Acute Myeloid Leukemia with Isolated Trisomy 4 Is Characterized By Frequent Mutations of TET2 and a Distinct DNA Methylation Signature

Background: Trisomy 4 (+4) is a rare cytogenetic abnormality found in less than 1% of acute myeloid leukemia (AML) either as sole abnormality (isolated AML+4, iAML+4) or associated with other genetic lesions. However, iAML+4 is inadequately studied due to its rarity and little is known about its molecular profile.

Aim: The aim of this study was to characterize the molecular profile of iAML+4, including gene mutations, gene expression and DNA methylation changes.

Methods: For the molecular characterization, we have performed targeted amplicon sequencing (n=68 target genes), gene expression profiling (SCRB-seq) and DNA methylation profiling (Illumina Methylation EPIC arrays) on 11 iAML+4 patients from German AML study groups (AML-CG 1999 and AML-CG 2008) and 105 patients with cytogenetically normal AML (CN-AML).

Results: By targeted sequencing, 35 mutations were detected in 11 genes with a median of 3 mutations per patient (range 1-7, Figure 1A). Recurrently mutated genes were TET2 (n=9 patients), NPM1 (n=5) and FLT3-ITD (n=4), DNMT3A (n=2), and WT1 (n=2). Of note, in 8/9 patients with TET2 mutations, a truncating mutation has been detected. Thus, the frequency of truncating TET2 mutations was exceptionally high (8/11; 73%) in iAML+4, as compared to CN-AML with 12/105 patients mutated (11%, P=2.4e-5, Fisher exact test), which is in line with previous findings in the literature. Moreover, 3 patients harbored multiple TET2 mutations. We have further performed targeted amplicon sequencing in 8 AML patients with non-isolated +4, where trisomy 4 was present along with other cytogenetic abnormalities. In non-isolated +4 AML, TET2 mutations were less frequently found, as compared to iAML+4 (2/8, 25%, P=0.07). Gene expression profiling revealed a distinct gene expression pattern in iAML+4 as compared to CN-AML patients. Across all chromosomes, differentially expressed genes were detected; with the highest numbers of overexpressed genes located on chromosome 4, followed by chromosomes 2, 12, 17 and 18, as well as a substantial number of downregulated genes, particularly on chromosomes 11 and 19, indicating a trans-regulatory effect of +4 (Figure 1B). DNA methylation analysis revealed a specific hypermethylation signature among iAML+4 patients, as compared to CN-AML (Figure 1C). A global increase of CpG methylation levels in iAML+4 may be likely due to the high frequency of truncating TET2 mutations, as TET2 is known to be involved in hydroxymethylation of the DNA. However, the hypermethylation signature was confirmed even when comparing iAML+4 to CN-AML within TET2 mutated patient samples, indicating DNA hypermethylation beyond the effect of truncating TET2 mutations. Hierarchical clustering of iAML+4 and CN-AML samples resulted in a separate cluster with a distinct hypermethylation signature, which is strongly enriched for iAML+4 patients. Of note, besides TET2 truncating variants, no additional mutation was found to be enriched in that cluster, indicating that +4 is the major (cyto) genetic driver mediating this pattern.

Conclusion: Our comprehensive multi-omics analysis revealed a homogenous molecular profile of iAML+4 with high frequency of truncating TET2 mutations indicating oncogenic cooperativity, a global pattern of differential gene expression as well as a uniform DNA hypermethylation signature, which is generally not associated with truncating TET2 mutations but a specific feature of trisomy 4 in AML.

Metzeler:Jazz Pharmaceuticals: Consultancy; Novartis: Consultancy; AbbVie: Honoraria; Celgene/BMS: Consultancy, Honoraria, Research Funding; Daiichi Sankyo: Honoraria; Pfizer: Consultancy; Astellas: Honoraria.