Role of Human Papilloma Virus in the Aetiology of Sinonasal Cancers

Abstract

Sino nasal carcinogenesis is attributed to numerous factors, namely tobacco, alcohol and snuff as the most common. Human Papilloma virus (HPV) has been associated with aetiopathogenesis of malignancies in the upper aerodigestive tract (UADT). The prevalence of HPV in Sino-nasal malignancy (SNM) which is closely related to UADT in location is not known. Hence, this study aims to explore if there is any such association between HPV and Sino nasal malignancies. This study aims to explore the association between HPV and SNM. A prospective case control study using tumour tissue specimens from 40 Sinonasal carcinoma patients and benign nasal tissue specimens from 50 cancer-free controls were conducted. Histopathological analysis and DNA extraction (DNeasy® Tissue kit) and polymerase chain reaction for detection of HPV was done. Positive samples underwent sequencing to detect the HPV serotype and statistical correlation was performed using SPSS software. All 90 patients were tested for HPV and we found that none of the patients had any association with HPV. Sino-Nasal malignancy in the Indian Sub-continent may not be related to HPV primarily.

Introduction

The viral aetiology of cancer is an area of new found interest, ever since Human papilloma virus (HPV) has been found to cause cervical cancer. HPV 16 & 18 [1] are classified as high risk species. The HPV associated malignancies are HPV 16—in the upper aerodigestive tract [2] and HPV 18 in genital tract [3]. The low risk genotypes namely HPV 6 and 11 are associated with benign conditions such as genital warts and laryngeal papillomatosis [4]^. Apart from the well documented horizontal mode of HPV transmission through sexual contact [5], newer theories suggest spread through vertical transmission from mother to the child [6] and to a lesser extent horizontally by autoinoculation [7]. HPV infection is now an emerging risk factor for benign and malignant lesions in young patients with no history of any addictions [8]. An interesting finding to note is that the presence of high HPV titres in oropharyngeal malignancy was associated with improved treatment outcomes and better disease free 5-year survival rates [9]^.

One of the recent advances in head and neck oncology was in the area of oropharyngeal cancers with the discovery of HPV associated malignancies. Prevalence of HPV in oral mucosa of normal adults is as low as 1–2% and is mostly low risk genotypes [6]. However high-risk genotypes 16 and 18 were seen in oropharyngeal cancers, mostly squamous cell carcinoma. It has been shown that the areas of the upper aerodigestive tract where there is a transition of epithelium (from squamous to columnar) in the limen vestibule, tonsillar pillar, nasopharyngeal surface of the soft palate, epiglottis, ventricle, carina and the lower end of the oesophagus are predisposed to HPV infection similar to the cervix [5]. Similarly, it is believed that the transition from squamous to columnar epithelium in the nasal cavity makes this region a likely site of HPV associated malignancies. Hasegawa M et al. found 7% of patients with chronic sinusitis, 27% in squamous cell carcinoma and 43% in inverted papilloma to be HPV positive [9]. Bharathi et al. have studied the role of HPV in benign sinonasal polyps and they have not found any association between benign polyps and HPV [12]. The role of HPV in benign sinonasal papilloma especially inverted papilloma is well established [8, 13], and its propenceity towards malignant transformation (5–15%) and high recurrence rates despite adequate surgical clearance [14]. A review study has shown that the prevalence of HPV ( genotypes 16 and 18) in malignant sinonasal tumors ranges from 20 to 30% [15]^.

Although HPV positivity in oropharyngeal malignancy have shown to have better clinical outcome and survival rates, our knowledge on the behaviour of HPV associated non-oropharyngeal head and neck malignancies is very limited. Data regarding the aetiology of sinonasal tumors in India is very few probably due to rarity of such cases. Hence, this study attempts to find the association of HPV in malignant sinonasal tumors in a cohort of Indian population to better understand the prognosis and outcomes in these patients.

Materials and Methods

A prospective case control study was conducted in the ENT department – the outpatient and inpatient services at a tertiary care hospital, between December 2013 to July 2015 after Institutional review board clearance (number).

Participants

Patients diagnosed with malignancy in the sino nasal region and undergoing surgery for their disease were consented and recruited and patients undergoing routine surgery for benign sinonasal pathology such as polyps and chronic rhinosinusitis were consented to be controls.

Surgical Specimen Collection Protocol During the surgical procedure tissue obtained from the surgical site were directly transferred to containers containing virus transporting medium (VTM).

VTM is a balanced isotonic solution at physiological pH, maintains the virus in a viable state as it contains foetal calf serum and antibiotics. These containers are then transported in ice containers maintaining cold chain to the virology lab. In the lab, the sample were transferred to 1.5 ml Eppendorf tube and stored at − 80 degree Celsius until further testing in the freezer.

DNA extraction protocol DNeasy tissue kit (Qiagen GmgH, Hilden, Germ [16] was used as for DNA extraction. The tissues specimen was cut into 25-g pieces and were digested by ATL buffer and Proteinase K at 56 degree Celsius. Once digested, an equal amount of lysis buffer -AL buffer and ethanol were added. The DNA precipitated is then washed twice with buffers AW1 and AW2 and then eluted with elution buffer. The extract containing DNA was stored at − 20 degree Celsius.

HPV DNA Detection The extracted DNA underwent PCR with a known positive control and beta globulin as internal control. HPV detection was be done by THE PGMY primer system. The PGMY primer PCR system amplifies the L1 region of the HPV genome resulting in a 450 base pair amplicon. Along with this, an internal control histocompatibility leukocyte antigen (HLA), beta globulin is amplified producing a 230 base pair amplicon confirming that DNA is extracted in sufficient amounts and that there are no inhibitors in the sample. The presence or absence of target bands (450 bp), the sample was interpreted as positive or negative. Beta globulin positivity was essential for analysis of sample.

Sequencing The “positive” detected were amplified by PCR and products purified by Millipore filtration and sequenced using an ABI prism big bye terminator cycle sequencing ready reaction kit. The sequences obtained were compared with GenBank HPV sequences. The HPV positive samples are sequenced to know the HPV genotype.

Statistical Analysis The data of all the patients were collected systematically with the software EPIDATA version 3.1 Statistical analysis was performed using statistical software SPSS (version 13.0). Chi square test was used to find association.

Results

A total of 90 patients were included in the study of these 40 were patients diagnosed with sino-nasal malignancy (SNM) and 50 controls (benign sino-nasal pathology). Patients with malignancy were predominantly male (Male: Female: 5:1) which was found statistically significant (p < 0.05). However, in the control arm this was not the case (Male: Female:2:1, p = 0.9). The age distribution was between 50 and 60 years for SNM. The median duration of common symptoms such as nasal obstruction, facial swelling, epistaxis was seen as 3 months (IQR = 2–6) for SNM (IQR = 8–42) for benign lesions, (p < 0.001-MannWhitney U test) (Fig. 1).

Fig. 1.

Distribution of Symptoms for sino nasal malignancies

Risk factors such as smoking, consumption of alcohol and the use of nasal snuff were studied. The use of nasal snuff and malignancy was found to be significantly associated with SNM(p < 0.05) in our cohort of patients. Tobaccos smoking was found to increase the risk 1.5 times though this was not statistically significant. (Table 1). The most common histological subtype of SNM seen in the study population was epithelial tumours, followed by haematolymphoid tumours. Squamous cell carcinoma was the common histological type seen (n = 15) among the epithelial tumours. The most common site is nasal cavity (n = 18) and maxillary sinus(n = 18). In this cohort of 90 patients, we found that HPV was not present in any group (Table 2). In our study there were 4 patients in the case arm with history of multiple sexual partners unlike the control arm where there were none. However, these four cases did not show HPV positivity. Therefore we conclude that probably HPV has no role in the etiology of sinonasal malignancies in Indian Subcontinent.

Table 1.

Risk factors and their association of cases and controls

Variables	Case	%	Control	%	p Value
Smoking
Present	26	65	24	48	0.346
Absent	14	35	26	42
Snuff
Present	15	38	8	16	0.024
Absent	25	62	42	84

Table 2.

Table showing HPV positivity in cases and controls

HPV	Present	Absent
Cases	0	40
Controls	0	50

Discussion

Sino nasal malignancies constitute about 3% of all head and neck malignancies [17]^. The most common malignancy seen in the sinonasal tract is squamous cell carcinoma [17]^. Literature search shows incidence from 35 to 70% worldwide [14].The epithelium of the sinonasal tract over the vestibule is lined by stratified squamous epithelium and the nasal cavity proper with pseudo stratified columnar respiratory epithelium with an area of squamo-columnar junction, at the limen vestibule. This area is predisposed to develop HPV infection.

In our study, the clinical symptomatology was similar in the case and control arms, the complaints in patients with sinonasal malignancies were of short duration. The symptom of facial swelling was found to be a good predictor of malignancy (p < 0.05).This finding can be explained with increased percentage of addictions and also the work pattern in male gender which can predispose to malignancy. In Indian population, data shows that there are more men who smoke and consume alcohol as compared to females [18].Several studies have showed that increased prevalence of sinonasal malignancy in the population due to tobacco [18] and nasal snuff [19].Statistical analysis in our study revealed that usage of nasal snuff increases the risk of developing sinonasal carcinoma by three times (OR – 3, p- < 0.05). Similar to previous literature it was also seen that smokers are at a higher risk to develop sinonasal malignancies (OR -1.5, p-0.2). [15].

In a study done by Kumaraswamy et al., HPV associated head and neck squamous cell carcinoma was seen in the younger age group with high risk sexual behaviours such as multiple sexual partners, early age of sexual intercourse and unnatural sexual habits [22].Though these tumors were poorly differentiated, they were found to have better prognosis [18].In our study, of the total number of the cases of sinonasal malignancy that were enrolled, 34 (86%) were married whereas 6 (14%) were unmarried. Sexual practices determine the mode of HPV transmission. Patients were not forthcoming with sexual history because of their conservative cultural, religious and ethnic background. Despite these limitations, in our study there were 4 patients in the case arm. However, these four cases did not show HPV positivity.

Studies in oropharyngeal malignancies have shown that 80% of these malignancies were positive for high risk HPV such as HPV 16 and 18 [19]. These HPV associated oropharyngeal malignancies were seen to have a better treatment outcome and improved five-year survival rates. However Indian studies gives inconsistent data on the prevalence of HPV in oropharyngeal malignancies [20].The interest in studying the association of HPV in sinonasal malignancies was due to HPV positive benign conditions such as inverted papilloma in the early 80s [25, 26]. In a study done by Bishop et al. on sinonasal malignancy, 21% (34 cases in 161 patients) of sinonasal malignancies were found to have HPV which was biologically active [24]. They also found that they were all squamous cell type histologically and was not seen in all the other variants. It was localized to non-keratinising and basaloid squamous cell carcinoma. Similar finding was also noted in oropharyngeal malignancy [22].Beigh Etal proposed that low risk HPV types 6 and 11, showed an association with sinonasal papillomas (5 out of 38) whereas HPV types 16 and 18( 2 out of 10) are associated with squamous cell carcinoma [28]. In a study done by Alos et al., HPV associated sinonasal malignancies were seen to have a better outcome with less rates of recurrence. They opined that these HPV associated malignancies have less chance of field cancerization and better response to radiotherapy which can explain the improved survival rates [28]. Further studies are warranted in HPV positive cases to analyse this trend. HPV has been implicated in the aetiology of head and neck carcinoma particularly in squamous cell carcinoma [2]. It is significant in patients with no known risk factors such as smoking, tobacco chewing or habituation to alcohol.

Limitations

Sinonasal malignancies are rare tumours. Our study was a two-year time bound study. Due to the limitation of time we could not attain a larger sample size. However, the data on the attained sample size gives us background information regarding the topic of interest. Because of the conservative cultural, religious and ethnic background in our subcontinent, it was difficult to obtain a detailed sexual history. We would suggest a longer prospective study with a larger sample size to study the association between HPV and Sino nasal malignancy.

Funding

Funding was provided by Fluid Research (Grant IRB No: 8589).