The Aberrations of Cytogenetics and Molecular Genetics in Core Binding Factor Acute Myeloid Leukemia

Objective:

1. To investgate the clinical, cytogenetic, and molecular genetic characteristics of 873 patients with de novo core binding factor acute myeloid leukemia (CBF-AML).

2. To evaluate the prognostic value of additional chromosome abnormalities, mutations, and the transcriptional levels of RUNX1-RUNX1T1, CBFβ-MYH11 in patients with CBF-AML.

3. To identify up-regulated or down-regulated microRNAs in CBF-AML by the Bead-based miRNA expression profiling and Q-PCR and analyze their biological effects.

Methods

1. All the samples were studied by R-band karyotypic analysis after using direct method and/or short-term culture for chromosome preparation. The clinical, laboratory, cytogenetic and molecular genetic characteristics of CBF-AML were evaluated. A variety of CBF-AML related mutations were evaluated by PCR amplification and direct DNA sequencing, namely: KIT, FLT3-TKD, FLT3-ITD, N-RAS, K-RAS, CBL, JAK2, CEBPA, NPM, ASXL1, IDH1, IDH2, WT1, EZH2, TET2 and DNMT3A.

2. We identified a cluster of up-regulated or down-regulated microRNAs in CBF-AML by the Bead-based miRNA expression profiling and Q-PCR. The pathologic role of these miRs in primary cells and leukemia cell lines of CBF-AML was studied by multiple in vitro medthods.

Results:

1. After reviewing the cytogenetic and molecular analysis database, 873 cases admitted to the Jingsu Institute of Hematology between June 1985 and January 2013 fulfilled WHO-2008 criteria for CBF-AML, including 767 patients with t(8;21) /RUNX1-RUNX1T1 and 106 with inv(16)/t(16;16)/CBFβ-MYH11. This cohort comprises 497 males and 57 females. The median age was 31 years. The patients with inv(16)/t(16;16)/CBFβ-MYH11 had a significantly higher median WBC, Hb, and Blast than those with t(8;21)/RUNX1-RUNX1T1 (P<0.05). About 71.6% of patients with t(8;21) were classified as M2 according to the FAB creteria, while 45.3% of patients with inv(16)/t(16; 16) were classified as M4Eo.

There were 452 (52.1%) CBF-AML patients had at least one additional chromosomal abnormality (ACA) besides t(8;21) or inv(16)/t(16; 16), 72 (8.3%) patients had two or more ACAs. The frequency of t(8;21) patients with ACA was higher than patients with inv(16). The most common ACA in t(8;21)-AML was loss of sex chromosomes (either X or Y) and del(9q), while the most common ACA in inv(16)-AML was +22. There were 63 cases presented with normal karotype and positive fusion transcripts showed by Q-PCR or/and FISH.

Mutation analysis was perfomed in 258 CBF-AML patients for whom genomic DNA and RNA were available. Overall, 138 patients (53.5%) were found to have at least one mutation, classified with: KIT (34.1%), FLT3 (12.5%), TET2 (11.7%), RAS (9.7%), WT1(6.7%), NPM1(3.3%), CBL (3.2%), CEBPA (2.3%), EZH2(1.7%)、ASXL1 (1.7%)、IDH2(1.7%)、DNMT3A (1.7%) and JAK2V617F (1.0%).

2. The mutations in exon 17 of the KIT and FLT3 genes had negative impact on overall survival (OS) and event-free survival (EFS) in CBF-AML patients. However, RAS mutations and ACAs had no impact on the outcome of CBF-AML patients. A higher than 3-log MRD reduction after first consolidation had positive impact on OS, but not on EFS.

3. The bead-based miRNA expression profiling was performed in 157 de novo AML samples. We identified a cohort of up-regulated or down-regulated microRNAs in CBF-AML. The expression level of miR-99a/100 was downregulated in the primary leukemia cells from CBF-AML patients and several CBF-AML cell lines (Skno-1, Kasumi-1, and ME-1) and associated with better outcome. The expression level of miR-130a is upregulated in the primary leukemia cells from CBF-AML patients and CBF-AML cell lines (Skno-1, Kasumi-1, and ME-1).

Conclusion

1. The most common ACAs in t(8;21)-AML was loss of sex chromosomes, while the most common ACA in inv(16)-AML was +22. The most common mutation in t(8;21)-AML patients was KIT mutation (especially exon17),but in inv(16)-AML was exon8.

2. The mutations in exon 17 of the KIT and FLT3 genes had negative impact on the outcome of CBF-AML patients. A higher than 3-log MRD reduction after first consolidation had positive impact on OS of CBF-AML patients.Sex should be considered too.

3. The expression level of miR-99a/100 was downregulated in CBF-AML and associated with better outcome. The expression level of miR-130a is upregulated in CBF-AML and may plays important role in the leukmogenesis of RUNX1-RUNX1T1 by downregulating HOXA10 and PTEN.