Background
Acute myeloid leukemia (AML) represents a group of myeloid malignancies with remarkably heterogeneous outcomes [
1]. Finding effective prognostic biomarkers has been one of the most urgent clinical needs and research hotspots. So far, a few prognosticators have been established, including mutations in
NPM1 and double
CEBPA that are associated with favourable outcomes; whereas
FLT3-
ITD is associated with poor prognosis. High expression levels of
WT1 [
2],
miR-
155 [
3,
4],
ERG [
5,
6],
BAALC [
6], and
MN1 [
7] have also been shown to be poor prognostic factors in AML.
V-ets avian erythroblastosis virus E26 oncogene homolog 2 (
ETS2), a downstream target for both the Ras/Raf/MAP kinase and phosphatidylinositol 3-kinase/Akt pathways.
ETS2 is one of the founder members of the E26 transformation-specific (
ETS) family located on human chromosome 21 [
8].
ERG, one of the classic prognostic markers in AML, also belongs to the
ETS family.
ETS2 and
ERG had been shown to be overexpressed in AML patients with complex karyotypes involving chromosome 21 [
9]. Although
ETS2 was initially characterized as a proto-oncogene acute megakaryocytic leukemia (AMKL) [
10], however, the clinical impact of
ETS2 expression in AML remains unknown.
In recent years, many studies suggest that
ETS2 exhibit both tumor-promoting and tumor-suppressive effects in malignancies. For example,
ETS2 has been found to be an oncogene in patients with AML [
11], but it also has tumor-suppressive effects in non-small cell lung cancer [
12]. Here, we demonstrate
ETS2
high as an adverse prognostic biomarker for AML based on analysis of two separate datasets and indicate
ETS2
high may guide treatment decisions towards allogeneic HCT; we also explore the distinctive gene/microRNA patterns associated with
ETS2 expression.
Methods
Patients
The first cohort was derived from The Cancer Genome Atlas (TCGA) dataset, including 200 clinically annotated adult de novo AML samples [
13]. In this cohort, RNA sequencing for 179 samples and microRNA sequencing for 194 samples had been previously reported. Detailed descriptions of clinical and molecular characteristics were also provided. All these data were publicly accessible from the TCGA website. The study was approved by the human studies committee at Washington University with written informed consent obtained from all patients.
The second cohort was derived from a whole AML cohort (n = 329) diagnosed and collected at Erasmus University Medical Center (Rotterdam) between 1990 and 2008, approved by the institutional review boards at Weill Cornell Medical College and Erasmus University Center, and all subjects provided written informed consent in accordance with the Declaration of Helsinki. Microarray expression profiles were obtained by Affymetrix Human Genome 133 plus 2.0 and U133A Gene Chips from
GSE6891 data. All experiments’ design, quality control and data normalization were in line with the standard Affymetrix protocols. All clinical, cytogenetic and molecular information as well as microarray data of these patients were publicly accessible at the Gene Expression Omnibus (GSE6891,
http://www.ncbi.nlm.nih.gov/geo) [
14]. All patients were uniformly treated under the study protocols of Dutch-Belgian Cooperative Trial Group for Hematology Oncology (HOVON, details of therapeutic protocol available at
http://www.hovon.nl).
Statistical analyses
OS was defined as the time from the date of diagnosis to death due to any cause. EFS was defined as the time from the date of diagnosis to removal from the study due to the absence of complete remission, relapse or death. RFS was defined as the time from the date of diagnosis to removal from the study due to relapse.
Patients with higher than median ETS2 expression values of all patients were classified as ETS2
high, and those with lower than median expression values were classified as ETS2
low. To investigate the associations between ETS2 expression levels and clinical, molecular characteristics, the Fisher exact and Wilcoxon rank-sum tests were used for hypothesis testing with categorical and continuous variables, respectively. The associations between ETS2 expression and the OS, EFS and RFS were analyzed by the Kaplan–Meier method and the log-rank test. Multivariate Cox proportional hazard models were employed to study the associations between ETS2 expression levels and OS, EFS and RFS in the presence of other known risk factors. Student’s t test and multiple hypothesis correction (False Discovery Rate, FDR) was used to identify different gene/microRNA between ETS2
high and ETS2
low groups. The statistical cutoff values were an absolute fold-change (FC) ≥1.5 and an adjusted P value ≤0.05. All analyses were performed by the R 3.1.1 software packages.
Discussion
Identifying the prognostic factors for AML is important for the development of new targeted therapies and risk-stratified treatment strategies. Recent studies had shown that high expression of
ERG and
ERG amplification, the most frequent copy-number alteration (CNA), are all the worse prognostic markers in AML patients [
5,
6,
33].
ETS2, one of the members of the
ETS family as
ERG, was previously characterized as a proto-oncogene in AMKL children that is Down-syndrome and non-Down-syndrome-related [
10], but the expression and clinical prognosis of
ETS2 in AML remains unknown. Here, we have demonstrated the aberrant expression of
ETS2 in AML patients. First, we found that
ETS2 expression was up-regulated in AML cohorts and was overexpressed in the NCCN intermediate- and poor-risk groups of patients, compared to the good-risk group. These findings indicated that
ETS2 might promote leukemogenesis. We also found that
ETS2 showed higher expression in monocytes using publicly available expression data which suggest that
ETS2 might play an important role in the function of monocytes [
34] (Additional file
1: Figure S1). Second, in the first cohort, our study demonstrated that
ETS2
high was associated with shorter OS and EFS. Notably,
ETS2
high patients had longer OS and EFS after receiving allogeneic HCT than chemotherapy-only, but similar differences between treatment modules were not observed in
ETS2
low patients. Its presence may direct treatment decisions towards allogeneic HCT.
To further confirm the prognostic significance of ETS2, we analyzed the second cohort of uniformly treated AML patients. ETS2
high also acted as an independent poor prognostic factor in the entire cohort, NCCN Intermediate-risk subgroup, CN-AML subgroup, as well as the ELN Intermediate-I subgroup. The above results denoted that ETS2
high was an independent, poor prognostic factor in AML. It could be employed to improve the risk stratification of ELN Intermediate-I category and NCCN Intermediate-Risk group.
Gene and microRNA-expression profiles derived from the first cohort gave us some insight regarding the role of
ETS2 in AML leukemogenesis. Tumor protein 53 (
TP53) is one of the most frequently inactivated tumor suppressor genes in human cancer and its mutations predict a poor prognosis in patients with acute myeloid leukemia (AML) [
35]. Recent studies have shown that mutations in the
TP53 (mTP53) protects
ETS2 from degradation and mTP53 disrupts ETS family target gene regulation, promoting cancer [
36]. In our study, we found that
ETS2
high was associated with mTP53.
The expression of
miR-
155 has been found to be independently associated with poor clinical outcome in AML [
3,
4]. In addition, we found that
ETS2
high was associated with over-expression of
miR-
155HG,
miR-
155-
3p and
miR-
155-
5p. This result is in accordance with recent studies which have found that
ETS2 is an important transcription factor regulating
miR-
155 [
37].
Conclusions
In summary, ETS2
high is an independent poor prognostic factor in AML patients and its presence should favor allogeneic HCT over chemotherapy-only in AML. In AML patients, distinctive gene/microRNA expression profiles associated with ETS2 expression may explain the role of ETS2 in the leukemogenic process.
Authors’ contributions
JS, LF, KX and XK designed the study and wrote the manuscript. HF, QW and YP performed statistical analyses. KX, LZ and JQ analyzed the data. XK, KX and JS coordinated the study over the entire time. All authors read and approved the final manuscript.