Genetic basis of calcifying cystic odontogenic tumors

Abstract

Calcifying cystic odontogenic tumors (CCOTs) are benign cystic tumors that form abnormally keratinized ghost cells. Mutations in CTNNB1, which encodes beta-catenin, have been implicated in the development of these tumors, but a causal relationship has not been definitively established. Thus, mutational hot spots in 50 cancer genes were examined by targeted next-generation sequencing in 11 samples of CCOT. Mutations in CTNNB1, but not in other genes, were observed in 10 of 11 cases. These mutations constitutively activate beta-catenin signaling by abolishing the phosphorylation sites Asp32, Ser33, or Ser37, and are similar to those reported in pilomatrixoma and adamantinomatous craniopharyngioma. In contrast, BRAF or NRAS mutations were observed in 12 and two control samples of ameloblastoma, respectively. In HEK293 cells, overexpression of mutated CTNNB1 also upregulated hair keratin, a marker of ghost cells. Furthermore, ghost cells were present in two cases of ameloblastoma with BRAF and CTNNB1 mutations, indicating that ghost cells form due to mutations in CTNNB1. The data suggest that mutations in CTNNB1 are the major driver mutations of CCOT, and that CCOT is the genetic analog of pilomatrixoma and adamantinomatous craniopharyngioma in odontogenic tissue.

Fig 1. Summarized landscape of gene mutations in CCOT and ameloblastoma.

Filled boxes indicate relevant gene mutations detected by next-generation (cases #1–18) and/or Sanger sequencing (cases #19–25). Note that only CTNNB1, BRAF, and MEK2K1 hot spots were examined in cases #19–25, while hot spots in 50 cancer genes were examined in cases #1–18.

Fig 2. Electropherogram in CCOT and ameloblastoma.

A, Electropherogram of a TCT>TGT substitution at c.98 in CTNNB1, resulting in a Ser33Cys missense mutation in case #4. B, Electropherogram of a GTG>GAG substitution at position c.1799 in BRAF, resulting in Val600Glu missense mutation in case #25. Guanine is indicated by a black line, cytosine is indicated by a blue line, adenine is indicated by a green line, and thymine is indicated by a red line.

Fig 3. Photomicrographs of CCOT and ameloblastoma.

A, and D, Representative photomicrographs of CCOT (case #6) and ameloblastoma (case #25) specimens stained with hematoxylin and eosin. B, C, E, and F, Immunostaining for (B and E) BRAF Val600Glu (clone name VE1) and (C and F) beta-catenin. Scale bars; 20 μm.

Fig 4. Photomicrographs of ghost cells in CCOT and ameloblastoma.

A, and B, Representative photomicrographs of ghost cells immunostained for hair cortex keratin (clone name AE13) in (A) CCOT (case #6) and (B) ameloblastoma (case #20). Scale bars; 20 μm.

Fig 5. CTNNB1 induces hair keratin expression in human embryonic kidney 293 cells.

A, Western blot for beta-catenin, hair keratin, and GAPDH. Cells were transfected with mock plasmid, CTNNB1, or mutant CTNNB1, and proteins were extracted 48 h after transfection. Protein extracted from a 1-mm fragment of hair was used as a positive control for hair keratin (clone name AE13). B, Immunofluorescent imaging of cytoplasmic and nuclear beta-catenin (green) in cells transfected with mutant CTNNB1, as well as expression of hair keratin (green) in DAPI-stained cells (blue). Original magnification 40x.