ETV6 Aberrations Are a Recurrent Event in Pediatric Acute Myeloid Leukemia with Poor Clinical Outcome
Authors: de Rooij Jasmijn , Beuling Eva , Fornerod Maarten , Obulkasim Askar , Baruchel André , Trka Jan , Reinhardt Dirk , Sonneveld Edwin , Zimmermann Martin , Pieters Rob , van den Heuvel-Eibrink Marry , Zwaan C. Michel 
Published: October 2019

Abstract
Pediatric acute myeloid leukemia (AML) is a heterogeneous disease and 30-40% of the patients still die. Prognosis is dependent on relevant genetic aberrations. Although many driving genetic alterations causing AML have been defined, in ~20% of the pediatric AML patients the oncogenic events remain unidentified. The ETS-Variant gene 6 (ETV6) encodes a transcription factor that functions as a tumor suppressor gene and is required for proper hematopoiesis in the bone marrow niche. Point mutations, deletions and translocations can lead to silencing of the gene, resulting in loss of transcriptional repression activity. ETV6 aberrations strongly associate with leukemia. In pediatric B-cell precursor acute lymphoblastic leukemia, translocation ETV6/RUNX1 occurs in ~25% of cases. Mutations in ETV6 are identified in ~25% of early immature T-cell ALL and also reported as event in adult AML (Van Vlierberghe et al, J Exp Med 2011; Barjesteh van Waalwijk van Doorn-Khosrovani et al, Oncogene 2005). We previously reported that pediatric AML patients can be divided in three clusters based on HOX-expression; (1) low HOXA/B expression, (2) high HOXA and low HOXB expression, and (3) high HOXA/B expression, and identified new repetitive genetic abnormalities in the third cluster, especially in NUP98. Cluster 1 is mainly represented by core-binding factor (CBF) AML, but in ~20% of these cases we did not find specific genetic abnormalities. Helton et al presented ETV6 aberrations in pediatric CBF-AML at ASH 2011, identified with whole genome sequencing, and with poor clinical outcome. We hypothesized that ETV6 aberrations might reduce the number of patients without known driving abnormality, especially in the low HOXA/B cluster.
We screened a large representative de novo pediatric AML cohort for ETV6 mutations in exons 2-8 with direct sequencing, for ETV6 deletions by multiplex ligation-dependent probe amplification and for ETV6 translocations using split signal FISH, and analyzed outcome.
In a well-characterized de novo pediatric AML cases with available gene-expression data, 6/275 (2.2%) patients had mutations affecting the predicted amino acid sequence of ETV6 and one had a silent mutation, 4/259 (1.5%) had an ETV6 deletion and 6/65 (9.2%) patients an MNX1/ETV6 translocation. Additionally, we identified 3 cases with a positive split signal FISH suggestive of a break in which ETV6 is involved, and a similar gene expression profile was found in these three cases. The aberrations of ETV6 were seen in patients of all three HOX-groups; n=9, n=6 and n=4 for cluster 1, 2 and 3 respectively.
In patients with an ETV6 mutation (n=6) or deletion (n=4) 13 and 38 genes, respectively, were significantly up-regulated, including CLDN5,DPEP1 and BIRC7. This is consistent with the up-regulated genes in functional studies silencing ETV6 in LOUCY cells (Van Vlierberghe et al, J Exp Med 2011). High expression of BIRC7 has been associated with poor prognosis in adult acute leukemia (El-Mesallamy et al, Leuk Res 2011).
The median age of patients with an ETV6-mutation or deletion (n=10) was 11.3 years (range 4.0-15.3) and 40% were female. Median WBC was significantly lower (15.1x109/L vs 47.0x109/L, p<0.01) in comparison to other pediatric AML cases. Other cytogenetic aberrations found in the ETV6-mutated or deleted cases were RUNX1/RUNX1T1 (n=3), PML/RARA (n=1), MLL/AF6 (n=1) and one case with an NPM1-mutation. Six out of ten patients encountered a relapse and one patient died of treatment-related mortality. The median age for patients with an ETV6-split signal FISH (n=9) was significantly lower compared to other pediatric AML cases (median 1.1 years vs 9.8, p<0.01), median WBC and sex did not differ. Five out of 9 encountered relapsed/refractory disease whereas 2 cases died of treatment-related mortality. The 3-yr pOS for all ETV6-aberrated patients taken together (n=19) was 37±11% vs 65±3% for the other pediatric AML patients (n=242, p<0.01); the 3-yr pEFS was 26±10% vs 46±3% (p=0.07), and 3-yr pCIR 47±13% vs 37±3% (p=0.24).
We conclude that ETV6 aberrations are rare but recurrent in pediatric AML. ETV6 aberrations predict a poor survival, although there was no evidence for an increased relapse incidence in this small cohort.

Disclosures
No relevant conflicts of interest to declare.