RASSF 1 A Methylation Status and BRAF V600E Immunohistochemical Expression in Odontogenic Lesions

Background: The etiology and pathogenesis of odontogenic lesions remain to be determined. Previous studies have identified epigenetic and genetic alterations that may be relevant to lesions progression and development. Hypermethylation of the Ras association domain family protein 1A (RASSF1A) has been observed in a variety of human cancers. However, the methylation status of RASSF1A in odontogenic lesions remains unknown. Thus, the aim of this study was to investigate the prevalence of RASSFA promoter hypermethylation and v-raf murine sarcoma viral oncogene homolog B V600E mutant (BRAF V600E) expression as well as the correlations between these alterations and clinicopathological features of patients with odontogenic lesions. Methods: We subjected 66 formalin-fixed, paraffin-embedded odontogenic lesions [ameloblastoma (AM), 21; ameloblastic carcinoma (AC), 6; odontogenic keratocyst (OKC), 19; and dentigerous cyst (DC), 20] to methyla-tion-specific polymerase chain reaction to determine RASSF1A hypermethylation and immuno histochemistry to detect BRAF V600E protein expression. Results: We observed RASSF1A hypermethylation in 20% (4/20; methylation could not Con-clusions: Concomitant RASSF1A methylation and BRAF V600E


Introduction
Odontogenic tumors and cysts are a heterogeneous group of diseases derived from the cells of the tooth forming apparatus and their remnants [1] [2]. Ameloblastoma (AM) is among the most common benign odontogenic tumors and is clinically characterized by features such as slow infiltrative growth mainly involving the mandible, an asymptomatic presentation, and high recurrence rate when treated with conservative surgery [2] [3]. In contrast, ameloblastic carcinoma (AC) is a rare malignant odontogenic tumor. It combines the histological features of AM with malignant cytologic features such as cellular pleomorphism, mitotic activity, and nuclear hyperchromatism. AC may cause extensive local destruction of the jawbone as well as lung metastasis in approximately a third of patients [2] [4]. Odontogenic keratocysts (OKCs) and dentigerous cysts (DCs) are common developmental odontogenic cysts. DCs arise from the crowns of impacted or unerupted teeth and are usually slow growing and asymptomatic [5]. In contrast, OKC has a unique presentation characterized by locally aggressive behavior and tendency to recur in cases of incomplete resection or in the presence of daughter cysts [2] [6].
Ras association domain family 1 isoform A (RASSF1A) is an important tumor suppressor gene located in the 3p21.3 region of the human genome [7]. RASSF1A has been implicated in the RAS signaling pathway and is involved in various biological events, such as cell cycle arrest and apoptosis, and may also reduce tumorigenicity in cancer cell lines [8]. Extensive investigations have revealed that RASSF1A is frequently hypermethylated in several tumor types, such as small cell lung cancer, breast cancer, prostate cancer (~88% -99% of samples), but is less frequently methylated in oral squamous cell carcinoma and ovarian cancer (~22% -40% and 26% -30% of samples, respectively) [8] [9].
BRAF is a member of the rapidly accelerated fibrosarcoma (RAF) family of serine/threonine kinases [10]. Specifically, this kinase activates the RAF/MEK/ERK cascade to induce cell cycle progression and cell growth [11] [12]. BRAF is commonly mutated in a variety of human cancers [10]. The most frequent mutation is a missense thymine to adenine transversion in exon 15, resulting in amino acid change in BRAF protein at codon 600 from valine (V) to glutamic acid (E) (V600E) [12]. BRAF V600E mutations have been reported in various tumors, such as malignant melanomas, thyroid papillary carcinomas, and right-sided colorectal cancers [10] [12].
Currently, the etiology and pathogenesis of odontogenic lesions remain un-clear [13], although previous studies have reported various transcriptional changes and genetic alterations relevant to lesion progression and development [14] [15] [16]. In AM, for example, BRAFV600E mutation has been detected in 62% -83% of both multicystic and unicystic cases [17], and strong agreement was observed between immunohistochemical analyses of BRAF V600E and the molecular detection of BRAFV600E mutation [18]. BRAF V600E expression has been detected in 46% -89% of cases of mandibular AM [19] [28]. To our knowledge, however, no information is available regarding the RASSF1A methylation statuses of these lesions and the corresponding associations with clinical variables. This study aimed to assess the frequency of RASSF1A promoter methylation and BRAF V600E immunohistochemical expression in various types of odontogenic lesions (AM, AC, OKC, and DC) and to explore potential correlations between these changes and clinicopathological variables. Potentially, the study findings could provide a better understanding about the roles of RASSF1A and BRAFin the development of odontogenic lesions. We based conclusive diagnoses of AC samples on malignant morphological features and a Ki-67 proliferation index > 10% [29]. The clinicopathological characteristics of all samples are summarized in Table 1.

DNA Extraction, Bisulfite Modification, and Methylation-Specific Polymerase Chain Reaction (MS-PCR)
We extracted genomic DNA from all 66 FFPE odontogenic lesions using the QIAamp DNA FFPE Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. Next, we modified 20 µl of DNA with sodium bisulfite to convert unmethylated cytosines to uracil while preserving methylated cytosines, according to the manufacturer's instructions. Bisulfite-converted DNA was eluted in a total volume of 12.5 µl and stored at -20˚C until use. We detected the methylation status of the RASSF1A promoter region using MS-PCR with specific primers, as described previously by Helmbold et al. [30].
The primers for methylated sequence (forward: , and 72˚C for 30 s (Extension); and a final extension at 72˚C for 10 min. We used universal unmethylated and methylated DNA (Qiagen) as positive controls for unmethylated and methylated genes, respectively. A reaction in which DNA was replaced by water was used as a negative control. We separated all PCR products on 2% Tris-borate EDTA agarose gels, stained with ethidium bromide, and evaluated them under ultraviolet light.

Immunohistochemistry
For immunohistochemical analysis, all FFPE odontogenic lesions were cut into

Statistical Analysis
We examined differences in the RASSF1A methylation frequency and BRAF

RASSF1A Methylation Statuses of Odontogenic Lesions and Associations with Clinicopathological Features
To investigate the frequency of RASSF1A methylation in odontogenic lesions, we analyzed the RASSF1A promoter methylation statuses of 21 AM, 6 AC, 19 OKC, and 20 DC samples by MS-PCR. Notably, we detected partial methylation in 4/20 (20%; one sample could not be detected), 6/6 (100%), 5/19 (26.3%), and 1/20 (5%) AM, AC, OKC, and DC samples, respectively. Notably, the frequency of RASSF1A methylationwas significantly higherin AC samples than in AM, OKC, and DC samples (p < 0.001), and significantly higher in OKC and AM samples than in DC samples (p < 0.001 and p = 0.004, respectively) ( Table 2; Figure 1). RASSF1A hypermethylation in the odontogenic lesions did not correlate significantly with patient sex or age (≤50 vs. >50 years) or with tumor status (primary/recurrent) or location (p > 0.05 for all). In AM and AC samples, RASSF1A promoter hypermethylation also did not correlate with histological subtype (follicular/plexiform; p > 0.05). In OKC samples, no significant correlation was observed between RASSF1A promoter hypermethylation and histology (daughter cyst present/absent; p > 0.05). Table 3

Discussion
The pathogenesis of odontogenic tumors and cysts has not been well established, although epigenetic and genetic alterations appear to play important developmental roles [13]. In this study, we investigated the potential correlations of the RASSF1A promoter methylation status and BRAF V600E immunohistochemical expression with various clinicopathological characteristics in AM, AC, OKC, and DC samples. Despite differences in both markers between the various lesion types, we did not identify any significant correlations with clinicopathological features.
It has been suggested that RASSF1A heterodimerizes Novel Ras effector 1 and may mediate the tumor-suppressive effects of Ras cascade [7]. As noted previously, RASSF1A hypermethylation is a common event in the development of several cancers [8]. In this study, we reported aberrant RASSF1A hypermethylation in all six tested AC samples, but low to moderate RASSF1A methylation frequencies in other odontogenic lesions. The methylation of other tumor suppressor genes has also been reported in odontogenic lesions. For example, p16 methylation was observed in 25% -53% of AM and 100% of AC cases [26] [28].
The presence of p16 alteration in AM may predispose the lesions to malignant features in the future [28]. p21 methylation was present in 30% of OKC samples, suggesting that p21 methylation might implicate in the OKC development [25].
In this study, we observed a much higher frequency of RASSF1A methylation  [32]. Other studies also reported higher frequencies of RASSF1A promoter methylation in patients with  high-grade and advanced-stage nasopharyngeal carcinoma, breast cancer, and ovarian cancer, suggesting that RASSF1A promoter methylation may lead to a more aggressive tumor phenotype and disease progression [33] [34] [35]. We further investigated the associations between the RASSF1A methylation statuses of the odontogenic lesions and various clinicopathological variables, including patient sex or age, tumor status or location, histological subtype (AM and AC), and histology (OKC). We did not observe significant associations of RASSF1A hypermethylation with any of these variables, although this might be attributable to the small sample size. To our knowledge, this was the first study to evaluate the potential correlations between the RASSF1A methylation statuses of odontogenic lesions and clinicopathological features. Consequently, we cannot directly compare our results to those of other studies. Further research is needed to assess the associations between clinicopathological characteristics and RASSF1A promoter hypermethylation in odontogenic tumors and cysts.
Previous studies demonstrated that the overexpression of RASSF1A promotes apoptosis and cell cycle arrest in various cancer cell lines [8]. Joo et al. demonstrated that exogenous RASSF1A upregulated p27 and downregulated cyclin D1 expression and decreased retinoblastoma protein phosphorylation (Ser 608) in human gastric cancer cell lines, thus inhibiting cell proliferation [36]. Moreover, Wang et al. demonstrated that exogenous RASSF1A could increase the percentage of cells in the G0/G1 phase of cell cycle, inhibit cell proliferation, and induce apoptosis in a nasopharyngeal carcinoma cell line [33]. Therefore, the epigenetic silencing of RASSF1A by promoter methylation has been implicated in the development of many human cancers [33]. Some recent studies have identified the biological functions of RASSF1A methylation in tumorigenesis. Schirosi et al. reported a positive correlation between RASSF1A methylation and cytoplasmic and nuclear β-catenin expression, which resulted in the proliferation and metastasis of colorectal cancer cells [37]. Dubois et al. suggested that RASSF1A methylation could downregulate GEF-H1 and RhoB activity and thus promote the nuclear accumulation of YAP, resulting in the metastasis of non-small cell lung cancer cells [38].
Reports suggest that BRAF V600E mutation plays a key role in the tumorigenesis of various cancers, particularly melanomas, thyroid cancers, and hematopoietic malignancies, and a less frequent role in colorectal and lung adenocarcinomas [39]. Recent studies demonstrated that activating BRAF V600E mutations occur in 62% -83% of AM and AC cases [16] [17]. Moreover, BRAF V600E immunohistochemistry is highly sensitive and specific for the detection of BRAF V600E mutation in thyroid cancers and ameloblastomas, with a level of reliability comparable to that of molecular techniques [18]  who reported non-specific BRAF V600E immunostaining in the epithelium and plasma cells in some dentigerous and radicular cysts [17]. Taken together, immunohistochemical BRAF V600E expression was observed at higher frequencies in AM and AC samples than in OKC and DC samples (p < 0.001).
Moreover, we did not observe any correlations of immunohistochemical BRAF V600E expression with patient sex or age, tumor location or status, or histological subtype in the AM and AC samples. This finding was consistent with a report by Kurppa et al. [19]. However, other studies of AM cases have reported significant correlations of BRAF V600E expression with recurrent disease, a younger age (≤50 years), and tumor location in the posterior region of mandible (vs. the anterior region) [20] [21] [22]. These inconsistent results might be attributable to small sample sizes and ethnic differences. Additional large-scale studies are needed to clarify the associations of BRAF V600E expression with clinicopathological features of AM patients.
Finally, previous studies have reported a correlation between RASSF1A methylation and KRAS/BRAF mutation in some tumors. Specifically, RASSF1A promoter hypermethylation occurs predominantly in sporadic colorectal, pancreatic, and thyroid cancers that habor wild type KRAS/BRAF [42] [43]. Interestingly, KRAS/BRAF mutation may occur concomitantly with RASSF1A hypermethylation in malignant melanomas and sporadic MSI colorectal cancers, suggesting a possible synergistic effect [44] [45]. In this study, we observed that 83.3% of tested AC lesions harbored concomitant RASSF1A methylation and BRAF V600E-positive expression, suggesting a synergistic result between the inactivating of RASSF1A and the genetic alteration of BRAF V600E expression. The majority of AM cases (70%) exhibited a preferential single genetic alteration leading to positive BRAF V600E expression. Our observations of infrequent RASSF1A methylation and BRAF V600E expression in OKC and DC samples suggest that genetic/epigenetic alterations in these genes may not play a vital role in the pathogenesis of these lesions.
A limitation of this study is the small sample size. Further studies with larger samples are needed to confirm our preliminary results and elucidate the role of RASSF1A methylation in the tumorigenesis of odontogenic lesions. In addition, we tried to confirm the results of RASSF1A methylation status presented in this study by analyzing the RASSF1A expression with reverse-transcription PCR (RT-PCR) and immunohistochemistry. However, in the majority of RASSF1A unmethylated cases, RASSF1A expression could not be detected by both techniques. It is possible that the routine decalcification procedure may diminish RASSF1A mRNA and protein in our samples. Thus, the selection of a weaker acid-decalcifying agent might provide better results of molecular and immunohistochemical studies of this gene.

Conclusion
In summary, we investigated the RASSF1A tumor suppressor gene methylation statuses of various odontogenic lesion types, and observed that RASSF1A promoter methylation was significantly more frequent in AC than in other odontogenic lesions. However, we did not observe a significant correlation between RASSF1A hypermethylation in these lesions and clinicopathological variables.
Moreover, we confirmed that positive BRAF V600E expression was frequently observed in mandibular AM and AC lesions (>80% of tested cases), but was absent in OKC and DC lesions. However, BRAF V600E expression did not correlate with clinicopathological features in either AM or AC cases.