Abstract
Background:
The invasive nature of ameloblastoma necessitates tumour-free surgical margins to minimise recurrence and thereby ensure improved prognosis. The vast majority of ameloblastomas originate centrally, and surgical management results in significant loss of affected structure, and therefore often requires extensive reconstruction. Recently, it has been shown that the mitogen activated protein kinase (MAPK) pathway plays an important role in the pathogenesis of ameloblastoma. B-Rapidly Accelerated Fibrosarcoma gene (BRAF, also known as B-Raf proto-oncogene, serine/threonine kinase) (HGNC:1097) is essential in the MAPK pathway physiology. Mutation involving BRAF V600E has been demonstrated in melanoma as well as in ameloblastoma. Although BRAF mutation has been shown in ameloblastoma, its implications relating to tumour stromal angiogenesis and proliferation remains elusive. This study aimed to assess the expression of BRAF V600E mutation in ameloblastoma using immunohistochemistry (IHC) and the correlation of the findings of these results with stromal vascularity and lesional proliferation. The results were then contextualized in relation to clinico-pathologic parameters.
Aim of the study: The aim of this project was to profile BRAF mutation in ameloblastoma and to correlate this with clinical and histological parameters and patient outcomes.
Objectives:
Firstly, to assess the expression of BRAF V600E mutation by means of IHC in tissue sections derived from cases of ameloblastoma. Secondly, and to correlate these results with stromal tissue vascularity determining the micro-vessel density (MVD) using CD34 and lesional proliferation using Ki-67. Finally, to correlate the presence of the mutant protein to the clinic-pathologic parameters.
Materials and Methods:
Formalin-fixed paraffin-embedded samples of ameloblastoma were retrieved. These samples were stained with antibodies against BRAF V600E (Ventana; 06918727001), Ki67 (Ventana; 05278384001) and CD34 (Ventana; 05278210001). The immunoglobulin G (IgG) isotype was used for negative control. Hotspot locations in the epithelium and connective tissue (n=3 each) from each specimen were photomicrographed and semi-quantitatively analysed for the intensity score while the proportion score was assessed qualitatively through the use of ImageJ plugin. Intensity and proportion scores were used to calculate the immunoreactive score (IRS). The microvessel density (MVD) and the proliferative index (PI) was assessed through the use of the ImageJ plugin gridlines over the hot spot areas that had been photomicrographed and the micro-vessels were then counted with the cell counter. Clinico-pathologic data was entered into an Excel spread sheet and data analysis using Fisher’s Exact and Pearsons tests were performed in GraphPad PRISM software. The p-value of less than 0.05 (p <0.05) denoting statistical significance was referenced.
Results:
A total sample of 44 ameloblastoma cases were included in this study. Most of the samples were predominately of the follicular pattern of ameloblastoma (59.1%) with a smaller proportion of lesions showing plexiform histological patterns (29.5%). The mean age at diagnosis was 36 years (range of 10-73). The male to female ratio was 1.2: 1 with 89% of the cases affecting the mandible. The neoplastic epithelium in 93% of cases was BRAF positive (BRAF+) and 27% of these displayed high reactivity (IRS>5). 81.8% of mandibular lesions were BRAF+. CD34 MVD and Ki-67 PI corresponded with higher BRAF reactivity. There was an independent statistically significant association observed between the expressed mutant protein and the recurrence of ameloblastoma. A significant association between ameloblastoma recurrence together with higher MVD and PI was also observed.