Milea Timbergen

69 Hierarchical cluster analyses based on Wnt target gene expression does not discriminate between wild-type and CTNNB1 mutant desmoids The total dataset contained 54613 probes, of which 107 probes were selected as mammalian Wnt targets and 47 were selected as mesenchymal Wnt targets. The hierarchical clustering using mammalian Wnt targets (Figure 2A) did not discriminate WT and mutated DTF (T41A and S45F) tumours, nor showed a discriminative pattern based on sex, age at the time of diagnosis, tumour size or tumour status (primary or recurrent tumour). An additional hierarchical clustering, using expression data from all 128 desmoid samples (including WT tumours as well as T41A, S45F, S45P mutated tumours, rare mutations classified as “other”, and tumours with an unknown mutation type), showed similar results (Supplemental Figure 1A). Cluster analyses using putative mesenchymal Wnt targets 36 in a subgroup containing samples that are CTNNB1 WT, T41A and S45F (Figure 2B) and the cluster analysis using all samples (Supplemental Figure 1B), also showed that the desmoid samples did not cluster according to CTNNB1 mutational status, neither did the observed cluster appear to be driven by clinicopathological parameters like sex, age at time of diagnosis, tumour size or tumour status (primary or recurrent tumour). Discussion This study evaluated whether the potentially more aggressive clinical behaviour of S45F mutated DTF, compared to T41A mutants and WT desmoids, could be explained by a differential Wnt signalling activity. As aberrant Wnt/β-catenin signalling plays a pivotal role in the initiation and development of DTF, variable activity of Wnt signalling may affect cancer-related biological processes like cancer stem cell maintenance and invasiveness in a different manner 37-39 . We therefore examined and compared Wnt target expression levels using different independent DTF sample sets that included DTF designated as WT as well as T41A and S45F CTNNB1 mutants. No evidence was found that WT tumours, T41A and S45F tumours have a differential activation of Wnt/β-catenin signalling. This finding contrasts with Hamada et al. who reported a significant increase of AXIN2 , CCND1 and c-MYC transcript levels in S45F DTF isolated single cell lines compared to human skin fibroblasts and WT and T41A cells 33 . A study conducted by Meneghello et al. showed upregulated (two to six fold) mRNA expression of AXIN2 measured by RT-PCR but also reported that desmoid cells had downregulated mRNA expression of CCND1 (two-four fold) when comparing DTF samples with connective tissue of non-desmoid patients 17 . In addition, Jilong et al. compared β-catenin 3