The association between high‐risk human papillomavirus and oral lichen planus

Oral lichen planus (OLP) is a cell‐mediated inflammatory mucosal disorder and is classified as an oral potentially malignant disorder. Some research has shown that apoptosis in OLP cells is similar to a viral infection such as human papillomavirus (HPV). So, the aim of this case‐control study was to investigate the association of high‐risk HPV with OLP.


| DNA extraction
The DNA of all 50 paraffin-embedded samples was extracted with a high pure polymerase chain reaction (PCR) template preparation kit (Cat. no: 11796828001, Roche Diagnostics GmbH, Penzberg, Germany), according to the manufacturer's instructions for FFPE tissue specimens.
The quality and quantity of extracted DNA were evaluated by NanoDrop 2000 (Thermo Fisher Scientific, Waltham, United States of America) and stored at −20°C for molecular applications.

| PCR assay
PCR was used to investigate the presence of HPV16 and HPV18 in 200 ng of DNA template by previously described primers (Table 1).
Each PCR reaction contained 12.5 µl of 2× Taq  universal gradient thermocycler (PEQLAB, GmbH, Germany) was used for DNA amplification, starting with the initial denaturation step for 5 min at 94°C, followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 56°C for 30 s and extension at 72°C for 45 s. In the end, the final extension was completed at 72°C for 10 min. HPV16 and HPV18 genomic DNA were used as positive controls. An image was taken of the PCR products under UV light following 2% agarose gel electrophoresis. The PCR product size (bp) was determined by comparing it with 1 kb DNA ladder ( Figure 1).

| Statistical analysis
All statistical analyses were run using the SPSS software version 22 (Chicago, United States of America). Associations were determined by using the Chi-Square test or Fisher's exact test. Statistical significance was assumed if p < .05. All reported p values are two-sided. Table 2 summarizes the demographic data of the studied samples (Table 2). 56% of OLP specimens were of the non-erosive-atrophic (non-EA) subtype, and 44% were of erosive-atrophic (EA) subtype.

| RESULTS
Twelve of 25 (48%) of OLP tissue samples were positive for HPV16, compared with 6 of 25 (24%) of normal control tissue samples; although there was not any significant difference between studied groups based on the frequency of HPV16, the difference was borderline (p = .07). Our results showed the frequency of HPV16 in the OLP group was twice as high as the control group (Table 3). 120 Syrjänen et al. (1991) On the other hand, 3 of 25 (12%) of OLP tissue samples were positive for HPV18, compared with 1 of 25 (4%) of normal control tissue samples; there was not any significant difference between studied groups based on the frequency of HPV18 (p = .3).
However, the total frequency of both HPV16 and HPV18 were 14 of 25 (56%) and 7 of 25 (28%) in OLP and normal control tissue, respectively; there was a significant association between the presence of high-risk HPVs and OLP (p = .04). Simultaneous presence of HPV16 and HPV18 was detected in a sample of the non-EA OLP subgroup.
There was no significant association between the prevalence of high-risk HPVs in studied groups based on gender ( Table 4).
Comparison of the OLP subgroups showed that the high-risk HPVs were more prevalent in EA-OLP as compared to non-EA, although no statistically significant association was found (p = .13; Table 5).

| DISCUSSION
This study aimed to investigate the association of high-risk HPV with OLP; although the difference in HPV16 prevalence in OLP and control groups was not significant, it was borderline. The difference in HPV18 prevalence in the studied groups was not significant.
However, there was a significant association between both high-risk  (Syrjänen et al., 2011). However, in some studies, there was no evidence of HPV presence in the normal oral mucosa (Campisi et al., 2004;González et al., 2007;Sameera et al., 2019;Sand et al., 2000). In Sand et al.'s (2000) study, HPV DNA was detected in 27% and 0% in the OLP and healthy control groups, respectively; in the Swedish cohort, 83% of OLP samples were positive for HPV18 by PCR method. In another study on the Indian population, the HPV18 detection rates were 86% of OLP samples and 0% of controls (Sameera et al., 2019). Older studies have found no significant association between the presence of HPV and OLP (Miller et al., 1993;Young & Min, 1991 were more prevalent in EA-OLP as compared to non-EA, although no statistically significant relationship was found. EA-OLP has more malignant potential than non-EA OLP. In a study conducted by Campisi et al. (2004), HPV DNA was found in 18.5% of non-EA OLP and 20.4% of EA OLP, without any significant association. In a study by Mattila et al. (2012), HPV was detected in 15.9% of EA-OLP samples, but the importance of the presence of HPV in EA-OLP was somewhat obscure due to the predominance of low-risk HPV genotypes.
According to the contradictory results obtained from previous studies regarding the association between HPV infection and OLP, in recent years (especially the last 5 years), more and more studies have focused on investigating this association. A summary of these studies is presented in Table 6 to get a better understanding of the current situation in this field (Table 6)