TET2 Mutations Are Highly Associated with RUNX1-RUNX1T1 Translocations and NPMc+ in Childhood AML: a Report from Children's Oncology Group AAML03P1, AAML0531 and NCI/COG Target AML Initiative
Authors: Kutny Matthew A. , Alonzo Todd A. , Wang Yi-Cheng , Ries Rhonda , Farrar Jason E. , Guidry Auvil Jaime M. , Smith Malcolm A. , Gerhard Daniela S. , Davidsen Tanja M. , Gesuwan Patee , Hermida Leandro C. , Marra Marco A , Mungall Andrew J. , Moore Richard , Long William , Ma Yussanne , Zong Stuart , Kolb E. Anders , Gamis Alan S. , Meshinchi Soheil 
Published: October 2019

Abstract
Abnormalities of epigenetic regulatory genes including DNMT3A, IDH1, IDH2 and TET2 are common in adults with AML. The prevalence of these abnormalities appears to increase with older age, but their impact in pediatric AML is less certain. The Children's Oncology Group (COG) has previously reported that mutations of DNMT3A, IDH1 and IDH2 are very rare in pediatric patients (Ho et al Leukemia 2010; Ho et al Pediatric Blood and Cancer 2011). In the current study we examined the prevalence and prognostic significance of TET2 gene mutations in a large cohort of pediatric patients with AML.
We screened for genomic mutations in the TET2 gene using DNA extracted from diagnostic specimens of 949 pediatric de novo AML patients treated on the COG studies AAML03P1 (N=226) and AAML0531 (N=723). The COG trial AAML03P1 was a phase III pilot study with non-random assignment to gemtuzumab ozogamicin in combination with multi-agent chemotherapy. AAML0531 was a phase III trial with randomization to gemtuzumab ozogamicin. The entire coding sequence of TET2 and was amplified and sequenced. Mutational data was correlated with clinical characteristics and outcome data in both univariable and multivariable analyses.
Mutations of the TET2 gene were found in 26 of 949 samples (2.7%). Mutations were found across TET2 gene exons 3-11 from amino acid 121 to 1920. There were 14 missense mutations, 8 nonsense mutations, 2 insertion/deletion (in/del) resulting in frame shift and 2 samples with multiple mutations (1 with missense mutation and in/del frameshift; 1 with nonsense mutation and in/del frameshift).
There was no significant difference in the age, gender, race or ethnicity of patients with or without TET2 mutations. Patients with TET2 mutations had a higher prevalence of normal cytogenetics than those without a TET2 mutation (44% vs. 22%, P=0.011). There was also a significant association of TET2 mutations with core binding factor leukemia, although the direction of the association differed between t(8;21) and inv(16). Patients with TET2 mutations compared to those without these mutations were more likely to have t(8;21) (53% vs. 17%, P= 0.021) but less likely to have inv(16) (0% vs. 21%, P=0.039). Molecular mutations currently used in risk stratification of pediatric AML were evaluated. There was no association with FLT3/ITD or mutations of CEBPA, but there was a strong association with NPMc+ which is known to confer a favorable prognosis. TET2 mutant patients had a 32% prevalence of NPMc+ compared to only 7% NPMc+ among TET2 non-mutant (P=<0.001). With cytogenetic and molecular data taken together, there was no difference in distribution of TET2 mutant patients among the 3 genetic risk groups utilized by the COG (Low Risk= t(8;21), inv(16), NPMc+, or CEBPA mutant; High Risk= Monosomy 7, Monosomy 5/del 5q, or FLT3/ITD; Standard Risk= all others).
Comparing patients with and without TET2 mutations, there was no significant difference in overall survival (OS) (5 yr: 77% vs. 65%, P=0.194), event free survival (EFS) (5 yr: 58% vs. 50%, P=0.411) or relapse risk (40% vs. 37%, P=0.772). We performed multivariable analysis of OS and EFS for TET2 mutation status, risk grouping, NPMc+ status and treatment exposure to gemtuzumab ozogamicin. TET2 mutation status was not predictive of outcome in this multivariable analysis. However, genetic risk group was significantly associated with both OS and EFS and treatment with gemtuzumab ozogamicin was associated only with EFS (HR 0.824 for patients treated with gemtuzumab ozogamicin). Due to the strong association of TET2 mutation and NPMc+, we further evaluated Kaplan Meier estimates of EFS of the 4 groups of patients stratified by both TET2 mutation and NPMc+ status and determined that co-occurrence of TET2 mutation does not modify the clinical significance of NPMc+. (Figure 1)
This study demonstrates that TET2 gene mutations are not common events in childhood AML but are highly associated with NPMc+ and may hint at potential cooperation of genetic and epigenetic factors that mediate myeloid leukemogenesis.
The authors would like to gratefully acknowledge the important contributions of the late Dr. Robert Arceci to the AML TARGET initiative.

Disclosures
Off Label Use: Arsenic Trioxide in pediatric patients with newly diagnosed APL. Gemtuzumab Ozogamicin in Pediatric AML..