Detection of human papillomavirus (HPV) DNA in benign laryngeal lesions and role of cigarette smoking as an inducing factor

Abstract

The association of human papillomavirus (HPV) in laryngeal malignancies is largely known. This study evaluated the association between HPV and benign laryngeal lesions and also the role of smoking and alcohol consumption in increasing the prevalence of HPV in such benign lesions. Seventy-eight specimens of benign laryngeal lesions including 26 polyps, 26 dysplasia, and 26 other lesions such as nodules and cysts were enrolled in this study. Polymerase chain reaction (PCR) technique was used to detect HPV DNA in the tissues. The role of smoking and alcohol consumption in the prevalence of HPV was also evaluated through appropriate statistical tests. This study showed that the prevalence of HPV in benign laryngeal lesions was not statistically significant. The Cohen’s effect size for comparing polyps vs. other lesions was nearly 0.7, indicating that HPV prevalence in laryngeal polyps may be clinically meaningful. Another finding in our study is the role of smoking in increasing the HPV prevalence in laryngeal polyps (P = 0.034). In benign laryngeal polyps, HPV prevalence may be clinically important. Smoking acts as a co-factor to induce HPV infection in laryngeal polyps in our study.

Introduction

The association of the human papillomavirus(HPV) and head and neck cancers has been well established in many studies[1].The papilloma virus DNA has been extracted from the malignant tissues of the oropharynx, oral cavity and to a lesser extent from the laryngeal carcinoma[1–3].There are more than 200 identified genotypes of HPV, about 13 to 15 of which are known to be high-risk and have the potential to cause cancer in humans. Genotypes are divided into low-risk and high-risk groups based on their role in inducing cervicogenital carcinoma. Head and neck tumors are more likely to be caused by the type 16 virus [3–7].

The prevalence of HPV in laryngeal cancer varies between 5 and 23% in several studies[2, 3].Evidence has shown that HPV-related head and neck cancer patients have better prognosis than non-HPV-related cases[2, 3, 8].One of the diseases in which the role of human papillomavirus has been well identified is recurrent respiratory papillomatosis, which is caused by type 6 and 11 viruses. Malignant transformation has been reported in rare cases of long-standing head and neck papilloma [9–11].Few studies have shown the presence of the DNA virus in benign laryngeal lesions such as polyps, nodules, and dysplasia. HPV is also found in benign lesions of the oral cavity papilloma[12–15].

Although the role of vocal trauma and misuse of voice is known to cause polyps and nodules in the larynx, it is possible that other factors may predispose the patients to develop this lesion. In this study, we investigated the prevalence of HPV in benign vocal lesions aiming to detect any possible association that may affect our knowledge and future treatment modalities. We also evaluated the habit of smoking and alcohol consumption in our patients to define any predisposing correlation.

Materials and methods

Patient selection

A total of 78 tissue specimens of benign laryngeal lesions including 26 polyps, 26 dysplasia, and 26 samples of other non-malignant cases such as nodules, laryngitis, fibrosis, amyloidosis, and cysts were included in the current study.

Sample size was determined by means of NCSS statistical software with a 5% error and80% power based on a previous similar study[12].All specimens were obtained through microlaryngeal techniques from patients with stroboscopic examination, and histopathologic examination confirmed the diagnosis of a benign lesion. Respiratory papillomatosis was not included in our study. Demographic data of patients and also habits of smoking and alcohol consumption were recorded to determine any further correlation. The specimens were sent to virology laboratory for HPV DNA detection using polymerase chain reaction (PCR) technique.

DNA extraction technique

The tissue specimens were fixed in 10% formalin, embedded in paraffin, and sectioned with a thickness of 10 × 5 µm stained with hematoxylin–eosin. Each specimen was stored in a 1.5 cc separate eppendorf tube. In order to prevent any possible contamination, at any stage, we washed all instruments with HCL solution, and for each sample, tissue preparing was conducted in a separate setting. To extract the HPV DNA, we employed a method including dewaxing, extraction, amplification and gel-electrophoresis. For each tube, we added 1200 μl xylol and incubated at room temperature for 5 min. Then, centrifuging at 14000 rpm for 5 min and adding of 1 cc ethanol100% was done. After that, re-centrifuging for 5 min and discarding the floating layer were done. All the steps were repeated and finally after evaporating the alcohol, the extracted DNA was stored in a −20 °C for PCR examination.

PCR technique

The extracted samples were tested for quality with beta globulin (PCO3/PCO4) PCR and beta actin PCR and then HPV PCR was conducted.PCR was performed in a total volume of 25 μl, including the followings:1 mM Mg C12(CinnaGen Co. Iran), 200 μM of dNPTs(CinnaGen Co. Iran), 200 μM of 1X reaction buffer(CinnaGen Co. Iran),1U of the enzyme Tag DNA polymerase (CinnaGen Co. Iran).Then, 1 μM of each specific primer including PCO3 and PCO4 was added. Samples with good quality were subjected to PCR for the presence of HPV DNA. The PCR testing for β-globulin was performed as follows: initial denaturation at 94 °C for 10 min, 35rounds of denaturation at 94 °C for 45 s, annealing at 44 °C for 45 s, extension stage at the temperature of 72 °C for 1 min, and final extension for 10 min at the temperature of 72 °C. Specific HPV primers included two MY09 and MY11 gene sequences that were amplified and then loaded in the L1 region. Sequences of PCR primers MY09/MY11 system for HPV DNA detection are listed below:

MY09 5'-CGTCCMARRGGAWACTGATC-3'.

MY11 5'-GCMCAGGGWCATAAYAATGG-3'.

Statistical analysis

The collected data were analyzed using Statistical Package for Social Sciences (SPSS), version 19.0(SPSS Inc. Chicago, Illinois, USA),Chi-square and t-test. P values < 0.05 were considered as statistically significant.

Results and discussion

Patient characteristics

A total of 78 specimens of benign laryngeal lesions from 52 male and 26 female patients with a mean age of 44.24 years were evaluated for HPV by PCR. Data of patients, smoking habits and alcohol consumption in different histopathological groups are shown in Table 1.

Table 1.

Distribution of HPV-positive cases of benign laryngeal lesions based on age,sex,alcohol consumption and cigarette smoking

Alcohol consumption N (%) At least 1drink per day	Cigarette smoker N (%) Current smoker	Age	Sex		Number	Kind of Lesion
		Mean (Min–Max)	Female N (%)	Male N (%)
4 (15%)	10 (38%)	40.57 (18–60)	9 (34.7)	17 (65.3)	26	Polyp
1 (3.8%)	10 (38%)	47.20 (18–80)	9 (34.7)	17 (65.3)	26	Displasia
1 (3.8%)	9 (34%)	46.23 (22–78)	8 (30.7)	18 (69.3)	26	Other Lesions
6 (7.7%)	29 (37.2)	44.25 (18–80)	26 (33.3)	52 (66.7)	78	Total

A total of 10(12.8%) patients had a positive and 68(87.2%) patients had a negative HPV result. In negative HPV patients, 20(29.4%) cases were in the polyp group, 24(35.3%) cases were in the dysplasia group, and 24(35.3%) cases were in the other lesion group. In patients with laryngeal polyps, 6(23.1%)cases had positive HPV detection.

HPV prevalence

The findings of this study demonstrated that the prevalence of HPV in the three groups of benign laryngeal lesions was not statistically significant. However, the results showed that the proportion of positive HPV in polyp and other groups was 23.1% and 7.7% which was at the margin of statistical significance by the Chi-square test in the polyp group (P = 0.055).Fig. 1

Fig. 1.

Comparison of the HPV + patients among studied groups (p = 0.055)

A total of 29 patients were smokers, of whom 22(75.9%) were HPV-negative and 7(24.1%) were HPV-positive. Of the 49 non-smokers in the study, 46(93.9%) were negative and 3(6.1%) were positive for HPV. There is a significant difference in terms of statistics in the prevalence of HPVDNA detection between smoker and non-smoker groups (P = 0.034).(Fig. 2).

Fig. 2.

Comparison of the HPV + patients among smoker and no smoker groups (p = 0.034)

There was no significant association between the prevalence of HPV in different groups of benign laryngeal lesions in relation to age, sex, and alcohol consumption (P = 0.823, P = 1.000, P = 0.168, respectively).

The association between laryngeal malignancies and HPV infection is relatively well established. In this study, the association between HPV and benign laryngeal lesions was evaluated. Our study showed that although this association was not exactly significant statistically, clinically it is important and meaningful for laryngeal polyp patients. It should be noted that the power of the Chi-square test is highly dependent on the sample size. Hence, a moderate or large difference between two proportions in Chi-square test may not be statistically significant, given a small or moderate sample. In this research, we used the effect size index to quantify the magnitude of differences, which may be practically or clinically meaningful. In the present study, the Cohen’s effect size measure for comparing two proportions (polyp versus others) was nearly 0.7, indicating that the difference between the two proportions are clinically meaningful, even though it was not statistically significant.

HPV and smoking

An important finding in this study was the role of cigarette smoking in increasing the rate of HPV infection in laryngeal polyps.Although phonotrauma plays a key role in the development of benign laryngeal lesions such as nodules and polyps, HPV may be one of the predisposing factors for these lesions[16].However, other predisposing factors such as smoking[17], alcohol consumption[18], gastroesophageal reflux[19–21], and allergies [22, 23] are also considered to have a role in causing benign laryngeal lesions. Among the predisposing factors mentioned, the role of smoking is the most important and well-known[24].

Our study showed that smoking played a significant role as a predisposing factor for HPV infection in laryngeal polyps. In other benign lesions such as nodules and dysplasia, this association was not significant.

Cohort studies have demonstrated that smoking is a co-factor to HPV infection and subsequent precancerous and cancerous cervical lesions[25, 26]. Other studies have also shown the same association between smoking and increasing HPV detection in laryngeal pre- neoplastic lesions[25, 26].Smoking can impair both the cellular immunity and antibody production against HPV infection[27, 28]. The studies show that smoking contributes to impaired immunity by interfering with tumor suppressor miRNA and immunity mediators. MicroRNAs(miRNA) are a group of noncoding RNA molecules that function in RNA silencing and regulation of gene expression[27, 28].Researchers demonstrated that smoking down-regulated the expression of microRNA, especially miRNA-133a-3p[29].This down-regulation interferes with cell cycle and induce tumor growth through increasing the expression of epithelial growth factor receptor. These changes in immune system are considered a cofactor for induction or progression of HPV infection and upcoming malignant and benign transformation. Association between smoking and an increasing HPV infection opens a novel area of interest in HPV-related disease research[29–32].

In a study conducted by Lacco et al.[12], the prevalence of HPV in the laryngeal nodules was 5%, which is somewhat similar to the results of our study in vocal nodules. This study did not find a significant relationship between smoking and prevalence of HPV in the nodules. However, in our study, laryngeal polyps in smokers showed a higher level of prevalence of HPV. There may be differences in the mechanisms of polyp formation compared to nodules which makes this difference.

In another study with a smaller sample size, the prevalence of HPV in benign laryngeal lesions other than papilloma was about 19%[13].In terms of virus prevalence, the results of this study were similar to our research.

The strength of this study is finding the association between cigarette smoking and increase of HPV infection in laryngeal polyps. The weakness of this study is the lack of typing for the virus, which cannot define any association between high-risk and low-risk type of virus and benign lesions of the larynx. More studies are needed in the future with more sample sizes and determination of virus types.

Compliance with ethical standards

Conflict of interest

Authors declared that they do not have any conflict of interest.