Detection of Human PapillomaVirus and its Association with Potentially Malignant Disorders and Oral Squamous Cell Carcinoma: A Retrospective Study.

Aim: This study was carried out with the objective of recognizing the involvement of human papillomavirus (HPV) in conditions of oral squamous cell carcinoma (OSCC) and oral potentially malignant conditions and lesions. Materials and
Methods: OSCC samples from thirty patients were collected and thirty paraffin-embedded, oral premalignant specimens were collected. It was decided to use as a negative control samples gingiva from healthy individuals who had no prior history of oral proliferative leukoplakia or OSCC. The samples were then processed using a paraffin-embedded tissue block method. Deoxyribonucleic acid will be extracted from the paraffin-embedded tumor sample of sixty case subjects and tested for the presence of HPV DNA using polymerase chain reaction.
Results: In study Group 1, 24 samples were taken from males and 6 from females. In study Group 2, 26 cases were males and 4 were females. In Group 1, 16 cases of moderately differentiated and 14 cases of well differentiated each were present. No case of poorly differentiated were found. There was statistically no significant difference present between male and female when Group 1 and Group 2 was considered. In study Group 1, 0 samples had positive HPV DNA detection, whereas 30 samples had negative HPV DNA detection. In study Group 2, 0 samples had positive HPV DNA detection, whereas 30 samples had negative HPV expression.
Conclusion: According to our findings, certain incidences of OSCC may be linked to HPV, however, this was not the case for all of the cancers. Copyright:

INTRODUCTION

One in every 563826 new cases (including 274,850 oral cavity cancers, 159,363 laryngeal cancers, and 52,100 oropharyngeal cancers), as well as the 301,408 fatalities caused by human squamous cell carcinoma (HNSCC), occur each year.[12] Squamous cell carcinomas of oral cavity, oropharynx, larynx, and hypopharynx make up the majority of cases of head-and-neck cancer. There has been an increase in the occurrence of oropharyngeal squamous cell carcinoma (OPSCC), especially among younger males, that involves the palatine and lingual tonsils. Tobacco and alcohol use are two of the most important risk factors for HNSCC.[3]

It was first hypothesized in the 1980s that human papillomavirus (HPV), specifically OPSCC, could be a cause of high-grade nonsmall cell lung cancer in people who do not smoke or drink alcohol.[45] More and more evidence from the fields of epidemiology and molecular biology points to the possibility that certain types of high-risk human papillomavirus, particularly types 16 and 18, are to blame for the emergence of certain types of head-and-neck cancer, particularly OPSCC.[67]

The rate of detection of HPV in premalignant lesions is believed to raise as the severity of the abnormality of disease increases. Some recent research has shown that HPV is present in Oral Squamous Cell Cancer. To better understand the diagnosis and treatment of oral squamous cell carcinoma (OSCC) and other potentially malignant lesions in the mouth caused by HPV, further study is required.[8910] Therefore, this study was carried out with the objective of recognizing the involvement of HPV in conditions of OSCC and oral potentially malignant conditions and lesions with the help of paraffin-embedded blocks.

MATERIALS AND METHODS

This review was endorsed by the Institutional Ethical Committee of PCDS and RC. OSCC tests from thirty patients were gathered from Jawaharlal Nehru Cancer Hospital and Research Center and thirty paraffin-inserted, oral premalignant examples from the Department of Oral Pathology, People's College of Dental Sciences, PCDS, and RC. Negative control tests were chosen as typical tissue examples from gingiva from patients without a background marked by oral proliferative leukoplakia (OPL) or OSCC. The paraffin-inserted tissue squares of analyzed instances of OSCC related with (n = 20) and without (n = 20) tobacco as well as liquor use and the oral mucosa of solid controls (n = 20) without tobacco or potentially liquor use were gathered from the files of the Department of Oral Pathology and Microbiology, People's College of Dental Sciences, Bhopal. DNA will be extracted from the paraffin-embedded tumor sample of sixty case subjects. The DNA of samples will be tested for the presence of HPV viral DNA [Figure 1] using polymerase chain reaction (PCR).

Figure 1

Thermal cycler polymerase chain reaction

Procedure

Weigh 2 g of agarose powder and mix in 100 ml of 1 × TAE buffer (for 2% agarose)

Boil with gentle stirring till homogenous clear solution is formed

Cool for some time. Add 2 μl of ethidium bromide (0.5 μg/ml)

Pour into gel mold and place the comb. Allow to settle for at least 20 min

Remove the comb carefully. Keep the gel in electrophoresis unit [Figure 2] (3B BlackBio, Bhopal) containing 1 × TAE buffer (200 ml). The gel should be completely submerged in the buffer

Figure 2

Gel Electrophoresis unit

Red color buffer in the amplified sample will act as loading dye

Take 20 μl of this amplified product and add carefully into the wells; Load molecular weight marker in the last well

Fix the electrode, turn on the power supply and adjust the current (16A)

Run the gel for 2 h

Then, take the photo of gel under UV light transilluminator and record the bands using gel documentation system (Major Science, USA) [Figure 3].

Figure 3

UV transilluminator (for gel analysis)

Single-strand conformation polymorphism

A single-strand conformational polymorphism was studied using (-p32) dCTP-specific activity, 4000 Ci/mmol; BARC, Mumbai, India, radiolabeling of PCR products after thirty cycles of regular amplification. The PCR mix contained (-p32) dCTP. Consequently, 100 L of regular PCR mix with 1 L of (-p32) dCTP was added to, and all other reaction components were left unchanged, for ten samples (10 L each). For 5 min at 95°C, 1 L of radiolabeled PCR product was heat denatured and then refrigerated for 5 min on ice before being mixed with nine volumes (ten times) of denaturing solution (95% formamide, 20-mM ethylenediaminetetraacetic acid (EDTA) pH – 8.,05% xylene cyanol, and. 05% bromophenol blue). It was decided to subject 3 L of this dilute sample to nondenaturing gel electrophoresis in 6% polyacrylamide gel with 10% glycerol. The gel was run in 0.5 XTBE (0.045M Tris borate, 0.001M EDTA pH – 8.0) for 12 h at 200V at 17 ± 1°C in a base sequencing gel device (Stratagene GmbH, Germany). Following a 48 h drying period, the gel was exposed to X-ray film using an intensifying screen and a 70°C temperature setting. The electrophoretic mobility of single-strand DNA bands was examined by making comparisons with controls that were healthy. The following diagram shows an example of this.

RESULTS

There were sixty tissue blocks from the Department of Oral Pathology's archives that were used for this investigation.

Group 1: thirty diagnosed cases of OSCC.

Group 2: thirty diagnosed cases of premalignancy.

Group 3: ten normal oral mucosa.

Gender distribution

In study Group 1, 24 samples were taken from males and 6 from females. In study Group 2, 26 cases were males and 4 were females.

Histopathological grading

In Group 1, 16 cases of moderately differentiated and 14 cases of well differentiated each were present. No case of poorly differentiated was found.

Men and women in Group 1 did not show any statistically significant differences [Table 1 for further information]. There was no statistically significant difference between male and female participants in Group 2 [Table 2].

Table 1

Human papillomavirus DNA detection and gender distribution in study Group 1

HPV	Gender		Total	P
	Male	Female
Positive	0	0	0	1
Negative	24	6	30
Total	24	6	30

HPV: Human papillomavirus

Table 2

Human papillomavirus DNA detection and gender distribution in study Group 2

HPV	Gender		Total	P
	Male	Female
Positive	0	0	0	>1
Negative	26	4	30
Total	26	4	30

HPV: Human papillomavirus

Association among study groups (Group 1, 2, and 3) between human papilloma virus and DNA detection

In study Group 1, 0 samples had positive HPV DNA detection, whereas 30 samples had negative HPV DNA detection. In study Group 2, 0 samples had positive HPV DNA detection, whereas 30 samples had negative HPV expression. In no way, shape, or form did the research, Groups 1, 2, and 3 have any connection to HPV DNA.

Association among study groups (Group 1, 2, and 3) between P53 mutations

In study Group 1 and 2, no samples showed the presence of any p53 mutation. In no way, shape, or form were study Groups 1, 2, or 3 linked to a p53 mutation [Table 3].

Table 3

p53 mutation status in study Group 1, 2

p53 mutation	Study group		Total	P
	Group 1	Group 2
p53 mutation present	0	0	1	>0.5
p53 mutation absent	30	30	60
Total	30	30	60

DISCUSSION

The HPV genome prevalence in OSCC varies from 2.9% to 49.5% because the basal cells are exposed on the oral mucosal surface, this region has the highest HPV risk. Squamous epithelium covers the oral mucosa; hence, it is crucial to examine the link between HPV infection and oral squamous cell cancer (OSCC).[6] There have been several studies showing a correlation between HPV and OSCC, however, the precise nature of this relationship to oral carcinogenesis is still unknown.[11]

The goal of this investigation was to look for HPV DNA in OSCC, PMDs, and normal mucosa. Observations on the prevalence of oral HPV in healthy individuals, PMDs, and OSCC have ranged from 0% to 100%, according to various studies.[9] The findings seemed to be influenced by the sample procedures, the patient's characteristics, the detection technologies used, and the anatomical position of the tumors. We used PCR with primers and probes specific to HPV to test thirty OPML and thirty oral SCCs for the presence of HPV DNA, which has been found often in cervical carcinomas. No HPV DNA sequences were found in any of the thirty samples from OSCC and OPMD. In this study Group 1 and 2, no samples showed the presence of any p53 mutation. In no way, shape, or form were study Groups 1, 2, or 3 linked to a p53 mutation. Preliminary results from studies looking at oral cancer samples using PCR-based approaches support this conclusion.[10]

According to Carey TE, there was no connection between HPV, OPL, and OSCC, which supported our results. Although HPV was shown to be much more prevalent in OPL than OSCC, Raj, AT et al. believe this might be an early marker of malignant transformation since it was found to be significantly more prevalent in OPL than OSCC. Diversification of the population is also thought to effect rate changes in several ways.[11] OSCC HPV detection rates are 0% in some countries, including India, Brazil, Japan, and Mozambique. Other reported detection rates include 1.54% in Thailand, 6.6% in America, 5% in Mexico, 39.4% in Spain, and 66.7% in Sudan. Study findings using traditional PCR tests in China vary widely, from 2.2% to 74% of the whole sample. There was a modest frequency of HPV (1.1%) in OPMD, according to Ha and colleagues. The variation may be due to differences in the types of samples or detection methods.[891011]

SCCs of the oropharynx were more likely to be infected with HPV than those of the oral or larynx. Gingival biopsies have also been shown to contain HPV, which suggests that the gingiva might be a potential reservoir for the virus. Latent HPV infection may be to blame for the presence of HPV in normal oral mucosa. There was no correlation between the prevalence of HPV infection and factors such as gender and age. This is consistent with previous research.[11]

CONCLUSION

HPV may have a role in the development of head-and-neck cancers, according to prior research. Molecular processes have recognized its capacity to disrupt crucial cellular components that regulate cell division and death. According to our findings, certain incidences of OSCC may be linked to HPV, however, this was not the case for all of the cancers.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.