The prevalence and genotyping of human papillomavirus in patients with oral tumors in health centers and clinics of Mazandaran in Iran

Abstract

Oral cancer is one of the most prevalent cancers in the world which contains many kinds of malignant neoplasms in the oral cavity. Due to the carcinogenicity of human papillomavirus (HPV) and its prevalence in cancer, including the oral cancer, this study was aimed at investigate the prevalence of HPV and its genotypes in patients suffering from oral tumors using PCR method. In this study, 83 samples of oral lesions were collected in the form of paraffin-embedded tissue. After extracting the DNA using DNA extraction kits, high-risk HPV positive samples were examined using special kits for genotyping, and low-risk types were sequenced after nested PCR. The results showed that 13.2% of samples was HPV positive. The result of PCR using genotyping kit indicated that high-risk types of 18, 31, 16, and 33 appeared in samples with prevalence rate of 27.2, 18.1, 9.09 and 9.09%, respectively. In this manner, the result of sequence indicated that the prevalence of HPV-6 genotype was 36.3% in the samples. The results of this study indicated that both low-risk and high-risk types of HPV are associated with the risk of oral tumors, so that Types 6 and 18 were reported as the most prevalent types in the samples.

Introduction

Oral cancer is known as the sixth most common cancer in the world, so that about 95% of cases are squamous cell carcinoma (SCC) [17, 24]. Epidemiological and molecular analyses indicated that human papillomavirus (HPV) is involved, as a factor, in oral cancer [15].

HPV is a tiny virus with a diameter of 45–55 nm belonging to papillomaviridae family. The genome of this virus contains a double-stranded DNA molecule with 8 kb size which is located in an uncoated protein capsid with cellular histones [3, 15, 17, 27]. Up till now, more than 200 different types of HPV have been isolated which are classified into high-risk and low-risk groups [15, 19, 22]. Low-risk types of HPV, such as HPV-6 and HPV-11, produce benign warts, and high-risk types, such as HPV-16 and HPV-18, can cause neoplasm precancer in squamous epithelial, that can progress to cancer [22, 23, 25]. About 90% of all HPVs associated with cancer are caused by HPV-16 [10, 11, 14] and can be connected with different kinds of cancers such as breast cancer, cervical cancer, laryngeal cancer, oral cancer and anal cancer [16, 18, 21, 26, 30].

These HPV viruses can enter the body through contacting small wounds of skin and cause hyperproliferation of skin and mucosa epithelial cells [32]. These viruses are one of the etiologic agents in the development of oral cavity cancer by contaminating basal cells of squamous epithelium and oral mucosa, and contribute 25.6% to the development of oral-pharyngeal carcinoma [4, 8, 15]. In the patients suffering from advanced stages of cancers, invasion to surrounding tissues, with lymph nodes and distant metastasis, has been identified, and there is a risk of second malignancy during the patient’s life time [13, 24]. Some of the risk factors for oral cavity cancer include: sexual intercourse behaviors, smoking, chewing areca and drinking alcohol [1, 12, 22, 31, 34].

The HPV prevalence rate varies in different areas from 0 to 91% [6, 29]. Furthermore, the development of oral carcinoma is increasing nowadays, so the aim of this study is to investigate the prevalence rate of HPV, and to appoint its genotypes in patients with oral tumors using PCR method.

Materials and methods

Sampling

In this study, 83 samples of paraffin-embedded tissues, were collected from patients from the beginning of 2006 to the end of 2016 at Bouali Sina hospital in Sari, Iran. In this study, the lesions of SCC, Squamous papilloma, Neoplasia, Dysplasia, Hyperplasia, Leukoplakia, Wart and Keratosis were investigated in the area of mouth anal lips. The patients’ clinical characteristics, such as age, sex, marital status and their occupations, were recorded in their dossier.

DNA extraction and electrophoresis

To extract DNA from the paraffin-embedded tissues; at first, 10 micron slices were prepared from the blocks, then they were transferred to microtubes, and to remove paraffin from the tissues, 1 ml of xylene was added to each sample. After removing paraffin, the samples were washed with 1 ml of ethanol 100%. They were placed in Thermo-mixer at 37 °C for 10–20 min to be dried. Then, Qiagen kit (made in Germany) was used to extract DNA from the tissues and the amount of OD of the extracted DNA was determined with spectrophotometry. Two pairs of primers (from Takapouzist Company) were used for PCR and the amplification of piece of the gene. A pair of PCO4.B.globin and GH2O.B.globin primers were used to ensure the presence of DNA that generated a PCR product with the length of 268 bp. The MY11 and MY09 primer pairs were also used to replicate a part of L₁ piece of HPV genome. The replicated pieces were used to detect papillomavirus, which produced a PCR product with 454 bp length. After performing PCR, its product was investigated on the 1.5% agarose gel.

Extraction of gel and sequencing

To determine the type of low-risk HPVs, samples were sequenced. Nested PCR was used For HPV positive samples. In this method, MY-11 and MY-09 primers were used in the first PCR, and MY-09 and GP-5 primers (to increase the sensitivity of PCR) were used in the second PCR, which produced a 407 bp length band. The sequence of primers are given in Table 1. Then, the nested PCR product was examined on 1.5% gel. After cutting the gel, it was extracted by YTA kit (Yekta Tajhiz Azma, Iran), and was sent to Bioneer Company in Korea for sequencing.

Table 1.

Features of primers used

Size	Sequences(5′-3′)		Primers
268 bp	5′-CAACTTCATCCACGTTCACC-3′	PCO4 (F)	β globin
	5′-GAAGAGCCAAGGACAGGTAC-3′	GH2O (R)
454 bp	5′-GCMCAGGGWCATAAYAATGG-3′	MY11 (F)	HPV
	5′-CGTCCMARRGGAWACTGATC-3′	MY09 (R)
407 bp	5′-CGTCCMARRGGAWACTGATC-3′	MY09 (F)	HPV
	5′-TTTGTTACTGTGGTAGATAC-3′	GP5 (R)

Genotyping and electrophoresis

The HPV positive samples were investigated by amplisens genotyping kit (made in Russia) which could identify high risk types (16,18,31,33,35,39,45,52,56,58,59,66). The kit contents which contained PCR-mix, PCR-buffer and Polymerase, were combined with HPV-DNA in a volume of 25 μl mixture and were prepared for PCR. The PCR was performed in a suitable condition and temperature program; initial denaturation at 95 °C for 15 min, followed by 42 cycles of denaturation at 95 °C for 30 s, annealing at 63 °C for 40 s, extension at 72 °C for 50 s with a final extension at 72 °C for 1 min. All these processes were performed with positive and negative controls existed in the kit. After all, the results were examined on 2% gel.

Result and discussion

Oral HPV detection

After extraction of the DNA and performing PCR for 83 samples, 40(48.1% female) and 43(51% men) it was indicated that all of the samples (100%) were positive for beta-globin gene, and 11 samples (13.2%) had HPV gene (Fig. 1), among these patients it is found that 7 (5.81%) and 4(3.32%) of them were married and single positive HPV respectively.of which five samples were SCC (45.4%), three samples were hyperplasia (27.2%), one sample was squamous papilloma (9.09%), one sample was neoplasia (9.09%) and one sample was both neoplasia and dysplasia (9.09%).

Fig. 1.

M: DNA marker (100 bp), NC negative control, PC positive control, 1 and 3: β.globin gene for positive samples, 5: β.globin gene for negative sample, 2 and 4: HPV positive samples, 6: HPV negative sample

Six of these samples were detected on the tongue (54.5%), three of them on the lips (27.2%), and the other two samples were observed on the other parts of oral cavity (18.1%). Six samples (54.5%) of all positive HPV-DNA were observed in females, and five of them (45.5%) were observed in males. The age of these people ranged between 8 and 83 years old with the average age of 46.2. The characteristics of patients are presented in the Tables 2, 3.

Table 2.

Distribution of HPV according to age group of patients

	HPV negative (72)		HPV positive (11)		Total (83)
	Number	Percentage	Number	Percentage	Number	Percentage
Age
6.02	5	4.1	3	18.1	2	< 15
8.4	7	8.3	6	9.09	1	16–31
20.4	17	18.05	13	36.3	4	32–47
34.9	29	40.2	29	0	0	48–63
19.2	16	18.05	13	27.2	3	64–79
10.8	9	11.1	8	9.09	1	80 <

Table 3.

Distribution of HPV according to occupation group of patients

	HPV negative (72)		HPV positive (11)		Total (83)
	Number	Percentage	Number	Percentage	Number	Percentage
Occupation
Housewife	3	27.2	20	27.7	23	27.7
Free	4	36.3	27	37.5	31	37.3
Employee	2	18.1	17	23.6	19	22.8
Student	2	18.1	8	11.1	10	12.04

The results of PCR with genotyping kit (Fig. 2), and the results of sequence (Fig. 3) are shown in Table 4.

Fig. 2.

The results of PCR with genotyping kit. a M: DNA marker (100 bp), 1: HPV-DNA positive (type 18 (425 bp)), C + 18: positive control of HPV-DNA typing 18 (425 bp), NC negative control. b M: DNA marker (100 bp), C + 31: Positive control of HPV-DNA Type 31 (520 bp), NC negative control, 1: HPV-DNA Positive Sample (Type 31 (520 bp)) c C + 16: Positive control of HPV-DNA typing 16 (325 bp), 1: HPV-DNA positive (Typing 16 (325 bp)), M: DNA marker (100 bp), NC negative control d M: DNA marker (100 bp), 1: HPV-DNA Positive sample (Type 33 (227 bp)), C + 33: Positive control of HPV-DNA Type 33 (227 bp), NC negative control

Fig. 3.

M: DNA marker (100 bp), 1 and 2: nested PCR for HPV-DNA positive (407 bp)

Table 4.

Characteristics of HPV types

Lesion location	Type of lesion	Percentage	Number	Type the virus
Tongue, lip, oral cavity	Hyperplasia, neoplasia	36.3	4	HPV-6
Tongue, lip	SCC, Squamous papilloma	27.2	3	HPV-18
Tongue	SCC, Neoplasia, dysplasia	18.1	2	HPV-31
Tongue	SCC	9.09	1	HPV-16
Oral cavity	SCC	9.09	1	HPV-33

HPV prevalence and HPV types

The results of the investigation proved the relation between human papillomavirus (HPV) and squamous cell carcinoma (SCC), so that, the low-risk type (HPV6) had the highest number, and the high-risk types (HPV16 and HPV33) had the lowest numbers.

The prevalence rate of oral diseases varies in different areas. According to a research conducted in 2013 with PCR-MA method in Michigan, the HPV prevalence rate in oral cavity (108 samples) was 9.6%, and the amounts of HPV in oropharynx and nasopharynx were 83% and 44% respectively, so that, the 16 type was reported as the dominant type. The HPV prevalence rate in oral cavity is partly similar to the current study, but the HPV prevalence rate in oropharynx and nasopharynx is more than the current study [33]. According to a study on paraffin-embedded tissue of SCC on the tongue of 50 people from Iran in 2017, the HPV prevalence rate was reported 14%, and none of the 16 and 18 types were reported. The HPV prevalence rate was similar to the current study [2].

According to another study that was performed in 2017 in Iran on 50 oral samples, the HPV prevalence rate was reported 36% that was more than the current study. And the prevalence rate of Types 18 and 11 were respectively reported 55.56 and 44.44% [28]. According to an experiment in the United States in 2014 on laryngeal cancer tissue (148 samples), it was indicated that 20.9% of samples were HPV positive, that was more than the current study. HPV16 (6.1%) and HPV33 (6.1%) were the dominant types in that experiment [20]. Another study was performed on sinus tissue (161 samples) in 2013 in the United States, using in situ hybridization method, HPV-DNA was detected 21% and types 16 (17%), 31/33 (2%), 18 (1%) were also detected [5]. In the experiment carried out in 2016 on formalin-fixed, paraffin-embedded (FFPE) on the head and neck in 29 countries of Europe, United States, Africa and Asia, the HPV-DNA prevalence rate in oral cavity was 7.4% (93 samples of 1264), that was less than the current study, and HPV16 (68.8%) was known as the most dominant type. Moreover, HPV-DNA was detected in oropharynx (24.9%), pharynx (21.4%), nose (7.9%), larynx (5.7%) and hypopharynx (3.9%), and the dominant genotype was HPV16 with the ratio of 75.2% [7]. In 2015 in the United States, the prevalence of oral HPV infection was reported 6.8% using PCR method. Although the 16 type was reported as the dominant type, it was still less than the current study. Most of the oncogenic HPV infections are related to sexual behaviors of men and women respectively 95.9 and 87.5%. The amounts of infection in men and women without sexual behavior were reported 0.3 and 0.2% respectively [9].

Risk factors for HPV infection

The difference in the rate of HPV prevalence in different studies can be due to the sensitivity of laboratory techniques and the type of diagnostic method. In addition, the prevalence rate of this virus is varies in different parts of the head and neck. It seems that the type of the selected sample is also an important agent for the inconsistency between the results. The difference between the results of the current study and the findings of other studies may be due to smoking and various sexual behaviors. The HPV prevalence rate varies in different countries, that may be due to geographical differences or the differences in environmental factors. The prevalence rate in Iran is less than most other countries, may be due to the greater awareness of the people about their sexual health, observance of the principles of prevention and well-timed referral to health centers, and also the existence of precise prevention programs and disease controls. Unlike most studies, in the current study, the prevalence rate of low-risk types are more than high-risk types, which can be due to the regional differences.

In conclusion, the presence of HPV in the samples suggest that there is correlation between HPV and oral tumors. This study show a high prevalence of HPV6 in oral lesions. The presence of high risk HPV16,33 detected at very low, that it might be related to geographic areas and sexual behaviors. however further examination is necessary to declare of clinical implication of these types of HPV.