Recurrent CTNNB1 mutations in craniofacial osteomas

Abstract

Osteoma is a benign bone forming tumor predominantly arising on the surface of craniofacial bones. While the vast majority of osteomas develops sporadically, a small subset of cases is associated with Gardner syndrome, a phenotypic variant of familial adenomatous polyposis caused by mutations in the APC gene resulting in aberrant activation of WNT/β-catenin signaling. In a sequencing analysis on a cohort of sporadic, non-syndromal osteomas, we identified hotspot mutations in the CTNNB1 gene (encoding β-catenin) in 22 of 36 cases (61.1%), harbouring allelic frequencies ranging from 0.04 to 0.53, with the known S45P variant representing the most frequent alteration. Based on NanoString multiplex expression profiling performed in a subset of cases, CTNNB1-mutated osteomas segregated in a defined "WNT-cluster", substantiating functionality of CTNNB1 mutations which are associated with β-catenin stabilization. Our findings for the first time convincingly show that osteomas represent genetically-driven neoplasms and provide evidence that aberrant WNT/β-catenin signaling plays a fundamental role in their pathogenesis, in line with the well-known function of WNT/β-catenin in osteogenesis. Our study contributes to a better understanding of the molecular pathogenesis underlying osteoma development and establishes a helpful diagnostic molecular marker for morphologically challenging cases.

Fig. 1. Results of the mutational analysis of the CTNNB1 gene in 36 osteomas.

A Clustered mutational profile of osteomas (n = 36). Alterations in CTNNB1 (rows) are indicated for each sample (columns). Clinicopathological information is summarized according to the legend. Cases with indicated sample IDs (framed squares) were included in the NanoString analysis. AF allelic frequency. B Schematic overview of β-catenin indicating relevant protein domains, phosphorylation sites, interaction partners and associated cellular functions. Detected mutations in the N-terminal domain and the Armadillo repeat region are indicated in red along with their frequency. NTD N-terminal domain, CTD C-terminal domain.

Fig. 2. Radiological and histological features of an example of osteoblastoma-like osteoma.

A CT scan and (B, C) H&E stained aspects of an osteoma with osteoblastoma-like features arising in the ethmoidal sinus involving the nasal cavity. Conspicuous osteoblastic and osteoclastic activity is observed against the background of a well-vascularized and moderately cellular and fibrous stroma in the marrow spaces (B, original magnification 10x; C, original magnification 20x). D Osteoid matrix-rimming osteoblasts display nuclear β-catenin positivity (original magnification 20x).

Fig. 3. NanoString multiplex gene expression profiling employing the human PanCancer Pathways CodeSet covering 770 genes.

A Exploratory heatmap of the normalized expression data generated via unsupervised clustering clearly distinguishes CTNNB1 mutated and CTNNB1 wild type osteomas according to their mRNA expression profile (red indicates high expression; green indicates low expression). B Heatmap of pathway scores presenting an overview of 13 essential hallmark-associated canonical pathways (MAPK, STAT, PI3K, RAS, Cell Cycle, Apoptosis, Hedgehog, WNT, Notch, DNA Damage Control, Transcriptional Regulation, Chromatin Modification, and TGF-β), indicating that CTNNB1 mutated osteomas exhibit, among others, similar WNT/β-catenin pathway activation profiles, as compared to the CTNNB1 wild type group (red indicates high pathway scores; green indicates low scores). C Volcano plot displaying each gene’s –log10 (p value) and log2 fold change as calculated from the differential expression profile in the set of CTNNB1 mutated osteomas vs. baseline expression in the set of CTNNB1 wild type cases. Statistically significantly regulated genes are shown in the upper part of the plot above the horizontal line, differentially expressed genes fall to either side. Genes related to WNT/β-catenin pathway activation profile are highlighted in red.