Thasvir Singh1,2*, Christopher Angel3, Sepehr Tabrizi4, Alyssa Cornall4,6, John Clement2 and Arun Chandu2
1Oral and Maxillofacial Surgery, the Royal Melbourne Hospital, Parkville, Victoria 3050, Australia
2Melbourne Dental School, Faculty of Medicine, Dentistry and Health Sciences, the University of Melbourne, Melbourne 3010, Victoria, Australia
3Peter MacCallum Cancer Centre - East Melbourne, St Andrews Place, East Melbourne 3002, Victoria, Australia
4Regional HPV Laboratory Network, Department of Microbiology and Infectious Diseases, The Royal Women’s Hospital, Victoria, Australia
5Department of Obstetrics and Gynecology, University of Melbourne, Parkville, Victoria, Australia
6Murdoch Children’s Research Institute, Royal Children’s Hospital, Flemington Road Parkville, Victoria, Australia
Received: 22 April, 2016; Accepted: 06 May, 2016; Published: 11 May, 2016
Thasvir Singh, Oral and Maxillofacial Surgery Office C/- 2 North, The Royal Melbourne Hospital, Parkville 3050, Victoria, Australia, Tel: +61 3 9342 8892; 61402579677; E-mail:
Singh T, Angel C, Tabrizi S, Cornall A, Clement J, et al. (2016) Ameloblastoma, Human Papillomavirus, and p16-is there An Association?. Arch Otolaryngol Rhinol 2(1): 020-024. DOI: 10.17352/2455-1759.000016
© 2015 Singh T, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Background: The aetiology of the ameloblastoma is still unclear. Several studies have searched for the presence of human papillomavirus (HPV) in ameloblastomas, however the results have been mixed. Our aim is to clarify this possible association, and to determine if p16 is a reliable surrogate marker for the presence of HPV in these tumours.
Methods: Forty-one cases of ameloblastoma and 1 ameloblastic carcinoma were identified and underwent immunohistochemical (IHC) testing for p16 and polymerase chain reaction (PCR) testing to detect HPV DNA.
Results: 80.5% of the benign ameloblastomas were ‘overall positive’ for p16 (56.1% strong positive, and 24.4% moderately positive) and 19.5% ‘overall negative’. The single case of ameloblastic carcinoma also stained strongly positive for p16. PCR did not detect HPV DNA in any of the 42 cases.
Conclusions: We did not find any positive HPV ameloblastomas within the samples evaluated. Despite most ameloblastomas being p16-positive, p16 cannot be used as a marker of HPV within these tumours.
Ameloblastoma is a benign odontogenic tumour with four main subtypes , solid/multicystic, unicystic, desmoplastic, and peripheral. In general, most ameloblastomas are locally aggressive, and can grow to a considerable size resulting in significant morbidity, and even mortality . They have a propensity to recur if treated inadequately, and thus radical surgical management is the mainstay of treatment [3-14]. Despite clinicians recognising the entity for over a century, the aetiology of this tumour is still unclear. It is thought to develop from epithelial remnants of the dental lamina/enamel organ, and various theories on causative agents have been proposed in the literature including trauma, dental extraction, severe oral sepsis, and viruses such as the human papillomavirus (HPV) [15-21].
HPV is a DNA virus that has come under renewed scrutiny due to its recently discovered association with oropharyngeal squamous cell carcinoma (OPSCC) [22-24]. HPV-positive OPSCC has an improved prognosis compared with HPV-negative tumours. With further research HPV-positive tumours may result in de-escalated chemoradiotherapy or bioradiotherapy treatment options, in an effort to reduce acute and long-term treatment related toxicities [25-29]. In OPSCC, p16 immunohistochemistry is used as a reliable surrogate marker for HPV infection [29-36].
A possible association between ameloblastoma and HPV has attracted some attention from researchers over the years. HPV may gain access to intraosseous lesions via contact with the overlying oral mucosa, surgical manipulation of the surrounding structures prior to tumour development, and early onset exposure to the HPV during invagination of the enamel organ. Although sexual contact would be extremely unlikely during the stages of dental development, perinatal or salivary transmission of HPV is possible [37-39]. Contamination of the tumour specimen by HPV either during the respective process, or post-operatively, is also possible. Several case reports and studies have explored this relationship (Table 1), but no firm conclusions have been reached. Use of p16 immunohistochemistry in ameloblastoma has not been thoroughly investigated.
This study aims to establish whether HPV DNA is associated with ameloblastomas, and to determine if p16 is a reliable surrogate marker for the presence of HPV in these tumours.
Materials and Methods
Ethical approval for this study was granted by The Melbourne Health Human Research Ethics Committee. Forty-nine cases of ameloblastoma were retrieved from The Royal Melbourne Hospital databases over an 11-year period (2001-2012). The histopathological slides and patient case notes were evaluated by an oral and maxillofacial surgical registrar and specialist oral and maxillofacial pathologist. Using the World Health Organization classification system , the diagnosis of each was confirmed and subtyped accordingly. Forty-two cases were included in the study, with 7 cases excluded due to insufficient clinical and/or pathological information.
p16 Immunohistochemistry (IHC)
Formalin fixed paraffin embedded (FFPE) tissue of the 42 ameloblastomas were cut into 4μm thick sections and then placed onto Superfrost® Plus Microscope Slides (Thermo Scientific, Lower Saxony, Germany) prior to being tested with p16 antibody (Roche CINtec®, Ventana Medical Systems, Inc, Arizona, USA) at a 1:3 dilution. Sections were dewaxed through a series of xylene solutions (x3) followed by absolute ethanol (x2), 70% ethanol and distilled water. Antigen retrieval was performed using a high pH antigen retrieval solution and IHC staining was performed on the Leica BOND-MAX™ automated IHC stainer (Leica Biosystems, Melbourne, Australia) using Bond™ Polymer Refine Detection system. Negative and positive controls (basaloid squamous cell carcinoma in lymphoid tissue) for were also processed using the same method for each specimen slide.
To grade the tumours for p16 positivity, 10 randomized areas of the tumour were evaluated for the number of positive cells under high magnification (400x) [40-42]. P16 negative cells were also counted in the same areas and a percentage formulated by dividing the number of positive cells by the number of total cells (1000). This percentage was classified at different levels to grade the tumour’s positivity (Table 2). To account for any inaccuracy in grading, tumours were also graded as overall negative (<25%) and overall positive (25 – 100%).
Polymerase Chain Reaction (PCR) detection of HPV DNA
The 42 FFPE tissue blocks were cut into three 7μm sections (without hydration) and underwent preparation for PCR. One of the sections from each block was de-waxed with histolene and ethanol, and digested with proteinase K on a heat block overnight . Where sections were resistant to complete tissue digestion, physical disruption of tissue with vortexing and beads were applied. DNA was extracted from digested tissues on an automated system - the MagnaPure LC (R) (Roche Diagnostics GmbH, Penzberg, Germany) using the DNA-I kit (blood cells high-performance protocol). An 110bp section of the human beta-globin gene was detected by quantitative real-time PCR to confirm successful DNA extraction . Samples were screened for the presence of HPV DNA using the DNA ELISA Kit HPV SPF10, version 1 (Labo Bio-medical Products BV, Rijswijk, The Netherlands) and ELISA-positive samples were further tested for HPV genotype on the RHA kit HPV SPF10-LiPA25, version 1 (Labo Bio-medical Products BV).
A database was constructed and Minitab® Statistical Software (Pennsylvania, USA) was used for statistical analysis. Fisher’s exact tests were prepared for the PCR results relative to the p16 grading, and statistical significance was determined by p < 0.05.
Forty-one cases of benign ameloblastoma were identified and one case of ameloblastic carcinoma (AC). Solid/multicystic ameloblastoma was the most common subtype (34 cases), followed by unicystic ameloblastoma (6 cases), and peripheral ameloblastoma (1 case). There were no established cases of desmoplastic ameloblastoma or metastasizing ameloblastoma. Males (63%) were affected slightly more than females, and the mandible was involved in 80.5% of the cases.
Of the 41 benign ameloblastomas, 33 (80.5%) were deemed overall positive for p16, including 23 (56.1%) strongly positive (Figure 1), and 10 (24.4%) moderately positive tumours. Only 8 tumours (19.5%) were graded as overall negative for p16, with 2 tumours (4.9%) being totally negative and 6 tumours (14.6%) weakly positive. Thus 95.1% of benign ameloblastomas had some positive reaction to p16 IHC staining, with the majority of these being strong or moderately positive (Figure 2).
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