Adenomatoid Odontogenic Tumor (AOT) and Ameloblastoma (AM) are benign epithelial odontogenic tumors affecting most commonly the tooth bearing areas of the jaws. Both tumors are composed of a proliferation of epithelial cells arranged in a way that reminds to some extent, to the enamel organ of a tooth germ [1]. AM is well-known for being locally infiltrating, for its continuous growth, its high rates of recurrences if not adequately removed and the possibility of undergoing malignant transformation [2]. On the contrary, AOT manifests clinically as a slow and self-limiting growth which does not require the aggressive surgical approach usually adopted for AM. In AOT, recurrences are extremely rare, even if it is partially removed [3]. The clinicopathological features of both tumors, allows to consider AM as a neoplasm, whereas there is a general agreement that AOT may represent a hamartoma [3–5], although this is a matter of debate.

Different genomic alterations, which includes chromosomal imbalances and genetic mutations, have been reported to be present in ameloblastomas. Mutations in genes that belong to the mitogen-activated protein kinase (MAPK) pathway are present in almost 90% of ameloblastomas, with BRAF V600E, being the most described mutation [6–10]. The prevalence of BRAF V600E in ameloblastoma ranges from 46 [6] to 90% [11], with a mean value of 68%. Other somatic mutations have been reported, either in MAPK or non-MAPK pathways [6, 8, 10, 12–16]. Some of these, such as mutations in PTEN, SMARCB1, EGFR, TP53, CTNNB1, and PIK3CA [6, 8–10], can occur in the background of the classical BRAF V600E mutation. Nevertheless, mutations in SMO, FGFR2, KRAS, HRAS, and NRAS have been reported to be mutually exclusive with BRAF V600E [8, 14]. Moreover, deletions in chromosome 22 [6, 17–19] and copy number alterations in BAG1, PPP2R5A, and PKD1L2 [20] have also been reported and could also be involved in the pathogenesis of the tumor.

Little information is known about the genetic background of AOT. Mutations in the β-catenin gene (CTNNB1) have been suggested, as strong cytoplasmatic expression of β-catenin is reported using immunohistochemistry [4, 21]. However, authors have failed to show alterations in CTNNB1 [4]. Nevertheless, other more recent studies have shown consistent mutations in KRAS [22–24] and copy number alterations [23] affecting IGF2BP3.

As both AM and AOT have different clinical behavior, suggesting a different biological nature, and there has been a significant interest in papers reporting their genetic alterations during the last years, the aim of this systematic review was to compare the genetic alterations of AOT with the ones reported in AM, in order to summarize the current genetic knowledge of these lesions and aid in the understanding of the genomic alterations underlying their development.

This systematic review was conducted following the PRISMA Statement guidelines.

The search results are outlined in a PRISMA flow diagram in Figure 1. The initial literature search identified 677 studies. Following duplicate removal (n = 396), 281 studies had their titles and abstracts screened by two of the reviewers (SN and CM) from which 205 articles were removed. Finally, 76 studies were included for full text evaluation to ensure they satisfied the inclusion and exclusion criteria. Thirty-four articles were excluded with the following reasons: reviews (n = 14) and wrong study design (n = 19). In total, 43 articles were included in this systematic review (Figure 1).

Odontogenic tumors (OT) arise from dental tissues or their remnants, and for decades, this statement was only supported by the histologic appearance of these lesions, which resembles the enamel organ, dental papilla or the dental follicle [1, 50, 51]. Increasing evidence showing mutations and/or chromosomal alterations in the same genes expressed during odontogenesis have confirmed this association, consolidating the close relationship between ontogenesis and oncogenesis [8, 9, 14, 16, 38, 52–60]. The ongoing development of the molecular aspects of odontogenic tumors has revolutionized the understanding of the ethiopathogenesis of these heterogenous group of lesions, allowing the proposal of novel molecular therapeutic targets. However, the exact mechanism underlying the tumorigenic process and possible causes for their different clinical behavior remains unknown.

Both AOT and AM are odontogenic tumors of epithelial origin, but their clinical behavior is diametrically different, resulting in different treatment approaches and prognosis. In the current systematic review, we aimed to compare the genetic alterations of AOT with the ones reported in AM, in order to summarize the current genetic knowledge of these lesions and aid in the understanding of the genomic alterations underlying their development and different behavior.

Our search identified six studies that analyzed the genetic aspects of AOTs (n = 59), in contrast to 37 that explored the genetic landscape of ameloblastoma (n = 530). Mutation in exon 12 of KRAS was found to be present in 76% of AOTs with G12V/R being the most found [22, 23]. On the other hand, mutations in BRAF were found in 71.1% of the samples, corresponding mainly to V600E [6, 25] (Figure 3). Interestingly, the proportion of AOTs with KRAS driver mutation, is similar to the proportion of driver mutations reported in ameloblastoma. Due to its high frequency, KRAS mutations were proposed as a driver mutation and signature marker of AOTs [22–24]. Because KRAS mutations are a recurrent finding in AOTs, Coura et al. [22], suggested the presence of KRAS G12V/R to help in the diagnosis of controversial cases of AOT, in the same way that BRAF V600E could be used in routine ameloblastoma diagnostics [14, 38].

The RAS oncogene family is comprised of three members, KRAS, NRAS, and HRAS, and plays an important role in normal development, but also for cancer development. Activated point mutations on RAS proteins are widely present across a different spectrum of human cancers [61, 62]. Our review showed that all KRAS mutations reported in AOTs have been found affecting codon 12 [22–24]. Mutations affecting this codon have been reported in non-small cell lung cancer and pancreatic ductal adenocarcinoma, being present in almost half of the cases for the former, and in 16% for the latter [63, 64]. KRAS corresponds to a small GTPase that transduces extracellular signals to intracellular signal transduction cascades [65] (Figure 3). It has been suggested that the mutation subtype may affect downstream signaling differently, which could be reflected clinically [66, 67]. Nevertheless, to date, this has not been demonstrated in AOTs. Coura et al., reported in their cohort of 38 AOTs, no statistically significant association between the presence of mutations (mainly KRAS G12V and G12R) and clinicopathological parameters (including patient's age, tumor size, location, follicular or extrafollicular variants, and fibrous capsule thickness) [22].

The activation of RAS/GTP complexes, can activate several downstream signaling pathways such as Raf-MEK-ERK, PI3K-AKT-mTOR, RalGDS-RalA/B, and the TIAM1-RAC1 [65]. To date, only the activation of the MAPK/ERK pathway has been demonstrated in AOT. With immunohistochemistry, Coura et al., demonstrated not only KRAS-mutated cases, but also wild-type KRAS cases to have strong pERK1/2 expression. This suggests that the MAPK pathway can be activated by other mechanisms rather than KRAS mutations [22]. Similarly, using immunohistochemical techniques, Bologna-Molina et al., demonstrated AOTs to express different proteins related to the MAPK/ERK pathway, including EGFR, KRAS, BRAF, CRAF, ERK, and MEK [24].

Apart from KRAS mutations, other somatic point mutations affecting SMO and AMBN [25, 26], and losses affecting 7p15.3 and 6q15 [23], were also found in AOTs. In a similar way, other somatic mutations have been reported in ameloblastoma, mainly affecting: SMO [6, 8, 10, 14], other MAPK pathway-related genes such as KRAS, NRAS, HRAS, FGFR2 [6, 8, 10, 12, 14, 16] and in a lower frequency, PTEN [10, 12, 34] and CTNNB1 [8, 10, 12, 13, 16, 34, 42, 44, 45] among others. Interesting results were found by Diniz et al., who reported one ameloblastoma negative for BRAFV600E, with greater number of CNAs and cnLOH encompassing genes directly related with RAF/MAPK pathway activation, suggesting an alternative mechanism of mimicking this pathway [20].

Recently, Bello et al. [68] proposed that the interactions between the adhesion proteins FAK, paxillin and PI3K may be relevant in the aggressiveness of AM compared to AOT, based on the observation that FAK expression was stronger in AM compared to AOT, and that one case of peripheral AM with strong expression of the three proteins had a history of two recurrences. Nevertheless, their conclusions should be carefully interpreted because were based on observations based on a small cohort of AOTs (n = 7).

The biological nature of AOT has been a constant matter of debate. In 2017, Reichart et al. [69], compared the immunohistochemical expression of different factors between AOT and AM, and proposed AOT to be a hamartomatous process rather than a true neoplasm [69]. Markers related to invasion, such as cytokeratin profiles and integrins, to proliferation, such as MDM2, p53 protein and metallothionein levels, were found to be higher in ameloblastomas compared to AOTs. Also, AOTs showed lower levels of matrix metalloproteinases (consistent with a reduced local aggressiveness), Ki76 and anti-apoptosis markers such as Bcl-2, and higher levels of β-catenin (suggesting greater cell adhesion properties) [69]. Similarly, there are publications about the strong cytoplasmatic expression of β-catenin on AOTs [4, 21], however no mutation in CTNNB1 was detected [4]. The proposal of Reichart et al. [69] was based purely on immunohistochemical findings without considering genetic aspect. Although our knowledge about the molecular background of AOT is still very limited, the genetic data collected by this review points to the direction that AOT harbor mutations in important oncogenic driver genes, such as KRAS, and based purely on this, some authors have proposed it as a neoplasm [22]. Nevertheless, until now, the presence of these genetic alteration seems not to have a direct impact on its clinical behavior. Thus, care has to be taken when interpreting these findings.

The low number of studies that have performed small to large-scale and/or “omics” techniques to characterize the molecular background of AOTs, the low frequency of AOT (accounting for <5% of odontogenic tumors) [70–72], limited clinical information availability, and the fact that most of the available studies come from single-institution series or case reports, limit the conclusions than can be drawed out of these findings. Also, current publications are all retrospective studies based on formalin-fixed paraffin-embedded samples (much of them subjected to decalcifications methods), which shows inherent limitations, mainly related to the quality of the nucleic acids for these purposes and the difficulty of retrieving a large cohort. Nevertheless, molecular pathology is demonstrating its utility in the diagnosis of challenging cases and for targeted therapy of disfiguring tumors such as ameloblastoma, avoiding considerable post-surgical morbidities.

The available genetic data reports that 75% of AOTs harbor somatic mutations in KRAS, a well-known oncogene. Nevertheless, the number of studies that have a assessed the genetic landscape of AOT is still very limited, not providing enough evidence to draw a conclusion regarding the relationship between the genomic alterations and its clinical behavior. There are a significant number of studies that have assessed the genetic aspects of ameloblastoma. Different genetic alterations have been reported, being the BRAFV600E mutation the most common. The relatively high frequency of ameloblastoma compared to other odontogenic tumors, such as AOT, has facilitated the performance of different sequencing techniques, allowing the discovery of different mutational signatures. On the contrary, the low frequency of AOTs is an important limitation for this. Thus, the presence of other mutational signatures with clinical impact, co-occurring with KRAS background or in wild-type KRAS cases, cannot be ruled out.

Since BRAF and RAS are in the same MAPK pathway, it is interesting that ameloblastomas, frequently associated with BRAFV600E mutation have aggressive clinical behavior, but in contrast, AOTs, frequently associated with RAS mutations have indolent behavior. Functional studies might be required to solve this question.

The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.

All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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