Cytogenetically normal acute myeloid leukemia (CN-AML) comprises nearly half of AML diagnoses annually, and historically, patients in this cytogenetic subgroup have been considered as being at intermediate risk for clinical outcomes. However, the outcome of these patients varies considerably based on the presence or absence of non-random genetic aberrations that can be used as risk stratification factors.1
The partial tandem duplication of the MLL gene (MLL-PTD) was the first somatic mutation associated with a trisomy aberration in AML1 and the first molecular prognostic marker identified in CN-AML2 where it was associated with shorter disease-free survival.2 – 4 We recently reported that the adverse prognostic impact of MLL-PTD in younger (<60 years) adults with de novo CN-AML may be abrogated when more intensive consolidation regimens are implemented.5 However, the patients with MLL-PTD who relapsed generally had a second adverse molecular marker, such as FLT3-ITD.5 In contrast, the clinical and biological impact of MLL-PTD in older patients with CN-AML is not yet established. Thus, we evaluated blood or bone marrow samples from 226 consented newly diagnosed de novo CN-AML patients aged ≥60 years and treated on Cancer and Leukemia Group B (CALGB) protocols for the presence of an in-frame MLL-PTD transcript using PCR/sequencing and confirmed using real-time PCR.2,5 Assays to detect other molecular aberrations with prognostic significance in CN-AML, and genome-wide gene and microRNA-expression profiling, were carried out centrally (Supplementary Information).
Baseline characteristics were compared between MLL-PTD and MLL wild-type (WT) patients using Fisher’s exact test for categorical and the Wilcoxon rank-sum test for continuous variables. Clinical endpoints were defined according to published recommendations (Supplementary Information). Achievement of complete remission was compared between MLL-PTD and MLL-WT patients using the Fisher’s exact test. For time-to-event analyses (that is, disease-free, overall and event-free survival), survival estimates were calculated using the Kaplan–Meier method. Survival data for MLL-PTD and MLL-WT patients were compared using the log-rank test. Statistical analyses were carried out by the Alliance for Clinical Trials in Oncology Statistics and Data Center.
MLL-PTD was present in 13 of the 226 (6%) patients analyzed. The frequency of MLL-PTD in the current study is slightly lower than that in our previous report of younger adults with CN-AML (10%)5 but is in line with reports analyzing exclusively older patients (4%)6 or both younger and older CN-AML patients as a single cohort (7.5%).7 Compared with MLL-WT patients, MLL-PTD patients had lower hemoglobin levels (P = 0.001; Table 1), lacked CEBPA and IDH2 R172 mutations, and 11 of the 13 MLL-PTD patients lacked NPM1 mutations (NPM1-WT) (P = 0.002; Table 1 and Supplementary Figure S1). Approximately one-third of MLL-PTD patients also harbored a FLT3-ITD, which is similar to our previous findings in younger CN-AML adults.5 However, contrary to another study reporting frequent co-existence of mutated RUNX1 and MLL-PTD in AML,8 we found these two molecular aberrations concurrently present only in three older patients with primary CN-AML. Aside from one patient, mutations in the genes encoding chromatin or epigenetic modifiers (DNMT3A, TET2 and ASXL1) appeared to be exclusive of each other in the MLL-PTD patients (Supplementary Figure S1). However, the majority of the older patients with MLL-PTD had at least one additional mutation in an epigenetics and/or chromatin remodeling-associated gene. This further supports the view that altered epigenetics and/or chromatin remodeling may constitute a crucial mechanism in AML leukemogenesis and is consistent with our previous report of an association between MLL-PTD and increased global DNA methylation.9 Importantly, therapeutic targeting of the involved epigenetic and chromatin remodeling factors and/or pathways can potentially prolong survival in these older adults who generally cannot tolerate current chemotherapies and/or transplantation. Other clinical and molecular features studied did not differ significantly between the MLL-PTD and MLL-WT patients (Table 1).
Table 1.
Comparisons of clinical and molecular features and the outcomes in overall patient cohort and within the ELN Intermediate I Genetic Group at presentation by MLL status
| Characteristic | MLL-PTD (n = 13) | MLL-WT (n = 213) | P-valuea |
|---|---|---|---|
| Age, y | 0.25 | ||
| Median | 65 | 68 | |
| Range | 60–77 | 60 – 83 | |
| Sex, no. (%) | 0.08 | ||
| Male | 10 (77) | 106 (50) | |
| Female | 3 (23) | 107 (50) | |
| Race, no. (%) | 0.61 | ||
| White | 13 (100) | 192 (91) | |
| Non-white | 0 (0) | 19 (9) | |
| Hemoglobin, g/dl | 0.01 | ||
| Median | 8.5 | 9.5 | |
| Range | 6.0 – 11.7 | 5.4 – 15.0 | |
| Platelet count, × 109/l | 0.41 | ||
| Median | 53 | 70 | |
| Range | 20 – 246 | 11 – 850 | |
| WBC count, × 109/l | 0.16 | ||
| Median | 9.1 | 28.4 | |
| Range | 1.3 –434.1 | 0.8 – 450.0 | |
| Blood blasts, % | 0.44 | ||
| Median | 32 | 52 | |
| Range | 0 – 96 | 0 – 99 | |
| Bone marrow blasts, % | 0.93 | ||
| Median | 47 | 68 | |
| Range | 17–97 | 4 – 97 | |
| FAB, no. (%)b | 0.68 | ||
| M0 | 0 (0) | 3 (2) | |
| M1 | 3 (33) | 32 (23) | |
| M2 | 1 (11) | 44 (31) | |
| M4 | 3 (33) | 31 (22) | |
| M5 | 2 (22) | 27 (19) | |
| M6 | 0 (0) | 3 (2) | |
| Extramedullary involvement, no. (%) | 2 (15) | 51 (25) | 0.74 |
| ELN Genetic Group, no. (%)c | 0.007 | ||
| Favorable | 1 (8) | 97 (47) | |
| Intermediate-I | 12 (92) | 109 (53) | |
| FLT3-ITD, no. (%) | 1.00 | ||
| Present | 4 (31) | 70 (34) | |
| Absent | 9 (69) | 137 (66) | |
| NPM1, no. (%) | 0.002 | ||
| Mutated | 2 (15) | 126 (61) | |
| Wild type | 11 (85) | 80 (39) | |
| CEBPA, no. (%) | 0.37 | ||
| Mutated | 0 (0) | 26 (13) | |
| Single mutated | 0 | 16 | |
| Double mutated | 0 | 10 | |
| Wild type | 13 (100) | 180 (87) | |
| RUNX1, no. (%) | 0.43 | ||
| Mutated | 3 (25) | 31 (16) | |
| Wild type | 9 (75) | 159 (84) | |
| WT1, no. (%) | 0.18 | ||
| Mutated | 2 (15) | 11 (5) | |
| Wild type | 11 (85) | 195 (95) | |
| FLT3-TKD, no. (%) | 1.00 | ||
| Present | 1 (8) | 22 (11) | |
| Absent | 12 (92) | 184 (89) | |
| IDH1, no. (%) | 1.00 | ||
| Mutated | 1 (8) | 25(12) | |
| Wild type | 12 (92) | 179 (88) | |
| IDH2, no. (%) | 0.51 | ||
| IDH2 | 4 (31) | 47 (23) | |
| R140 | 4 | 38 | |
| R172 | 0 | 9 | |
| Wild type | 9 (69) | 157 (77) | |
| TET2, no. (%) | 1.00 | ||
| Mutated | 3 (23) | 58 (29) | |
| Wild type | 10 (77) | 144 (71) | |
| ASXL1, no. (%) | 0.23 | ||
| Mutated | 4 (31) | 31 (15) | |
| Wild type | 9 (69) | 171 (85) | |
| DNMT3A | 1.00 | ||
| Mutated | 4 (33) | 65 (33) | (mut vs wt) |
| R882 | 3 | 38 | |
| Non-R882 | 1 | 27 | |
| Wild type | 8 (67) | 135 (67) | |
| ERG expression, no. (%)d | 0.47 | ||
| High | 3 (38) | 75 (54) | |
| Low | 5 (62) | 64 (46) | |
| BAALC expression, no. (%)d | 0.28 | ||
| High | 6 (75) | 69 (51) | |
| Low | 2 (25) | 66 (49) | |
| MN1 expression group, no. (%)d | 1.00 | ||
| High | 6 (55) | 69 (51) | |
| Low | 5 (45) | 67 (49) | |
| Complete remission rate, no. (%) | 9 (69) | 142 (67) | 1.00 |
| Disease-free survivale | 0.45 | ||
| Median, y | 0.7 | 0.8 | |
| Disease-free at 3 y, | 22 (3– 51) | 18 (12– 24) | |
| % (95% CI) | |||
| Overall survivalf | 0.38 | ||
| Median, y | 1.1 | 1.1 | |
| Alive at 3 y, % (95% CI) | 15 (2– 39) | 19 (14– 24) | |
| Event-free survival | 0.60 | ||
| Median, y | 0.6 | 0.6 | |
| Event-free at 3 y, | 15 (2– 39) | 12 (8– 16) | |
| % (95% CI) | |||
| ELN Intermediate-I Genetic Group | |||
| No. of patients | 12 | 109 | |
| Complete remission rate, no. (%) | 8 (67) | 62 (57) | 0.56 |
| Disease-free survivalg | 0.88 | ||
| Median, y | 0.6 | 0.6 | |
| Disease-free at 3 y, % (95% CI) | 13 (1– 42) | 10 (4– 19) | |
| Overall survivalh | 0.92 | ||
| Median, y | 1.0 | 0.8 | |
| Alive at 3 y, % (95% CI) | 8 (1– 31) | 10 (5– 17) | |
| Event-free survival | 0.88 | ||
| Median, y | 0.6 | 0.3 | |
| Event-free at 3 y, % (95% CI) | 8 (1– 31) | 6 (2– 11) |
Abbreviations: CI, confidence interval; ELN, European LeukemiaNet; FAB, French-American-British classification; FLT3-ITD, internal tandem duplication of the FLT3 gene; FLT3-TKD, tyrosine kinase domain mutation in the FLT3 gene; WBC, white blood cell.
P-values for categorical variables are from Fisher’s exact test, P-values for continuous variables are from Wilcoxon rank sum test and P-values for time to event variables are from the log-rank test.
FAB are centrally reviewed.
The ELN Favorable Genetic Group is defined as patients with mutated CEBPA or mutated NPM1 without FLT3-ITD; Intermediate-I Genetic Group is defined as patients that are not classified in the Favorable Genetic Group, that is, those with wild-type CEBPA who are either FLT3-ITD-positive with or without an NPM1 mutation or FLT3-ITD-negative with wild-type NPM1.
The median expression value was used as a cut point.
The median follow-up for those who have not had an event is 5.5 years, range: 4.6 – 11.6 years (n = 15).
The median follow-up for those alive is 5.5 years, range: 2.3 – 11.6 years (n = 19).
The median follow-up for those who have not had an event is 7.3 years, range: 5.5 – 7.4 years (n = 3).
The median follow-up for those alive is 7.3 years, range: 5.5 – 7.4 years (n = 3).
Complete remission rates were similar (69 and 67%) between patients with and without MLL-PTD, respectively (P = 1.00; Table 1). Median disease-free survival was 0.7 and 0.8 years for MLL-PTD patients as compared with the MLL-WT patients (P = 0.45) and the percentages of patients disease-free at 3 years were 22% and 18%, respectively. With a median follow-up for those alive at 5.5 years (range: 2.3–11.6 years) (n = 19), overall survival was also not significantly different between MLL-PTD and MLL-WT groups (median OS: 1.1 and 1.1 years, respectively; P = 0.38). By 3 years, the percent of MLL-PTD patients alive was 15%, whereas that of MLL-WT patients was 19% (Table 1).
Recently the molecular heterogeneity with respect to NPM1 and CEBPA mutations and FLT3-ITD has also been used by the European LeukemiaNet (ELN) to classify CN-AML patients into two distinct Genetic Groups for reporting and comparing AML studies.10 When we evaluated the prognostic significance of MLL-PTD in the ELN Genetic Groups, the MLL-PTD patients were more frequently classified in the ELN Intermediate-I Genetic Group, which is defined by the absence of CEBPA mutations and co-presence of mutated NPM1 and FLT3-ITD or presence of NPM1-WT with or without FLT3-ITD (P = 0.007). However, as in the overall analysis, the presence of MLL-PTD did not impact on outcome endpoints in this Genetic Group (Table 1). Although not directly comparable to ours, in a study that included both CN-AML patients and those with abnormal cytogenetics, Schlenk et al.6 found no prognostic impact of MLL-PTD in patients older than 60 years receiving all-trans retinoic acid in addition to intensive chemotherapy. Likewise, Steudel et al.7 reported that MLL-PTD did not impact on outcomes of a cytogenetically heterogeneous group of adults with AML. The lack of prognostic impact by the MLL-PTD in older de novo CN-AML patients may be related to the overall poor prognosis of this age group of patients regardless of the presence or absence of the mutation.
To gain insights into MLL-PTD-associated biology in older CN-AML patients, we performed microarray gene- and microRNA-expression profiling analyses. No gene expression signature was associated with MLL-PTD, despite the established transcriptional and epigenetics roles of normal and abnormal MLL protein in hematopoiesis and leukemia, respectively. However, the current finding is consistent with a study that included adult AML patients with normal and abnormal cytogenetics, in which no gene expression clusters or signatures associated with MLL-PTD were identified.11 Although the reasons of the failure to derive a MLL-PTD-associated gene expression signature are unknown, they might be related to the presence of other molecular markers with stronger biological impact on gene expression.
However, when we examined the expression of the eight MLL probe-sets on the microarray, three (212078_s_at, 212079_s_at and 1565436_s_at) were homologous to the regions encompassing the commonly duplicated exons in MLL-PTD patients (GenBank accession no. NM_005933). These three probe-sets showed evidence of upregulation in MLL-PTD patients relative to the MLL-WT patients (PTD:WT fold-changes of 1.41, 1.74 and 1.61, respectively; P<0.005, each; Supplementary Table S1). In contrast, none of the remaining five probe-sets were differentially expressed between MLL-PTD and MLL-WT patients (Supplementary Table S1). These results correlate well with PCR/sequencing of the same regions of the MLL transcript, validating our MLL-PTD assay.
Contrary to the global gene-expression analysis, a microRNA signature was obtained when comparing MLL-PTD and MLL-WT patients’ microRNA profiles (Table 2; global test of differential microRNA expression, P = 0.01). The microRNA-expression signature comprised 23 probes, representing 21 microRNAs, many of which have reported roles in hematopoiesis and/or leukemia. The minor transcript miR-196b*, underexpressed in MLL-PTD patients (Table 2), is derived from the antisense strand of the miR-196 gene located within the HOXA gene cluster. Although a strong correlation between HOXA9 expression and expression of the major transcript, miR-196b, and an association of upregulation of miR-196b with reduced overall survival in AML have been reported,12 the role of the related miR-196b* is unknown. The most underexpressed microRNA was miR-197 and in silico analyses predict ASXL1, a recently identified adverse prognostic marker in CN-AML (Supplementary ref. no. 8), to be targeted by miR-197.
Table 2.
Differentially expressed microRNAs associated with MLL-PTD in older, de novo CN-AML patientsa
| Target microRNA | Fold-change: MLL-PTD/MLL-WT | P-value |
|---|---|---|
| MicroRNAs downregulated in MLL-PTD patients | ||
| hsa-miR-96 | 0.59 | 0.0013752 |
| hsa-miR-130b*,b | 0.76 | 0.0037523 |
| hsa-miR-185*,b | 0.69 | 0.0009761 |
| hsa-miR-196b*,b | 0.70 | 0.0034725 |
| hsa-miR-197 | 0.65 | 2.45E-05 |
| hsa-miR-205 | 0.70 | 0.0045768 |
| hsa-mir-320a (prec) | 0.67 | 0.0008229 |
| hsa-mir-320a (prec) | 0.67 | 0.0009745 |
| hsa-miR-326 | 0.57 | 0.0015483 |
| hsa-miR-328 | 0.64 | 0.0003135 |
| hsa-mir-329-1 (prec) | 0.61 | 0.0004629 |
| hsa-mir-331 (prec) | 0.55 | 0.0011029 |
| hsa-mir-422a (prec) | 0.68 | 0.002089 |
| hsa-miR-497 | 0.62 | 0.0012652 |
| hsa-miR-596 | 0.67 | 0.0020937 |
| MicroRNAs upregulated in MLL-PTD patients | ||
| hsa-miR-26a | 1.65 | 0.0024263 |
| hsa-miR-26a | 1.75 | 0.0019854 |
| hsa-miR-122 | 1.32 | 0.0049319 |
| hsa-miR-142-5p | 1.56 | 0.0027454 |
| hsa-miR-150 | 2.14 | 0.000397 |
| hsa-miR-185 | 1.52 | 0.0010639 |
| hsa-miR-202 | 1.54 | 0.0017005 |
| hsa-mir-640 (prec) | 1.67 | 0.0006557 |
Abbreviation: prec, the precursor microRNA sequence is detected by the probe.
A total of 23 of 460 microRNA probes tested were significant (P<0.005; global test; P-value = 0.011). Probes are grouped by direction of fold-change and ordered by target microRNA.
An asterisk behind the microRNA’s symbol indicates that this microRNA is the minor sequence generated from the antisense strand; the asterisk is part of the standard nomenclature for naming microRNAs.
The most overexpressed was miR-150 (2.1-fold); this microRNA targets c-MYB translation associated with altered erythroid–megakaryocyte progenitor commitment and B-cell maturation.13 WT MLL and c-MYB proteins functionally interact to regulate HOXA9 gene expression,14 a known transcriptional target of MLL-PTD. Another overexpressed microRNA was miR-142, previously reported to be a transcriptional target of the MLL protein.15
We conclude that MLL-PTD does not have prognostic impact in older CN-AML patients treated with cytarabine/anthracyline-based chemotherapy. This may be related to the overall poor prognosis in this age group and/or simultaneous presence of other genetic aberrations that have stronger clinical impact that masks the influence MLL-PTD may have on outcome. Our work contributes to the understanding of the possible role of microRNAs in older AML by reporting the first microRNA-expression signature associated with MLL-PTD. The hope is that these data will aid in the development of novel approaches that improve the otherwise poor outcome of these older patients.
Supplementary Material
Acknowledgments
We thank Ms Donna Bucci of the CALGB Leukemia Tissue Bank and the Ohio State University Comprehensive Cancer Center’s Nucleic Acid and Microarray Shared Resources for their technical support. We thank participating institutions, medical professionals and AML patients for their valuable involvement with this study. This work was supported in part by the National Cancer Institute (Bethesda, MD, USA) Grants CA140158, CA101140, CA114725, CA31946, CA33601, CA16058, CA77658, CA089341 and CA129657, the Coleman Leukemia Research Foundation (to Dr Bloomfield) and the Deutsche Krebshilfe–Dr Mildred Scheel Cancer Foundation (to Dr Becker).
Footnotes
CONFLICT OF INTEREST
The authors declare no conflict of interest.
Supplementary Information accompanies the paper on the Leukemia website (http://www.nature.com/leu)
Contributor Information
MA Caligiuri, Email: michael.caligiuri@osumc.edu.
CD Bloomfield, Email: clara.bloomfield@osumc.edu.
References
- 1.Mrózek K, Marcucci G, Paschka P, Whitman SP, Bloomfield CD. Clinical relevance of mutations and gene-expression changes in adult acute myeloid leukemia with normal cytogenetics: are we ready for a prognostically prioritized molecular classification? Blood. 2007;109:431–448. doi: 10.1182/blood-2006-06-001149. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Caligiuri MA, Strout MP, Lawrence D, Arthur DC, Baer MR, Yu F, et al. Rearrangement of ALL1 (MLL) in acute myeloid leukemia with normal cytogenetics. Cancer Res. 1998;58:55–59. [PubMed] [Google Scholar]
- 3.Schnittger S, Kinkelin U, Schoch C, Heinecke A, Haase D, Haferlach T, et al. Screening for MLL tandem duplication in 387 unselected patients with AML identify a prognostically unfavorable subset of AML. Leukemia. 2000;14:796–804. doi: 10.1038/sj.leu.2401773. [DOI] [PubMed] [Google Scholar]
- 4.Döhner K, Tobis K, Ulrich R, Fröhling S, Benner A, Schlenk RF, et al. Prognostic significance of partial tandem duplications of the MLL gene in adult patients 16 to 60 years old with acute myeloid leukemia and normal cytogenetics: a study of the Acute Myeloid Leukemia Study Group Ulm. J Clin Oncol. 2002;20:3254–3261. doi: 10.1200/JCO.2002.09.088. [DOI] [PubMed] [Google Scholar]
- 5.Whitman SP, Ruppert AS, Marcucci G, Mrózek K, Paschka P, Langer C, et al. Long-term disease-free survivors with cytogenetically normal acute myeloid leukemia and MLL partial tandem duplication: a Cancer and Leukemia Group B Study. Blood. 2007;109:5164–5167. doi: 10.1182/blood-2007-01-069831. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Schlenk RF, Döhner K, Kneba M, Götze K, Hartmann F, Del Valle F, et al. Gene mutations and response to treatment with all-trans retinoic acid in elderly patients with acute myeloid leukemia. Results from the AMLSG Trial AML HD98B. Haematologica. 2009;94:54–60. doi: 10.3324/haematol.13378. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Steudel C, Wermke M, Schaich M, Schäkel U, Illmer T, Ehninger G, et al. Comparative analysis of MLL partial tandem duplication and FLT3 internal tandem duplication mutations in 956 adult patients with acute myeloid leukemia. Genes Chromosomes Cancer. 2003;37:237–251. doi: 10.1002/gcc.10219. [DOI] [PubMed] [Google Scholar]
- 8.Schnittger S, Dicker F, Kern W, Wendland N, Sundermann J, Alpermann T, et al. RUNX1 mutations are frequent in de novo AML with noncomplex karyotype and confer an unfavorable prognosis. Blood. 2011;117:2348–2357. doi: 10.1182/blood-2009-11-255976. [DOI] [PubMed] [Google Scholar]
- 9.Whitman SP, Hackanson B, Liyanarachchi S, Liu S, Rush LJ, Maharry K, et al. DNA hypermethylation and epigenetic silencing of the tumor suppressor gene, SLC5A8, in acute myeloid leukemia with the MLL partial tandem duplication. Blood. 2008;112:2013–2016. doi: 10.1182/blood-2008-01-128595. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Döhner H, Estey EH, Amadori S, Appelbaum FR, Büchner T, Burnett AK, et al. Diagnosis and management of acute myeloid leukemia in adults: recommendations from an international expert panel, on behalf of the European LeukemiaNet. Blood. 2010;115:453–474. doi: 10.1182/blood-2009-07-235358. [DOI] [PubMed] [Google Scholar]
- 11.Bullinger L, Döhner K, Bair E, Fröhling S, Schlenk RF, Tibshirani R, et al. Use of gene-expression profiling to identify prognostic subclasses in adult acute myeloid leukemia. N Engl J Med. 2004;350:1605–1616. doi: 10.1056/NEJMoa031046. [DOI] [PubMed] [Google Scholar]
- 12.Popovic R, Riesbeck LE, Velu CS, Chaubey A, Zhang J, Achille NJ, et al. Regulation of mir-196b by MLL and its overexpression by MLL fusions contributes to immortalization. Blood. 2009;113:3314–3322. doi: 10.1182/blood-2008-04-154310. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.García P, Frampton J. Hematopoietic lineage commitment: miRNAs add specificity to a widely expressed transcription factor. Dev Cell. 2008;14:815–816. doi: 10.1016/j.devcel.2008.05.011. [DOI] [PubMed] [Google Scholar]
- 14.Jin S, Zhao H, Yi Y, Nakata Y, Kalota A, Gewirtz AM. c-Myb binds MLL through menin in human leukemia cells and is an important driver of MLL-associated leukemogenesis. J Clin Invest. 2010;120:593–606. doi: 10.1172/JCI38030. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.Guenther MG, Jenner RG, Chevalier B, Nakamura T, Croce CM, Canaani E, et al. Global and Hox-specific roles for the MLL1 methyltransferase. Proc Natl Acad Sci USA. 2005;102:8603–8608. doi: 10.1073/pnas.0503072102. [DOI] [PMC free article] [PubMed] [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.