Molecular diagnostics in odontogenic tumors

Ameloblastoma (AM) is the most common benign epithelial odontogenic tumor, representing approximately 1% of all oral tumors and about 9 to 11% of all odontogenic tumors [1]. The tumors are classified into four groups: conventional, unicystic, extraosseous/peripheral, and metastasizing variants. In the recent World Health Organization (WHO) classification, adenoid ameloblastoma (AdAM) is a newly recognized entity separate from the AM group of tumors [2]. Although AMs are known as locally aggressive tumors with high recurrence rates, unicystic ameloblastoma (UAM) shows an indolent course different from the other variants. This unpredictable and distinct biological behavior of AMs has lent them priority to trigger molecular studies on understanding their pathogenesis. In the past decade, oncogenic mutations were discovered which constitutively activate signal transduction pathways relating to developmental stages of odontogenesis, including the mitogen-activated protein kinase (MAPK) and hedgehog pathways [6‐8]. Advanced next-generation sequencing (NGS) analyses identified the high frequency of BRAF V600E and SMO L412F mutations in all types of ameloblastoma [5‐8]. This is followed by KRAS (mostly p.G12R), NRAS, HRAS, FGFR2, and mutations reported in a few BRAF wildtype cases ([7‐9]; Fig. 1). Additionally, we reported EGFR mutations and the presence of other gene mutations, including somatic mutations in KRAS, PIK3CA, PTEN, FGFR, CDKN2A, and CTNNB1 on the background of either BRAF or SMO mutation-positive ameloblastomas, occurring exclusively in conventional AMs [9].

In line with reports about other neoplasms that harbor a malignant counterpart, the frequency of the BRAF p.V600E mutation is higher in ameloblastoma (64% in conventional, 81% in unicystic, and 63% in peripheral) than in ameloblastic carcinoma (35%) [5]. As both conventional AM and UAM have been found to harbor BRAF p.V600E mutations, aggressive and destructive tumors could be candidates for BRAF-targeted therapy that has the potential to reduce tumor size and ultimately enable a conservative surgical procedure. Preliminary data of biological treatment show effectiveness in selected cases [2, 3].

Adenoid ameloblastoma (AdAM) is a newly recognized entity separate from the AM group of tumors. AdAM is characterized by an aggressive biological behavior with local infiltration, and the recurrence rate is high (45.5–70%). BRAF p.V600E mutations, usually identified in AM/UAM, are absent in AdAM. Whether AdAM is a unique standalone tumor or a histologic variant of AM requires further investigation [2].

Adenomatoid odontogenic tumor (AOT) manifests clinically as a slow and self-limiting growth which does not require an aggressive surgical approach [1]. AOTs are characterized by frequent KRAS codon 12 (either p.G12V or p.G12R, and in a single case p.G12D) driver mutations, which occur in approximately 70% of cases [2, 5, 10]. Although they have not been connected to their clinicopathological features, molecular profiling is important for the differential diagnosis of this tumor from other lesions such as AdAM, adenomatoid odontogenic hamartoma, and adenomatoid dentinoma ([2, 5]; Fig. 2).

Calcifying epithelial odontogenic tumor (CEOT) is recognized to have three histopathological subtypes: clear cell, cystic/microcystic, and non-calcified/Langerhans cell rich. Mutations in tumor suppressor genes (PTEN, CDKN2A, PTCH1) and oncogenes (JAK3, MET) have been identified in CEOT; however, so far, these do not contribute to clinical properties or treatment decisions ([2, 5]; Fig. 2).

Odontoma is the most common odontogenic tumor and is composed of mesenchymal and epithelial components of the tooth [1]. WNT/beta-catenin pathway activation in embryonic SOX-2-positive dental stem cells can drive odontoma formation [2]. Ameloblastic fibrodentinoma (AFD) and ameloblastic fibroodontoma (AFO) are classified as developing odontomas, although the prevalence of BRAF p.V600E mutations in AFD and AFO is similar to ameloblastic fibroma (AF) but differs from odontoma, which lacks BRAF p.V600E mutations [11].

Ameloblastic fibroma (AF) is a rare benign odontogenic tumor with the potential for recurrence and malignant transformation to ameloblastic fibrosarcoma ([1]; Fig. 2). AFs are characterized by BRAF p.V600E mutations, like other ameloblastic tumors [2, 10, 11]. Early developing stages of odontomas may be comprised of soft tissue closely resembling dental papilla, with prominent epithelial strands and limited or no evidence of dental hard tissue induction. These features overlap with ameloblastic fibroma (AF), sometimes causing a problem differentiating between them. The differentiation between early odontoma and AF is important to avoid unnecessary potentially destructive surgery [1, 2]. Thus, detection of BRAF p.V600E mutations is important for differential diagnosis.

Odontogenic myxoma (OM) is a rare odontogenic tumor that arises from odontogenic ectomesenchyme. The tumor often behaves in a locally aggressive and infiltrating fashion, with a 25% recurrence rate [1]. Activating mutations in the MAPK/ERK signaling pathway have been identified in this tumor and may serve as targets for pharmacologic therapy.

Cemento-ossifying fibroma (COsF) became an integral part of the benign mesenchymal odontogenic tumors in the 2022 WHO classification. A minority of COsFs are linked to inactivating mutations in the tumor suppressor gene CDC73 (HRPT2), especially in those cases that are part of hyperparathyroidism–jaw tumor syndrome. COsF can also be part of gnathodiaphyseal dysplasia, which is characterized by GDD1 gene mutations [2, 11].

Ameloblastic carcinoma (AMCa) is a highly aggressive, malignant epithelial odontogenic tumor. It is now defined as an entity which is not related to ameloblastoma [2]. However, AMCas harbor BRAF p.V600E mutations like other ameloblastoma-related tumors, with varying prevalence from 25 to 40% [5]. Mutations in other genes which are not related to MAPK/ERK, such as TP53, CTNNB1, and APC, have also been reported inAMCas [2, 5, 11].

Clear cell odontogenic carcinoma (COdC) is a malignant tumor with high recurrence rate (40%). Its regional lymph node metastases are more common than distant ones and the death rate is about 11%. Differential diagnosis can be critical, which includes jawbone clear cell-containing tumors such as CEOT, amyloid-rich odontogenic fibroma, odontogenic carcinoma with dentinoid, primary or metastatic tumors of salivary glands (e.g., mucoepidermoid carcinoma, clear cell carcinoma, epithelial myoepithelial carcinoma), and metastatic tumors (i.e., clear cell renal carcinoma, melanoma). COdC is characterized by EWSR1 gene rearrangement in about 80% of cases. It has also been shown to harbor BRAF p.V600E in limited cases [2, 5].

Odontogenic sarcoma (OS) is a mixed tumor, histologically characterized by a benign ameloblastic epithelium within a sarcomatous mesenchymal component, with or without dentine and enamel. OS can arise de novo or emerge from a sarcomatous change in AF, and approximately one third of AFS (Ameloblastic Fibrosarcoma) cases stem from a recrudescent AF. Clinically, OS shows locally aggressive behavior. The tumor shows a high recurrence rate of approximately 37% [12]. The BRAF p.V600E mutation has been detected in 67–71% of reported cases. An NRAS mutation has also been reported in one case of OS in a mutually exclusive manner with BRAF p.V600E [5, 11]. Although the rarity of this tumor precludes extensive knowledge about its molecular pathology, the current results could support the role of the MAPK/ERK pathway in its pathogenesis and pave the way for further investigations on targeted therapy [5].