Oral lichen planus (OLP) is a chronic inflammatory disease in which the immunopathogenesis involves cell-mediated immune dysregulation [
OLP is classified as a potentially malignant disorder (PMD) of the oral mucosa with a transforming rate of 0–6.25% [
Human papillomaviruses (HPVs) are epitheliotropic DNA viruses with more than 150 genotypes. Clinically, HPV infection is usually characterized by hyperplastic, papillomatous, or verrucous lesions in the stratified epithelium. HPV classification has been based on the degree of HPV DNA homology. HPV has been detected in various types of oral lesions, ranging from benign to malignant [
A causal role of HPV has been reported for OLP and OSCC, but there are wide variations in disease prevalence with regard to different geographic populations [
Most HPV infections are asymptomatic; however, lesions can develop any time after infection. Currently, there is no clear understanding of HPV latency, reactivation, or subclinical infection without apparent disease [
The aims of this study were to investigate the presence of HPV DNA in OLP lesions of Thai patients using PCR and to characterize the virus type in those lesions by nucleotide sequencing.
Participants with a clinical diagnosis of OLP who visited the Department of Oral Medicine, Faculty of Dentistry, Chulalongkorn University from November 2008 to August 2010 were recruited into the study. Prior to enrollment, the patients signed informed consent forms approved by the Ethics Committee of the Faculty of Dentistry. None of the participants received any medication for their OLP before specimens were obtained. Specimens were collected from tissue biopsies for histopathological examination. Thirty-seven cases that were confirmed as OLP positive based on histopathological evaluation were selected for DNA extraction. Each specimen was transferred to a 1.5 mL sterile microcentrifuge tube containing 0.5 mL of virus transport media. Matching controls from each subject were obtained by scraping healthy normal mucosa into a 1.5 mL sterile microcentrifuge tube containing virus transport media. All samples were kept on ice, immediately taken to the laboratory, and stored at −80°C until further analysis.
DNA extraction and purification from both tissue biopsies and negative control cells were performed using the Qiamp DNA mini kit (QIAGEN, Valencia, CA) per the manufacturer’s instructions. DNA extraction from control sites was performed by standard organic extraction (phenol-chloroform) and alcohol precipitation. Purified DNA was re-suspended in deionized water to a final volume of 30
We assayed for HPV DNA using primers to both the E1 and L1 regions. The primer design, amplification, and sequencing were performed [
The sequences were aligned with the BioEdit program (version 7.0.4.1). The phylogenetic trees were constructed using the MEGA4 program with neighbor-joining analysis, and Bootstrapping applied with 1,000 replicates was used to support tree topologies.
The study comprised 37 participants, ranging from 19–78 years old, with a median and mean age of 48 and 49 (±12.58) years, respectively. The male to female ratio was 12 : 25. The 37 specimens were obtained from 12 hyperplastic-, 23 atrophic-, and 2 ulcerative-type OLP lesions. All subjects had lesions on both the buccal mucosa and gingiva at the time of biopsy. All specimens from the tissue biopsies had a confirmed diagnosis of OLP (Figure
Histopathology of tissue biopsies confirming oral lichen planus. (a) Magnification ×100 shows hyperkeratosis, a bandlike of lymphocytes infiltrate in the superficial lamina propria, and (b) magnification ×200 shows degeneration of basal cell layer, various degrees of parakeratosis, and orthokeratosis of the surface epithelium, dense infiltrate of lymphocytes subjacent to the epithelium, and degenerating keratinocytes.
Agarose gel electrophoresis result of human papillomavirus E1 nested PCR detection. M: Marker, DNA ladder (Fermentas), S: sample, P: positive control, and N: negative control. DNA band in S4 indicates the sample with positive result.
Phylogenetic tree in E1 region of HPV16 variants.
OLP affects 1.27% of the global population with a prevalence varying according to geographic locations [
The present study supports the finding of a previous report on HPV prevalence in OLP in Thai patients. A prior study in Thai patients did not find HPV in any of the sixteen OLP lesions examined [
A systematic review shows a statistically significant twofold difference (11% versus 23%) between HPV associated with OLP lesions and HPV found in normal tissue [
The
Efforts in exploring the correlation between HPV and OLP have mainly focused on epidemiological studies of different populations. It is noteworthy that there have been profound variations in the results found among geographically different populations [
The DNA sequence of the HPV found in the present study was identified as the high-risk-type 16. The lesion positive for HPV DNA was of the atrophic type. Mattila et al. [
A predisposition of the oropharyngeal mucosa to malignant transformation by HPV was first suggested when HPV16 was detected in tumors of the tongue, tonsil, and pharynx but not in control tissues [
OLP is an inflammatory immune-mediated disease associated with cell-mediated immunological dysfunction. Infectious agents have been proposed as one of the causes of OLP. Our data showed a low prevalence of HPV infection in OLP lesions. We conclude that HPV may not play a significant role in Thai patients with OLP. Genomic sequencing of the E1 gene demonstrated that the HPV-type 16 from the sole positive lesion was closely related to the East Asian type. Although it is likely that additional factors affect malignant transformation, identifying these will require the long-term followup of HPV-positive OLP patients.
This study was supported by the Thailand Research Fund MRG5280047, Center of Excellence in Clinical Virology, Department of Pediatrics, Faculty of Medicine, Chulalongkorn University, Chulalongkorn Memorial Hospital, Outstanding Professor Fund, Thailand Research Fund and The Higher Education Research Promotion, and National Research University Project of Thailand, Office of the Higher Education Commission (HR1155A). This study was conducted according to the ethical standards of the World Health Organizations Declaration of Helsinki (version 2002), and the study protocols were reviewed and approved by the Ethics Committee of the Faculty of Dentistry, Chulalongkorn University, reference no. 86/2008 with the understanding and with signed informed consent of the patients involved. The authors thank Dr. Pairoj Junyangdikul, for providing laboratory instruments. They would like to thank Associate Professor Kittipong Dhanuthai for the histopathology confirmation and Dr. Klawajee Kaetkaew and the staff at the Department of Oral Medicine for their assistance. The authors would like to express their gratitude to Dr. Kevin Tompkins for his critical editing.