|Year : 2016 | Volume
| Issue : 1 | Page : 206-209
Role of human papilloma virus in oral leukoplakia
Anuja Bhargava1, M Shakeel1, AN Srivastava2, Tasleem S Raza3, Saliha Rizvi3, Prasoon Varshney1
1 Department of Otorhinolaryngology, Era's Lucknow Medical College and Hospital, Lucknow, Uttar Pradesh, India
2 Department of Pathology, Era's Lucknow Medical College and Hospital, Lucknow, Uttar Pradesh, India
3 Department of Biochemistry, Era's Lucknow Medical College and Hospital, Lucknow, Uttar Pradesh, India
|Date of Web Publication||28-Apr-2016|
Department of Otorhinolaryngology, Era's Lucknow Medical College and Hospital, Lucknow, Uttar Pradesh
Source of Support: None, Conflict of Interest: None
Background: Controversy surrounds regarding the role of human papillomavirus (HPV) in oral precancerous and cancerous lesions in India where smokeless, tobacco consumption is rampant. Aims: The present study was carried out with an aim to investigate the presence and type of HPV infection in oropharyngeal leukoplakia and to determine the association of HPV positivity with various patient and lesion characteristics. Settings and Design: Prospective case series. Materials and Methods: A total of fifty confirmed cases of oral leukoplakia (OL), aged 18–55 years were enrolled in the study. Specimens were obtained from the site of lesion. All the specimens were subjected to histopathological analysis and HPV-16, 18 detection was done using real-time polymerase chain reaction technique. Results: Mean age of patients was 34.00 ± 8.85. Majority were males (62%), from middle social class (78%) and were educated up to graduation or above (54%). All the patients had one or more adverse habits (betel, chewing tobacco, smoking and alcohol) with mean duration of disease was 3.53 ± 3.05 years. Five patients (10%) had diabetes. Burning sensation (86%) and trismus (36%) were the reported complaints. Maximum were Stage II (50%), sized 2–4 cm2 (60%), homogenous (70%). Floor of mouth and tongue were most commonly involved sites (50%). Majority had mild dysplasia (58%). Acanthosis (66%), hyperplasia (62%), koliocytosis (40%), and keratosis (98%) were quite common. HPV (16/18) could not be detected in any specimen. Conclusions: The present study highlighted the fact that association of HPV with oral leukoplakia seems to be overestimated and needs to be reexamined with consensus Human papilloma virus (HPV) primers to detect HPV types with more valid empirical relationships.
Keywords: Human papillomavirus, leukoplakia, premalignant oropharyngeal lesions
|How to cite this article:|
Bhargava A, Shakeel M, Srivastava A N, Raza TS, Rizvi S, Varshney P. Role of human papilloma virus in oral leukoplakia. Indian J Cancer 2016;53:206-9
| » Introduction|| |
Human papillomavirus (HPV) is double-helical DNA structure, which taxonomically is placed in family Papillomaviridae. They were first discovered, isolated, and sequenced from cervical tumor specimens and have been attributed to be one of the important causative agents for the development of cervical cancer by zur Hausen et al.,,, which subsequently led him to win the Nobel Prize of Physiology and Medicine for the year 2008.
Following its discovery in cervical tumor specimen, its presence in cutaneous and mucosal tissues of the oral cavity, upper gastrointestinal tract, anogenital tract, and skin of hands and feet was also discovered. The presence of these viruses was also discovered in other mammals; however, their transmission from one species to another species is not established. Papillomaviruses have been shown to have a high degree of specific cellular tropism for squamous epithelial cells, and they have been linked with less severe clinical manifestations such as benign hyperplastic epithelial proliferative innocuous lesions to invasive cancer of genital tract, oropharynx, and esophageal regions.
Although the role of HPV in the causation of cervical cancer is well established yet controversy surrounds regarding their role in oropharyngeal malignant and premalignant lesions and has attracted the researchers throughout the world.,, Till date, several HPV types have been linked with malignancy of both genital tract and nongenital tract and put under the high risk (HR) category. These types include - HPV 16, 18, 31, 35, 39, 45, 51, 52, 56, 59, 66, 68, 69, 73, and 82.
In the present study, an attempt was made to evaluate the presence of HPV Type 16 and 18 in oral leukoplakia (OL) lesions among the specimen obtained from the patients of OL.
| » Materials and Methods|| |
Tissue specimens were obtained from fifty patients of OL attending the Department of Otorhinolaryngology of ERA's Lucknow Medical College, Lucknow. Institutional ethical clearance was obtained, and informed consent was taken from all the patients. Demographic details such as gender, age, personal habits (tobacco use, smoking, and alcohol) were obtained. Duration of complaints and clinical history [Figure 1] were collected from patients' interviews and medical records. Size and severity were obtained from clinical notes, pathology reports. Staging was done according to LSCP (L – lesion, S – site, C – clinical aspect, P – histopathological features) [Table 1] classification and staging system for OL.
|Figure 1: Leukoplakia as it appears at different sites – buccal mucosa lower lip|
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Oral biopsy from the lesion was sent as sample A for histopathological (HPE) examination and as sample B for real-time polymerase chain reaction (RT-PCR) analysis.
For HPE examination, the tissue was collected in 10% formalin and sent to HP laboratory. The tissue was processed through histokinette. Sections 4–5 micron thickness were cut on leica microtome and stained with hematoxylin and eosin (H and E) staining. Wherever necessary for diagnosis, other specialized stains such as H and E and Van Gieson were used. All these methods are time-honored and standardized in histopathology Department of ELMC.
For conducting RTPCR analysis tissue biospy samples were collected in normal saline. DNA extraction and HPV-16 and 18 detection was done using HPV-16 and 18 RT-PCR kit (Liferiver, Shanghai), following manufacturers protocol. RT-PCR was performed in 40 µl reaction mixture containing approximately 4 µl of extracted DNA and 36 µl of master mix. The master mix for each reaction was prepared by pipetting 35 µl of reaction mix (HPV serotype 16 and 18 reactions mix), 0.4 µl of enzyme mix (DNA polymerase), and then 1 µl of internal control ending up with a total of 36.4 µl of master mix. The RT-PCR cycling conditions included were initial one cycle at 37°C for 2 min, then one cycle denaturation at 94°C for 2 min, followed by 40 cycles at 93°C for 15 s and 60°C for 60 s. The detection of amplified HPV 16 and 18 DNA fragments was performed in fluorimeter channel FAM and HEX/VIC/JOE with the fluorescent quencher BHQ1 at 60°C.
Data were analyzed using Statistical Package for Social Sciences, Version 15.0. Chi-square test and Fisher's exact test were planned to be employed to find out the association between HPV positivity, patient characteristics, clinical presentation, stage and severity of disease.
| » Results|| |
Age of patients ranged from 18 to 55 years. Mean age of patients was 34.00 ± 8.85. Majority were males (62%), from middle social class (78%) and were educated up to graduation or above (54%) [Table 2].
All the patients had one or more adverse habits (betel, chewing tobacco, smoking, and alcohol) with mean duration of adverse habits was 3.53 ± 3.05 years. Betel tobacco chewing was the most common adverse habit (80%) followed by tobacco chewing and smoking (54%), smoking (36%), and alcohol use (36%). About 5 (10%) had diabetes. Burning sensation (86%) and trismus (36%) were the reported complaints [Table 3].
|Table 3: Personal Habits, Medical History and Complaints at Presentation|
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Maximum patients were of Stage II (50%). A total of 17 (34%) were of Stage I. Advanced stage (Stages III and IV) was seen in 8 (16%) patients. Mean duration of symptoms was 3.96 ± 2.17 years. Lesion size was 2–4 cm 2 in majority (60%). A total of 18 (36%) had lesion size >4 cm 2. In 2 (4%) patients, lesion size could not be specifically measured. Floor of mouth and tongue were most commonly involved sites (50%), in remaining half buccal mucosa, gingival and tongue borders were involved. In 35 (70%), the lesion was homogenous [Table 4].
Histopathologically, majority had mild dysplasia (58%) followed by those having moderate dysplasia (40%). None had severe dysplasia. Acanthosis (66%), hyperplasia (62%), koliocytosis (40%), and keratosis (98%) were quite common. Keratosis was differentiated as ortho in 14 (28%) patients, para in 18 (36%), hyper in 2 (4%), and in remaining 18 (36%), it could not be defined properly [Table 5].
HPV (16/18) could not be identified in any specimen.
| » Discussion|| |
In this study, HPV could not be identified in any of the fifty OL specimen examined. OL is considered an uncommon potentially malignant lesion of the oral mucosa. In 1978, Kramer et al. defined OL as “a white patch or plaque that cannot be characterized clinically or histopathologically, as any other disease.”, Observing only oral precancerous lesions, OL is the most frequent potentially malignant lesion of this mucosa, represents 85% of oral precancerous lesions. This lesion has a predilection to male gender,,, as also observed in this study. Despite the exact cause of OL still been unknown, tobacco intake is considered the most common risk factor for OL development., The findings of this study support this viewpoint. In this study, one or more adverse oral habits such as betel nut/tobacco chewing, tobacco chewing, smoking and alcohol use were seen to be present in all the cases, thus undermining the viral etiology and attributing the major etiology to these adverse oral habits – mainly tobacco intake as reported in earlier studies.
In this study, we focused on identification of HR HPV types. In some previous meta-analyses evaluating the role of HPV in oral precancerous lesions, a significant role of low-risk HPV types has been identified, however as far as HR HPV types are concerned, it still remains to be associated with oral squamous cell carcinoma than premalignant lesions. As such studies from India have shown a relatively varying prevalence of HPV 16/18 even in malignant lesions of head and neck region including oropharynx with prevalence rates ranging from 6% to 47%. Ringström et al. in their study of tumors of head and neck regions for prevalence of HPV positivity also reported it to be only 11.1% for the oral cavity lesions. In the literature, a significant association of HPV prevalence with leukoplakia severity has also been reported. However, in this study for both mild as well as moderate dysplasia groups as well as for lower and higher clinical stages, HPV could not be identified at all. One of the limitations of this study was the absence of severe dysplasia cases. In the higher clinical stage (III and IV) too, there were only 8 (16%) cases. Thus, the association of HPV with clinical and HPE severity could not be properly investigated.
The findings in present study further established the fact that etiological factors for oral precancerous lesions as observed in Indian population are quite different from their Western counterparts. While in western studies, HPV 16,18 has been recognized as a significant risk factor for cancerous lesions, its role in Indian population remains to be verified, specifically in premalignant oral lesions such as leukoplakia. Larger studies in Indian population with both evaluation of both low risk and high risk HPV types is recommended.
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Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]