FISH to identify cryptic chromosome aberrations.