The fusion transcripts of MLL rearrangement [MLL(+)] in acute myeloid leukemia (AML) and their clinicohematologic correlation have not be well characterized in the previous studies. We used Southern blot analysis to screen MLL(+) in de novo AML. Reverse transcriptase-polymerase chain reaction was used to detect the common MLL fusion transcripts. cDNA panhandle PCR was used to identify infrequent or unknown MLL partner genes. MLL(+) was identified in 114 (98 adults) of 988 AML patients. MLL fusion transcripts comprised of 63 partial tandem duplication of MLL (MLL-PTD), 14 MLL-AF9, 9 MLL-AF10, 9 MLL-ELL, 8 MLL-AF6, 4 MLL-ENL and one each of MLL-AF1, MLL-AF4, MLL-MSF, MLL-LCX, MLL-LARG, MLL-SEPT6 and MLL-CBL. The frequency of MLL-PTD was 7.1% in adults and 0.9% in children (P<0.001). 11q23 abnormalities were detected in 64% of MLL/t11q23 and in none of MLL-PTD by conventional cytogenetics. There were no differences in remission rate, event-free survival and overall survival between adult MLL-PTD and MLL/t11q23 groups. Adult patients had a significantly poorer outcome than children. The present study showed that cDNA panhandle PCR can identify all rare or novel MLL partner genes. MLL-PTD was rare in childhood AML. MLL(+) adults had a poor outcome with no difference in survival between MLL-PTD and MLL/t11q23 groups.
The MLL (mixed lineage leukemia) gene located at 11q23, is fused to a variety of partner genes through chromosomal translocations in acute leukemias.1, 2 Up to now, more than 40 different MLL partner genes have been identified.3, 4 MLL gene contains 100 kb of DNA, but nearly all breakpoints are clustered within a 8.3 kb region.5 Molecular analysis shows that fusion of the amino terminus of MLL to the carboxy terminus of partner genes generates the critical leukemogenic fusion proteins.2 The most common translocation involving 11q23 in acute myeloid leukemia (AML) is t(9;11)(p22;q23),6 which results in the formation of fusion transcript of MLL-AF9.7 The other common translocations involving 11q23 in AML are t(6;11)(q27;q23), t(10;11)(p12;q23), t(11;19)(q23;p13.1) and t(11;19)(q23;p13.3);8, 9, 10 these translocations result in the formation of fusion transcripts of MLL-AF6, MLL-AF10, MLL-ELL or MLL-ENL, respectively.11, 12, 13, 14 Apart from reciprocal chromosomal translocations, MLL gene is also rearranged to generate a partial tandem duplication of MLL (MLL-PTD) in AML that are cytogenetically silent and also undetectable by fluorescence in situ hybridization (FISH).15, 16
A variety of cytogenetic and molecular techniques can be used to detect MLL gene rearrangements in leukemic cells. The majority of the previous studies on MLL rearrangement were based on preselection of cases with cytogenetically 11q23 abnormalities17, 18, 19, 20 or specific FAB subtypes.21, 22, 23 Moreover, previous studied have shown that a substantial proportion of cases with MLL rearrangements detected by molecular analysis were missed by standard cytogenetic studies or FISH.15, 16, 18, 21, 22, 23, 24 This was especially true in patients who had cytogenetically silent MLL-PTD.15, 16 Furthermore, 11q23 translocations were frequently but not invariably associated with MLL rearrangement.25, 26, 27 In addition, the partner genes of MLL rearrangements have not been well characterized in the majority of previous studies.
Up to now, data comparing the clinical features and outcome between patients with MLL-PTD and other MLL translocations (MLL/t11q23) has not been available. The present study, using a variety of molecular technologies, was conducted to systematically investigate a large cohort of well-defined de novo AML patients. We aimed (1) to determine the frequency of MLL rearrangement, (2) to characterize the MLL fusion partners, (3) to correlate the MLL fusion transcripts with their clinicohematologic features and treatment outcome. To the best of our knowledge, this is by far the most comprehensive study on the MLL rearrangements in a large cohort of de novo AML patients.
Material and methods
Patients and samples
Between 1991 and 2004, 865 adult patients diagnosed with de novo AML at Chang Gung Memorial Hospital, and 123 children diagnosed with de novo AML between 1995 and 2004 at Mackay Memorial Hospital and Chang Gung Children's Hospital were investigated. The study was approved by the Institutional Review Board of Chang Gung Memorial Hospital. Bone marrow samples were obtained with informed consent. The mononuclear cells were isolated from bone marrow samples and cryopreserved until use. Morphologic classification, immunophenotyping and cytogenetic analysis were performed at initial diagnosis as described previously.28 Reverse transcriptase-polymerase chain reaction (RT-PCR) assays for detection of fusion transcripts of AML1-ETO, CBFβ-MYH11, PML-RARα and BCR-ABL were performed on the diagnostic samples as described previously.29 Samples positive for t(8;21)/AML1-ETO (N=104), inv(16)/CBFβ-MYH11 (N=38), t(15;17)/PML-RARα (N=126), or t(9;22)/BCR-ABL (N=2) were excluded from further examination. The remaining 718 unselected consecutive bone marrow samples were examined for MLL rearrangement.
For adult patients, the induction chemotherapy consisted of daunomycin 45 mg/m2/day × 3 and cytarabine 100 mg/m2/day × 7 (3+7), and a (2+5) regimen if no complete remission after first cycle. Before 1995, the (2+5) regimen was given monthly for 3 cycles after remission and then weekly maintenance with 6-thioguanine 80 mg/m2/day D1–4 and cytarabine 60 mg/m2 SC D5. Maintenance was interrupted every 3 months with the (2+5) regimen in which daunomycin was substituted by etoposide (100 mg/m2) when maximum cumulative doses achieved. After 1995, the postremission therapy consisted of monthly cytarabine 1 gm/m2 q12 h × 8 plus daunomycin (45 mg/m2/day) D1–2 or etoposide (100 mg/m2/day) D1–3, for 4–6 cycles. Six patients underwent allogeneic stem cell transplantation. Children with AML were treated with the nationwide Taiwan Pediatric Oncology Group (TPOG) AML 901 protocol (before 1997) and TPOG-AML-97 protocol (after 1997).30
DNA and RNA extraction
Genomic DNA was extracted from frozen bone marrow cells by use of a DNA extraction kit (Puregene Gentra System, Minneapolis, MN, USA) according to the manufacturer's instructions. RNA was extracted and reversely transcribed to cDNA as described previously.28
Detection of MLL rearrangement and common partner genes
Southern blot analysis was performed to detect MLL rearrangement.5, 28 Two sets of multiplex PCR assays were performed with nested protocol, using combination of the same MLL forward primer and different reverse primers labeled with different fluorochromes, one for detection of MLL-AF6, MLL-AF9 and MLL-ENLa1, the other for MLL-ENLa2, MLL-ELL and MLL-PTD.16, 31, 32 The PCR products were further analyzed with GeneScan analysis (ABI 377). The different partners of the MLL gene were identified by their characteristic fluorescent color.
Standard RT-PCR was used for detection of fusion transcripts of MLL-AF1, MLL-AF4 and MLL-AF10 with the MLL forward primer as for multiplex RT-PCR and specific reverse primers for AF1, AF4 and AF10, respectively.12, 33
Cloning of MLL fusion transcripts by cDNA panhandle PCR
cDNA panhandle PCR technology was used to identify the infrequent or unknown MLL partner genes according to the method of Megonigal et al. with minor modification.34, 35 RT-PCR was performed by use of an MLL forward primer and the reverse primers specific for the cloned partner genes to validate that the fusion transcripts were indeed present in the patients’ samples.35, 36
The Fisher's exact test, the χ2 analysis, the unpaired t-test, and the Wilcoxon's rank-sum test were used as appropriate to make comparisons between groups. Overall survival was defined as the length of time from diagnosis to death or last follow-up. Event-free survival was defined as the length of time from diagnosis to the date of failure (induction failure, relapse, or toxicity-related death) for patients who experienced failure or to the date of last contact for all others. Kaplan–Meier analysis was used to evaluate survival. Differences in survival were assessed using the log-rank test. Statistic analyses were performed using a SPSS software version 8.0 for Windows (SPSS Inc., Chicago, IL, USA). In all analyses, P-values were two-tailed and values <0.05 were considered statistically significant.
Incidence of MLL rearrangements and its correlation with FAB subtypes in de novo AML
MLL rearrangement [MLL(+)] was identified in 114 de novo AML patients by using Southern blot analysis. In all, 98 patients were adults and 16 were children. The ages ranged from 15 years to 85 years with a median age of 52 years in adults, and from 1 day to 5.5 years (median 1.3 years) in children. In all, 52 patients were males and 62 were females. MLL(+) occurred in 11.3% and 13.0% of adult and pediatric de novo AML, respectively, that is, 11.5% of total cohort of AML. The frequencies of MLL(+) with respect to FAB subtypes are shown in Table 1. The two MLL(+) M7 patients were both children. MLL(+) AML was most frequently found in AML-M5, both in adults (32.7%) and in children (52.6%).
Identification of common MLL partner genes
Common MLL fusion partners including AF6, AF9, AF10, ELL and ENL as well as PTD of MLL were identified in 107 patients by standard RT-PCR assay or multiplex nested PCR technology (Table 2). MLL-PTD was distributed in all FAB subtypes except M7. MLL-PTD was significantly more prevalent in adults than in children (P<0.001). Of patients with MLL-PTD, 45 had a duplication of exons 2–6 of MLL resulting in fusion of exon 6/exon 2 (e6e2) and the remaining 18 had e8e2. MLL-AF6 was more frequently present in M1 and M4, MLL-AF9 and MLL-AF10 most frequently occurred in M5, and MLL-ELL in M4 or M5.
Table 3 shows the genetic and cytogenetic findings of the 114 MLL(+) patients. None of patients with MLL-PTD had 11q23 abnormalities. Cytogenetic analysis failed to detect 11q23 abnormalities in five of the 13 patients with MLL-AF9, in four of 10 patients with MLL-ENL or MLL-ELL. Moreover, for those with detectable 11q23 abnormalities, their MLL chromosomal partners could not be precisely identified in nine patients.
Identification of rare or novel MLL partner genes
cDNA panhandle PCR technology was able to identify five rare or novel fusion transcripts, the breakpoints of MLL, the partner genes and their chromosomal locations as well as the clinicohematologic features and cytogenetics are shown in Table 4. In addition, the fusion transcripts of MLL-ENL with a novel breakpoint in ENL gene were cloned in two patients by cDNA panhandle PCR.
Correlation of subgroups of MLL fusion transcripts with clinicohematologic features
Viewing that the fusion transcripts of the 51 MLL(+)/t11q23 AML were derived from 12 different partner genes leading to a very small number of patients in each genetic subgroup; we, thus, grouped the MLL fusion transcripts into MLL-PTD and MLL/t11q23. A comparison of the clinicohematologic features between MLL-PTD and MLL/t11q23 groups was made (Table 5). In adult AML, patients with MLL-PTD were significantly older than those with MLL/t11q23 (P<0.001), MLL/t11q23 was associated with a lower platelet count and higher percentage of blasts in bone marrow compared with MLL-PTD group (P=0.045, and P<0.001, respectively). There was no difference in gender, hemoglobin level, white blood cell count, or percentage of blasts in peripheral blood between adult MLL-PTD and MLL/t11q23 groups. The distribution of FAB subtypes was significantly different between the two MLL subgroups (P<0.001). MLL-PTD was highly associated with AML-M1 or M2, whereas MLL/t11q23 was strongly associated with AML-M4 or M5. The distribution of FAB subtypes in patients with MLL/t11q23 was significantly different between children and adults (P=0.020). We again analyzed the correlation of the clinicohematologic features between MLL-PTD and MLL/t11q23 groups in the whole cohort of AML. The results remained similar as observed in adults alone, except for no difference in platelet count between MLL-PTD and MLL/t11q23 (P=0.262).
For the entire cohort of 114 MLL(+) patients, the median survival was 6.7 months with a survival of 3.8 months for MLL-PTD and 9.0 months for MLL/t11q23 patients. Of the 98 adults, 32 patients greater than 65 years old received palliative or no chemotherapy, seven patients presenting with hyperleukocytosis and infections died shortly after presentation prior to the initiation of chemotherapy, another three patients refused chemotherapy; the remaining 56 patients received combination chemotherapy. In all, 36 patients (64%) achieved a complete remission, 61% (19/31) in MLL-PTD group and 68% (17/25) in MLL/t11q23 group (P=0.780). The median overall survival was 11.1 months for adult patients with MLL-PTD compared with 12.0 months for those with MLL/t11q23 (P=0.451, Figure 1a). There was no difference in remission duration (P=0.434) and event-free survival (P=0.466) between MLL-PTD and MLL/t11q23 groups (Figure 1b). When the outcome of adult patients with MLL-AF9 was compared with that of other MLL/t11q23 patients, no difference was observed in the remission duration (P=0.804), event-free survival (P=0.809) and overall survival (P=0.887) between the two groups.
Of the 16 pediatric MLL(+) AML, the one with congenital AML did not receive therapy, all of the remaining 15 children achieved a complete remission, with a median follow-up of 44.2 months, the median event-free survival, and median overall survival were 24.3 months and 27.3 months, respectively. Children with MLL(+) AML had a significantly better outcome in terms of remission rate (P=0.0073), remission duration (P=0.0013), event-free survival (P=0.0013), and overall survival (P=0.0061) than adult MLL(+) AML patients.
AML with 11q23 (MLL) abnormalities comprises one category of recurrent genetic abnormalities in the WHO classification.37 Studies on 11q23 abnormalities in the previous series had been mainly based on cytogenetic analysis. Furthermore, cytogenetics can barely discriminate between 11q23 with MLL rearrangement and 11q23–q25 not actually involving MLL gene.25, 26, 27 FISH analysis with an MLL probe is more sensitive than cytogenetics in detecting MLL(+) cases, however, FISH cannot precisely identify the involved partner genes or MLL-PTD. It highlights that molecular analysis is required for precise identification of 11q23 (MLL) abnormalities. In the present series, the frequency of MLL rearrangement in adult de novo AML detected by Southern blot analysis was higher than the reported incidence of 3–7% detected by cytogenetic analysis;20, 27, 38, 39, 40, 41, 42 the frequency of MLL(+) pediatric AML was comparable to those reported previously.39, 43, 44, 45
By use of a combination of primers for the common MLL fusion partners in the multiplex RT-PCR assays, we could identify the great majority of various MLL fusion transcripts. Further, cDNA panhandle PCR allowed us to identify all the infrequent or novel MLL partner genes; of which, the two novel MLL-CBL and MLL-SEPT6 have recently been reported.35, 36 MLL-MSF, MLL-LCX and MLL-LARG were described by others.46, 47, 48 CBL and LARG were located at 11q23, with LARG being telomeric to the CBL gene;35, 48 MLL-CBL and MLL-LARG were both missed by cytogenetic analysis. Very recently, Meyer et al.,4 using a universal long-distance inverse-PCR approach, were also able to identify unknown MLL partner genes.
MLL-PTD was the most common genetic lesion of adult MLL(+), whereas it was very rare in children. The frequency of MLL-PTD in de novo AML in the present series was 6.4% (63/988) compared with 3.4–5.5% reported by other groups.49, 50, 51 The duplication spanned exons 2–6 or exons 2–8 of MLL with frequent multiple splicing as described by other investigators.16, 49, 50, 51, 52 The frequency of MLL-PTD ranged between 4 and 12% for each FAB subtype, with no difference among FAB subtypes. The MLL/t11q23 group consisted of 12 partner genes: with 5 subtypes of MLL-AF9, MLL-AF10, MLL-ELL, MLL-AF6 and MLL-ENL accounting for 94%, of the remaining seven different MLL fusion transcripts, one subtype occurring in one patient. As demonstrated in the present series and also by others,18, 23, 24 even 11q23 abnormalities were detected, the precise identification of MLL translocation partners was usually difficult.
It has been described that AML with MLL-PTD could be discriminated from AML with MLL/t11q23 based on the global gene expression profiling, suggesting that these two groups of MLL(+) leukemia are biologically different (Schnittger S et al., Blood, 2002; 100: 320a; abstract). The current study showed that the clinicohematologic features with respect to age, distribution of FAB subtype, and percentage of bone marrow blasts were different between MLL-PTD and MLL/t11q23 groups.
Previous studies have shown that AML with 11q23/MLL rearrangements was associated with adverse outcome in both adult and childhood AML.20, 42, 43, 45 We found that children with MLL rearrangement had a significantly higher remission rate, longer remission duration, favorable event-free survival and overall survival than adults. We failed to find a favorable survival in patients with MLL-AF9 as observed by some groups.38, 42, 45 MLL-PTD has been described with an unfavorable outcome in unselected patients with AML or AML with normal karyotypes;49, 50, 51 however, no data comparing the difference in outcome between MLL-PTD and MLL/t11q23 have been available. We found no difference in complete remission rate, remission duration, event-free survival or overall survival between MLL-PTD and MLL/t11q23 subgroups. The definitive assessment of prognostic relevance for each genetic subtype of MLL/t11q23 requires a large number of patients in each subgroup in prospective trials with uniform protocol; however, it is difficult for this relatively rare genetic category of AML.
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We thank Dr Iou-Jih Hung, Dr Chao-Ping Yang and Dr Hung Chang for providing their patients’ samples. We also thank Ms Siew-Hoon Tech for the technical assistance, Ms Hsiu-I Wang for the statistical analysis and Ms Yu-Feng Wang for the secretarial assistance. This work was supported by NHRI-EX90-9011SL, NHRI-EX91-9011SL, NHRI-EX92-9011SL, NHRI-EX93-9011SL from the National Health Research Institute, Taiwan, Grant CMRP860 from Chang Gung Memorial Hospital, Taiwan, and Grant MMH-E-94009 from Mackay Memorial Hospital, Taiwan.
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Acquired persistently complete remission by decitabine-based treatment for acute myeloid leukemia with the MLL-SEPT9 fusion gene
Leukemia & Lymphoma (2019)
International Journal of Laboratory Hematology (2019)
Molecular Case Studies (2018)
Cellular and Molecular Life Sciences (2018)