Milea Timbergen

92 Abstract Introduction The majority of desmoid-type fibromatosis (DTF) tumours harbour a β-catenin mutation, affecting specific codons in CTNNB1 exon 3. S45F tumours are reported to have a higher chance of recurrence after surgery and more resistance to systemic treatments compared to wild-type (WT) and T41A tumours. The aim of this pilot study was to examine the genome- wide DNA methylation profiles of S45F and T41A mutated DTF, to explain the observed differences in clinical behaviour between these DTF subtypes. Material and methods Genome-wide analysis of DNA methylation was performed using MeD-seq on formalin fixed, paraffin embedded primary DTF samples harbouring a S45F (n = 14) or a T41A (n = 15) mutation. Differentially methylated regions (DMRs) between S45F and T41A DTF were identified and used for a supervised hierarchical cluster analysis. DMRs with a fold change ≥1.5 were considered to be differentially methylated and differences between S45F and T41A tumours were quantitatively assessed. The effect of DMRs on the expression of associated genes was assessed using an mRNA expression dataset. Protein-protein interactions between wild-type β-catenin and mutant variants and DNA methyltransferase 1 (DNMT1) were examined by immunoprecipitation experiments. Results MeD-seq analyses indicated 354 regions that displayed differential methylation. Cluster analysis yielded no distinct clusters based on mutation, gender, tumour site or tumour size. A supervised clustering based on DMR between small (≤34 mm) and large (>87 mm) DTF distinguished the two groups. Only ten DMRs displayed a fold change of ≥1.5 and six of them were found associated with of the following genes: NLRP4 , FOXK2 , PERM1 , CCDC6 , NOC4L and DUX4L6 . The effects of DMR on gene expression yielded a significance difference (p < 0.05) in the expression between S45F and T41A for CCDC6 and FOXK2 but not for all Affymetrix probes sets used to detect these genes. Immunoprecipitation did not reveal an association of wild-type β-catenin or mutant variants with DNMT1. Conclusion This study demonstrated that S45F and T41A DTF tumours did not exhibit gross differences in DNA methylation patterns. This implies that distinct DNA methylation profiles are not the sole determinant for the divergent clinical behaviour of different DTF mutant subtypes. 4