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Abstract
Introduction: Ameloblastoma is a benign epithelial odontogenic neoplasm that constitutes approximately 1% of all oral tumors and about 9 to 11% of all odontogenic tumors. They are slow-growing, locally invasive, and demonstrate a potential for metastasis and malignant transformation. The molecular pathogenesis of ameloblastoma is attributed to aberrant activity of the signal transduction pathways relating to developmental stages of odontogenesis including the mitogen-activated protein kinase (MAPK) pathway. The BRAF V600E mutation was identified as the most frequently mutated gene in this neoplasm. Studies have shown that use of BRAF inhibitors in patients diagnosed with ameloblastomas led to a significant reduction in tumor volume. Aims: To detect the expression of BRAF V600E mutation in ameloblastomas in an Indian population using immunohistochemistry. To compare the difference in the occurrence of the BRAF V600E mutation between mandibular and maxillary cases. Materials and Methods: Thirty-three formalin-fixed paraffin-embedded tissues of histopathologically proven cases of ameloblastoma were assessed for the BRAF V600E mutation by immunohistochemistry using the BRAF V600E monoclonal antibody. Patient data such as age, sex, anatomical site, recurrence were documented. Statistical Analysis: The statistical analysis was performed using the Pearson Chi-square test and Student's t-test. Results: The present study revealed a high expression of the BRAFV600E mutation in mandibular cases of ameloblastoma among Indians irrespective of the age, sex, site, recurrence or histological pattern. Conclusions: The identification of this driver mutation opens the possibility of an adjuvant therapeutic modality to reduce the significant facial disfigurement and morbidity following surgical management.
Keywords: Ameloblastoma, BRAF V600E mutation, immunohistochemistry
How to cite this article: Introduction 
Ameloblastoma is a true neoplasm of enamel organ type tissue that does not undergo differentiation to the point of enamel formation.[1] The tumor was defined by Robinson in 1937 as 'usually unicentric, nonfunctional, intermittent in growth, anatomically benign and clinically persistent'. Ameloblastomas are unique in that though essentially benign, they are characterized by unpredictable behavior, potential for malignant transformation, and metastasis. Ameloblastomas constitute approximately 1% of all oral tumors and about 9 to 11% of all odontogenic tumors.[2] Due to their complex growth pattern, most ameloblastomas are treated by surgical resection, often resulting in facial deformity and significant morbidity. More conservative approaches tend to result in a high recurrence rate, ranging between 55% and 90% necessitating further mutilating surgery.[3]
The molecular pathogenesis of ameloblastoma is attributed to aberrant activity of the signal transduction pathways relating to developmental stages of odontogenesis including the mitogen-activated protein kinase (MAPK) and the hedgehog pathways.[4],[5],[6] The BRAF V600E mutation was identified as the most frequently mutated gene (40-80% of cases).[7] BRAF-activating transversion (T > A) in the kinase domain at exon 15 (position c. 1799) is the most frequent BRAF mutation, and it results in a valine (V) to glutamic acid (E) substitution at codon 600. BRAF V600E was reported to be associated with parameters of a more aggressive behavior.[8] Studies have shown that use of BRAF inhibitors in patients diagnosed with ameloblastomas led to significant reduction in tumor volume.
The ameloblastoma is the most commonly reported odontogenic tumor in Indian populations. The prevalence of ameloblastoma in India was 56.3-67.69%.[9],[10] However, there is only one study published on the detection of the BRAF V600E mutation in ameloblastomas in the Indian population including an institutional study which was carried out on cases of mandibular ameloblastomas where a low frequency of BRAF V00E immunoexpression was detected.[11] Thus, there is a need to determine the expression of this mutation within the various populations of India.
[TAG:2]Materials and Methods[/TAG:2]
This retrospective study was approved by the Ethical Committee of Goa Dental College and Hospital, Bambolim-Goa. It evaluated a total of 33 cases of histopathologically proven cases of ameloblastoma-unicystic and solid, retrieved from the archives of the Department of Oral and Maxillofacial Pathology, Goa Dental College and Hospital. Patient data such as age, sex, anatomical site, recurrence were documented. Acid decalcified tissues and hybrid odontogenic tumors were excluded.
In order to confirm the diagnosis of the 33 archival tissue specimen, fresh sections were made for each selected tissue block using a semi-automated microtome and stained by Harris Hematoxylin and 1% Eosin Y staining protocol. Once, the diagnosis for all 33 selected samples was confirmed, the same tissue blocks were re-sectioned for the purpose of immunohistochemical analysis. ThermoFisher BRAF Monoclonal Antibody (RM8) MA5-24661 was used in the dilution of 1:50. Standard immunohistochemistry procedure was carried out. 3-4 μm sections were floated from the water bath onto bar coded (Dako Seymour System™) positively charged- hydrophilic slides. Antigen retrieval was performed using the Heat Induced Epitope Retrieval (HIER) system (DAKO PTLink™) and Dako target retrieval solution (pH 6). The Dako AutoStainer and Dako reagents were used to carry out the immunohistochemical staining procedure. The BRAF V600E antibody was applied to the tissue sections for 20 min and the diaminobenzidine substrate chromogen solution was applied for 10 min. The sections were then counterstained with hematoxylin and washed with phosphate buffer solution, to remove the excess stain. Lastly, the slides were dehydrated in 70%, 96% and 99% alcohol (each for two minutes), cleared in xylene (single dip) and mounted using DPX (Dibutyl Phthlate Xylene) mounting media. Papillary thyroid carcinoma tissue specimen was used as the positive control and dentigerous cyst tissue specimen was used as the negative control.
The stained slides were examined under scanner view (40×), low power (100×) and high power (400×) of the microscope by two observers. BRAF V600E immunoexpression were assessed in a blind fashion, regardless of the patient's identity and clinical history. Five high-power fields were selected randomly and images of the five fields were captured and positive cells were counted. The images were analyzed using Image J (Version 1.52a) image analysis software, using the multipoint tool. The extent and intensity of stained tumor cells were assessed by a semi-quantitative scale in each sample. The number of positive cells per high-power field was assessed according to the Allred scoring guidelines.[12] Only those cells that showed positive immunohistochemical staining and were defined by cytoplasmic reactivity were considered as positive cells.[6] Such cells were considered to have BRAF V600E mutation.
Statistical analysis
Data was analyzed using SPSS (Statistical Package for Social Sciences) version 20 and Microsoft Excel. The statistical analysis was performed using the Pearson Chi-square test and Student's t-test. The level of statistical significance was accepted at P < 0.05.
Results Cytoplasmic expression of BRAF V600E mutation in Follicular Ameloblastoma, Acanthomatous Ameloblastoma, Plexiform Ameloblastoma, Granular Cell Ameloblastoma, Unicystic Ameloblastoma- Luminal variant, Unicystic Ameloblastoma- Transmural variant, Positive control, Negative control is seen in [Figure 1] and [Figure 2]. Data from a total of 33 cases of ameloblastomas are shown in detail in [Table 1]. Of these, there was a slight male predominance (57.6%), with median age of 37.06 years. The posterior region of the mandible was the most commonly affected site (66.7%). As for the histological classification, the most common variant was unicystic ameloblastoma (54.5%). 3 cases were recurrent (9.1%). In our study, 29/33 (87.9%) cases were positive for BRAF V600E immunoexpression. 18/19 (94.7%) males and 11/14 (78.6%) females were positive for the BRAF V600E mutation. The average age of individuals carrying the mutation was 37.9 while the average age of individuals negative for the mutation was 30.5. 26/30 (86.6%) mandibular cases and 3/3 (100%) maxillary cases were positive for the mutation. 12/13 (92.3%) follicular ameloblastomas, 1/2 (50%) plexiform ameloblastomas and 16/18 (88.9%) unicystic ameloblastomas were positive for mutation. 2/3 (66.7%) recurrent cases were positive for mutation and 1/3 (33.3%) recurrent case was negative for the mutation. No statistically significant difference in relation to age (P 0.455), sex (P 0.160), site (P 1.000), histopathological variants (P 0.470), and recurrence (P 0.330) with BRAF V600E expression were obtained.
Figure 1: Photomicrograph of IHC stained section showing cytoplasmic expression of BRAF V600E mutation in Follicular Ameloblastoma (400×) (a), Acanthomatous Ameloblastoma (400×) (b), Plexiform Ameloblastoma (400×) (c), Granular Cell Ameloblastoma (400×) (d)
Figure 2: Photomicrograph of IHC stained section showing cytoplasmic expression of BRAF V600E mutation in Unicystic Ameloblastoma- Luminal variant (400×) (e), Unicystic Ameloblastoma- Transmural variant (40×) (f), Positive control (100×) (g), Negative control (40×) (h) Discussion The ameloblastoma is a benign epithelial odontogenic neoplasm, thought to arise from SOX2-expressing dental lamina epithelium and remnants of the tooth-forming enamel organ.[13] The tumor is comprised of well-defined follicles or a plexus of peripheral polarized epithelial cells resembling pre-ameloblasts enclosing loosely arranged cells resembling stellate reticulum separated by a basement membrane from a bland fibrous connective tissue stroma. Ameloblastomas are slow-growing, locally invasive and demonstrate a potential for metastasis and malignant transformation. They can occasionally transform into ameloblastic carcinoma. According to the WHO 2017 classification of odontogenic tumors, ameloblastomas are categorized as ameloblastoma (conventional), unicystic, extraosseous/peripheral, and metastasizing variants.[14]
Current treatment modalities range from being conservative, which involves enucleation to radical excision and vary according to tumor subtype. A high recurrence rate (50–80%) has been observed in cases following a conservative treatment.[7],[15] The morbidity increases with either aggressive management or a recurrence necessitating a second surgery and extensive reconstruction.
The molecular pathogenesis of ameloblastoma is now attributed to dysregulation of the mitogen-activated protein kinase (MAPK) pathway, activated by BRAF, a member of the Raf kinase family[16] as illustrated in [Figure 3].
Figure 3: Schematic illustration of BRAF pathway that is involved in the pathogenesis of Ameloblastoma [Created with BioRender.com]In the year 2014, three independent reports identified BRAF V600E as the most prevalent mutation among recurring MAPK mutations in ameloblastoma analyzed by genomic analysis and immunohistochemistry. The predominant mutation in the BRAF gene involves thymidine to adenosine T > A transversion at exon 15 nucleotide 1799, resulting in replacement of valine (V) with glutamic acid (E) at position 600 of amino acid sequence (BRAF V600E), accounting for >90% mutations in BRAF.
Although these studies employed confirmatory molecular techniques, they also evaluated the usefulness of immunohistochemistry using anti-BRAF V600E antibody in ameloblastomas and unequivocally substantiated that BRAF V600E immunopositivity significantly correlated with BRAF V600E mutation status in ameloblastomas. Excellent concurrence of BRAF V600E immunohistochemistry with gene mutation has been established as the presence of mutated protein can be detected at a single-cell level.[6],[17]
The results of the immunohistochemical detection of the BRAF V600E mutation in ameloblastomas, among European populations revealed an expression ranging from 63 to 84%, 46.6% to 100% among Western populations and from 33.3% to 80% among Asians.
In our study, a comparable positive BRAF V600E immunoexpression in 29/33 (87.9%) cases of ameloblastomas were obtained as summarized in [Table 1].
Ameloblastomas occurring in the mandibular region comprised majority of cases in previous studies on BRAF V600E mutation [Table 2]. In our study, 30/33 (90.7%) were mandibular cases and 3/33 (9.1%) were maxillary cases of which 26/30 (86.6%) mandibular cases and 3/3 (100%) maxillary cases were positive for the BRAF V600E mutation. Sweeney et al.[5] Brown et al.[6] and Gültekin et al.[2] reported that BRAF V600E mutation showed greater predilection for the mandible than the maxilla. Whereas, Diniz et al.[19] found no association of BRAF mutation and tumor location. Ameloblastomas have a higher predilection for the posterior mandible as compared with the maxilla. The combined total of mandibular ameloblastomas evaluated in the foregoing studies far outnumbered the total number of maxillary ameloblastomas.
Table 2: Various studies with occurrence of BRAF positivity in mandibular and maxillary cases of ameloblastomaAn inherent bias is evident in these studies owing to an unequal distribution of the number of mandible and maxillary cases in the study samples.
The present study samples are likewise predominantly mandibular; hence a correlation of BRAF V600E immunoexpression could not be established between mandibular and maxillary ameloblastomas.
Similar to our study, Kurppa et al.[4] and Motta do Canto et al.[18] found no significant association of BRAF V600E mutation with age or sex, tumor histology and tumor recurrence. Shirsat et al.[11] did not observe any significant association between age and sex with BRAF V600E expression. However, Gültekin et al.[2] and Brown et al.[6] observed a significant correlation of BRAF V600E mutations with younger age.
In our study, 19/33 (57.5%) were males and 14/33 (42.4%) were females of which 18/19 (94.7%) males and 11/14 (78.6%) females were positive for the BRAF V600E mutation. The average age of individuals carrying the BRAF V600E mutation was 37.9 while the average age of individuals negative for BRAF V600E mutation was 30.5. Hence, no statistically significant difference relating age and sex (P = 0.160) with BRAF V600E expression were obtained.
Kurppa et al.[4] Fregnani et al.[8] and Diniz et al.[19] found no significant association between BRAF V600E and histological pattern which coincided with findings in our study. Chang et al.[23] reported a strong association of BRAF V600E expression with the follicular variant of ameloblastomas. However, Shirsat et al.[11] found a significant correlation of BRAF V600E expression with plexiform variant of ameloblastoma which is in complete contrast to findings of all previous studies. An analysis of data of Sweeney et al.[5] showed plexiform variant to be significantly common among BRAF-negative tumors.
The present study had 13/33 (39.4%) of follicular ameloblastomas, 2/33 (6.1%) of plexiform ameloblastomas and 18/33 (54.5%) of unicystic ameloblastoma of which 12/13 (92.3%) follicular ameloblastomas, 1/2 (50%) plexiform ameloblastomas, and 16/18 (88.9%) unicystic ameloblastomas are positive for BRAF V600E mutation. The results of our study were compared to a study conducted by Heikinheimo et al.[24] in which the study aimed to characterize the mutation profile of unicystic ameloblastoma and to compare it to conventional ameloblastoma. In the mandible, 94% of UAMs (29/31, including 8/8 luminal, 6/8 intraluminal, and 15/15 mural subtypes) revealed BRAF V600E mutations which was higher compared to the BRAF positivity seen in of conventional AMs (28/38 = 74% cases).
The present study is an immunohistochemical analysis to determine BRAF V600E immunoexpression in ameloblastomas amongst Indians. To the best of our knowledge, this is the second study to be carried out in an Indian population. The first study was carried out by Shirsat et al.[11] which was restricted only to mandibular ameloblastomas where the lowest frequency of BRAF V600E immunoexpression was obtained i.e., in 10/30 (33.3%) cases. However, in our study a high frequency of BRAF V600E immunoexpression was obtained i.e., in 29/33 (87.9%) cases which included both maxillary and mandibular ameloblastomas. Amongst the mandibular ameloblastoma cases, 26/30 (86.6%) cases showed BRAF V600E immunoexpression positivity.
Contrary to Brown et al.[6] who reported that earlier recurrences were associated with BRAF-negative cases, Fregnani et al.[8] and Shirsat et al.[11] demonstrated that positive BRAF V600E immunoexpression significantly correlated with the presence of recurrences.
Gültekin et al.[2] conducted a study wherein next-generation sequencing was applied to study 28 different genes: ARAF, BRAF, CDK4, CDKN2A, CTNNB1, DDR2, EGFR, ERBB2, FGFR2, FGFR3, GNA11, GNAQ, HRAS, IDH1, KEAP1, KIT, KNSTRN, KRAS, MAP2K1, MET, NFE2L2, NRAS, OXA1L, PDGFRA, PIK3CA, PTEN, RAC1, and TP53. In addition, SMO was analyzed by Sanger sequencing. They observed that BRAF mutations occurred almost exclusively in mandibular tumors, SMO mutations predominantly in maxillary tumors, and single mutations in EGFR, KRAS, and NRAS. KRAS, NRAS, PIK3CA, PTEN, CDKN2A, FGFR, and CTNNB1 mutations co-occurred in the background of either BRAF or SMO mutations. Multiple mutations were exclusively observed in European patients, in solid ameloblastomas and were associated with a very high risk for recurrence. In contrast, tumors with a single BRAF mutation revealed a lower risk or relapse.
In our study, statistically significant difference between recurrences in BRAF V600E immunoexpression positive cases versus recurrences in BRAF V600E immunoexpression negative cases was not obtained (P = 0.330). The reason could be that our sample size included only 3/33 (9.1%) cases of recurrent ameloblastoma of which 2/3 (66.7%) cases were positive for BRAF V600E mutation and 1/3 (33.3%) case was negative for the mutation.
Since ameloblastomas are aggressive tumors, resection of the tumors causes facial deformity and increased patient morbidity. Various case reports have shown that treatment of patients harboring the BRAF V600E genetic mutation with BRAF inhibitors like dabrafenib and vemurafenib led to reduction of the tumor volume by shrinkage of the lesion.[7],[15],[25] Hence, detection of this genetic mutation would help to significantly reduce patient morbidity.
The positive benefits of BRAF inhibitors must be weighed against the possibility of compensatory activation of the EGFR pathway and development of resistance. This underscores the need for combined detection of the BRAF V600E, SMO mutations and EGFR overexpression in order to deliver optimum therapy to individual patients.
Conclusion The present study revealed a high expression of the BRAFV600E mutation in mandibular ameloblastomas among Indians irrespective of the age, sex, site, subtype, or histological pattern; the largest number of ameloblastomas was the unicystic subtype. The study also confirmed that a subset of BRAF V600E mutation-negative ameloblastomas exist, including recurrent cases. The cytoplasmic expression detected by immunohistochemistry proved to be specific for the BRAF V600E mutation making it a viable option for incorporation into the routine diagnostic workup for ameloblastoma. BRAF inhibitors have been successful in promoting shrinkage of ameloblastomas. The identification of this driver mutation opens the possibility of an adjuvant therapeutic modality to reduce the significant facial disfigurement and morbidity following surgical management.
Acknowledgements
Schematic representation of the BRAF pathway was created with BioRender.com.
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Conflicts of interest
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References 
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CheckDOI: 10.4103/ijpm.ijpm_398_21
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