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Year : 2021  |  Volume : 13  |  Issue : 2  |  Page : 155-159

Study of HPV16 L1 Capsid Seropositivity in Patients with Oral and Oropharyngeal Cancers

1 Department of Microbiology, Guntur Medical College, Guntur, Andhra Pradesh, India
2 Department of Radiotherapy, Government General Hospital, Guntur, Andhra Pradesh, India

Date of Submission15-Nov-2021
Date of Acceptance15-Dec-2021
Date of Web Publication14-Jan-2022

Correspondence Address:
Dr. Shalini Kanaparti
Flat no: 103, Renuka Towers, Near Kunchanapalli, Tadepalli Mandal, Guntur, 522501, Andhra Pradesh
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jofs.jofs_258_21

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Introduction: Epidemiologic studies show that incidence of oral and oropharyngeal cancers (OPCs) is increasing worldwide over the last decades and the increase is more marked in men than in women. Several meta-analyses have suggested a rising prevalence of human papillomavirus (HPV) in the setting of OPC over time and have now reached epidemic proportions in many areas of the world. The aim of the study is to study the seroprevalence of HPV16-specific immunoglobulin (Ig) G and IgM antibodies and to determine whether the presence of these antibodies could be used as a potential biomarker in HPV16-related oral and OPCs. Materials and methods: Blood samples were collected from 70 patients with oral and OPCs. Samples were analyzed by indirect enzyme-linked immunosorbant assay (ELISA) to detect HPV16-specific IgG and IgM antibodies. Furthermore, the association of various other causal factors such as smoking, alcohol consumption, and betel nut chewing were also evaluated. Results: ELISA revealed that the HPV16-specific IgG antibodies were detected in 7 (10%) out of 70 patients analyzed and HPV16-specific IgM antibodies were not detected in all the cases (0%). High prevalence n = 4 (57.1%) of HPV16-related oral and OPCs was observed among males and in the 51 to 60 years age group who were smokers and alcohol abusers. Conclusion: Determining the HPV status in patients with OPC by serology might be an affordable option in the clinical settings to reduce the morbidity and mortality of patients with oral and OPCs by early intervention.

Keywords: HPV16, OPC, oral cancers, seropositivity

How to cite this article:
Kanaparti S, Kolli P, Jyothi P, Rao AY, Lokeshu T, Induluri J. Study of HPV16 L1 Capsid Seropositivity in Patients with Oral and Oropharyngeal Cancers. J Orofac Sci 2021;13:155-9

How to cite this URL:
Kanaparti S, Kolli P, Jyothi P, Rao AY, Lokeshu T, Induluri J. Study of HPV16 L1 Capsid Seropositivity in Patients with Oral and Oropharyngeal Cancers. J Orofac Sci [serial online] 2021 [cited 2022 Jul 4];13:155-9. Available from:

  Introduction Top

Oral cancer is the sixth most prevalent malignancy worldwide, causing severe illness leading to a significant death rate.[1] The World Health Organization (WHO) classification of oropharyngeal cancers (OPCs) includes the base of the tongue, tonsils, and adenoids. In contrast, the eighth edition of the American Joint Committee on Cancer Staging Manual includes a broader range of sites in the oral and OPCs, including the base of the tongue and tonsils (lingual and pharyngeal), soft palate, uvula, and lateral or posterior pharyngeal walls.[2] There is a wide variation in this disease’s global burden, with the highest incidence in India and across South and South-east Asia among the world.[3] At present, the incidence of oral and oropharyngeal cancers in the United States is 6.2/1,00,000 for men and 1.4/1,00,000 for women.[4]

The incidence of these cancers varies from country to country or from region to region, depending on personal habits such as the use of tobacco, chewing of betel nut, and consumption of alcohol.[1] It has been reported that oral cancers may develop even in the absence of these risk factors and are associated with other factors such as dietary habits, ionizing radiations, genetic predisposition, etc.[1]

One of the major risk factors was found to be human papillomavirus 16 and 18 (HPV16 and HPV18). HPV related to oral and OPCs has now reached epidemic proportions in many areas of the world, and the incidence is increasing substantially every year.[5] Even with modern medical treatments, it has been estimated that the overall mortality rate for oral and OPCs remain high.[1] A fundamental backdrop with regard to the survival rate of oral and OPC patients is that most of the cases were diagnosed in the advanced stage. In this regard, it is important that assessments to be developed for the easy identification of risk factors.[1]

The strong association between HPV and oral and OPCs also has implications for vaccination strategies. If early-stage disease could be detected, modern trans-oral surgical techniques would allow for localized treatment of the tonsils or base of the tongue, which reduces cancer morbidity and mortality, a hallmark of a successful screening program.[5]

Serum antibodies to HPV16 antigens have emerged as promising biomarkers that could aid in identifying individuals at high risk for HPV16-related oral and OPCs who could then go for further screening.[5] HPV16 L1 seropositivity has been proposed as a surrogate marker of past HPV exposure. The present study was conducted to estimate the seroprevalence, age- and gender-wise distribution of HPV16-specific immunoglobulin (Ig) G and IgM antibodies in oral and OPCs.

  Materials and Methods Top

This was a prospective observational study conducted for over a period of 18 months. Ethical clearance (No. GMC/IEC/154/2019) was obtained from Institutional Ethics Committee of Guntur Medical College, Guntur, Andhra Pradesh on January 25, 2019. A total of 70 patients who were clinically and histopathologically diagnosed as oral and OPCs were included in the study. Demographic data and history were obtained from all the cases. Adult human subjects of age above 18 years and of both sexes who can communicate effectively and willing to provide written informed consent were included in the study. Patients who are not willing to participate in the study were excluded. Blood samples were collected under aseptic conditions after obtaining written informed consent from each individual and serum was separated and stored at −80°C.

All the serum samples were subjected to HPV16 IgG and IgM enzyme-linked immunosorbant assay (ELISA). The HPV16 IgG and IgM ELISA kits (Cusabio, Wuhan, China) specifically quoted for L1 antigen were obtained. These kits included microtiter plates that were precoated with HPV16-specific L1 antigen and with horse-radish peroxidase (HRP) as a marker enzyme. ELISA procedure was conducted according to the manufacturer’s protocol, where serum samples were diluted to 2000-folds with sample diluent for IgM detection and 1000-folds for IgG detection. About 100 μL of each diluted sample was added to the microtiter wells and incubated at 37°C for 30 minutes. The wells were subsequently washed, and HRP-conjugated antihuman IgG/IgM was added and plates were incubated again at 37°C for 30 minutes. After thorough washing, tetramethylbenzidine substrate solution was added to each well and the enzyme substrate reaction was terminated by adding stop solution (HCL). The color changes were then measured spectrophotometrically at 450 nm using a microplate reader (Venchal Scientific, New York, NY, USA, model no. 512100).

  Results Top

Age- and gender-wise distribution of all oral and OPC cases (n = 70) was depicted in [Figure 1], which shows male predominance (n = 50) with highest number of cases (n = 27) among the 51 to 60 years age group.
Figure 1 Age- and gender-wise distribution of all oral and oropharyngeal cancer cases.

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[Figure 2] emphasizes distribution of all cases based on social habits. Among 70 patients with oral and OPCs, 45 (64.2%) were alcohol abusers, 19 (27%) were smokers, 14 (20%) were gutkha/khaini users, and 11 patients did not have any habits.
Figure 2 Distribution of all oral and oropharyngeal cancer cases based on social habits.

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Comparison of HPV16-specific IgG and IgM, positive cases were emphasized in [Figure 3], where male predominance (n = 6) was reported among the IgG-positive cases.
Figure 3 Comparison of human papillomavirus16-specific IgG and IgM-positive cases. Ig, immunoglobulin.

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Age- and gender-wise distribution of HPV16 IgG-positive oral and OPC cases was depicted in [Figure 4], which shows high prevalence (n = 4) in the 51 to 60 years age group.
Figure 4 Age- and gender-wise distribution of immunoglobulin G-positive oral and oropharyngeal cancer cases.

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[Figure 5] emphasizes the distribution of IgG-positive cases based on social habits, which shows that 57.1% (4) were alcohol abusers and 42.8% (3) were smokers. Among the seven IgG-ositive cases, three males were both smokers and alcohol abusers and one female case was a passive smoker.
Figure 5 Distribution of IgG-positive cases based on social habits.

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[Figure 6] depicts the topographical distribution of IgG-positive cases which shows that three (18.7%) patients had carcinoma on tongue, two (15.3%) patients on larynx/post cricoid, and two (11.1%) patients on the buccal mucosa.
Figure 6 Topographical distribution of IgG-positive cases.

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  Discussion Top

Oral squamous cell carcinoma (OSCC) is cancer originating from the squamous cell epithelium in the oral cavity, accounting for more than 90% of all tumors in the head and neck regions.[6] The global average incidence from the WHO/IARC global cancer statistics database (GLOBACAN 2012) shows “oral cancer" to be the sixteenth commonest cancer types, accounting for 3,00,200 new cases, which consisted of 1,98,900 and 1,01,300 new cases among males and females, respectively.[6] There is a marked variation with regard to the incidence and ethnic/racial groups. The WHO regards oral cancer as a significant public health challenge in India where the incidence of HPV in OPCs ranges from 26% to 39%.[7]

Risk factors for these cancers include chewing of tobacco, betel nuts, Ganja, consumption of alcohol, long-term exposure of the face to sunlight, genetic predisposition, previous diagnosis of oral cancers, and poor nutrition.[1] Low socioeconomic status is inter-related with factors such as nutrition, health care, living condition, and risk behavior which contributes to the development of oral cancer.[8]

Seroprevalence for HPV16-specific IgG and IgM antibodies in the present study was 10% (7) and 0% (0), respectively. Seroprevalence of IgG (10%) was correlating with study carried out by Pierce Campbell et al. (Florida, 2016) who reported 11.6%.[9] The presence of HPV antibodies indicates infection history, which can occur at various sites of the body, including genitals and oral cavity. It has been reported that cancer occurs after transformation of latently infected cells which may even take decades to develop. HPV16 seropositivity was present in patient’s serum for more than a decade before OPC was first diagnosed. Therefore, identifying HPV infection and understanding the role of HPV IgG and IgM antibodies might assist in the early detection of HPV-related cancers.[1] Zero prevalence for IgM antibodies in this study might be due to the reason that IgM antibodies are the first antibodies to appear in response to initial exposure to HPV16 antigen and are relatively short lived and disappear earlier than IgG.[10] Therefore, presence of HPV16-specific IgM antibodies represent acute or current exposure and presence of HPV16-specific IgG antibodies indicate past exposure to HPV16.[1] Though oral cavity is accessible for visual examination and the premalignant lesions and oral cancers have well-defined clinical diagnostic features, oral cancers are usually diagnosed in their advanced stages by the time which IgM antibodies may disappear.[8] It is unclear at what point the HPV16 antibodies are generated and are detectable because the immune response to HPV infection at oropharynx may induce seroconversion even in the absence of invasive lesions.[11] There is very little literature in India regarding the prevalence of HPV in OSCC, unlike western countries.[12]

In the present study, high prevalence of HPV16-related oral and OPCs was reported in the age group 51 to 60 years. According to Gunasekera et al., the rise of HPV16 seropositives in 51 to 60 years age group could be an indicator for the infection occurring in the fourth decade of their life.[13]

Predominance of OPCs in males might be because of changes in their life style, sexual behavior, and prolonged exposure to risk factors such as smoking, alcoholism, chewing of tobacco and tobacco-related products which exert their carcinogenic effect after several years of exposure and prolonged exposure to sunlight due to occupation.[14]

In the present study, 19 (27%) of cases were smokers, 14 (20%) were gutkha/khaini/pan parag users, and 13 (18.5%) were betel quid chewers. In countries such as India, bidi smoking is most common as they are cheaper than cigarettes and people of lower socioeconomic status and rural areas smoke bidis compared to cigarettes. Bidi smoking is considered as an important risk factor contributing to a considerable number of oral cancer cases, as they contain high content of nicotine and alkaloids and its poor combustibility due to the filter less design.[15]

Pan masala, gutkha, and khaini which are dried forms of tobacco, areca nuts which contain arecoline-specific nitrosamines, lime, and catechu, and combination of these products are strongly carcinogenic.[15] Alcohol has a synergistic effect with smoking and chewing tobacco-related products which might be the reason for higher incidence of oral and OPCs in people with habits of both smoking and alcoholism.[15]

Oral sex is one of the commonest risk factors for transmission of HPV16-related oral and OPCs. But cases included in the present study denied to provide the history of sexual behaviors. Hence, data regarding sexual behaviors were not able to provide in this study.

Poor oral hygiene is one of the risk factors for oral cancers. Poor oral hygiene-related attributable risk is around 32% for men and 64% for women in India.[8]

In developing countries such as India, most common sites involved in oral cancers are tongue and buccal mucosa. Gutkha/khaini/panparag/areca are kept in contact with buccal mucosa or gingivobuccal mucosa for a longer period and the extract is swallowed. Areca nut in combination with lime releases reactive oxygen radicals which contribute to oral carcinogenesis.[15]

In the present study, seroprevalence of IgG antibodies was high among males (n = 6, 85.7%) compared to females (n = 1, 14.2%). This study was correlating with studies carried out by Dahlstrom et al. (Texas, 2015) who reported 89.6%.[16] High prevalence of HPV16 IgG antibodies among men is high because of the difference in lifestyle including high risk sexual behavior such as earlier age of sexual debut, practice of premarital sex, number of sexual partners, and practice of oral sex.[13]

In the present study, 42.8% (n = 3) and 57.1% (n = 4) HPV seropositive cases were smokers and alcoholics, respectively. Similar findings for smoking was observed in the study carried out by Gunasekera et al. who reported 41.6%.[13] Higher prevalence of HPV16 oral cancers among smokers is due to the damage caused by tobacco which alters the cells making them more susceptible to infection, impairs mediators of immune system, and enhances DNA breaks thus promoting the integration of HPV DNA into the host DNA.[17]

In the present study, prevalence of HPV seropositive carcinoma of tongue, larynx, and buccal mucosa were 18.7% (n = 3), 15.3% (n = 2), and 11.1% (n = 2), respectively. Prevalence of HPV-related carcinoma of tongue was correlating with the studies carried out by Ellington et al. (USA, 2020) who reported 14%.[18] Higher prevalence of cancer buccal mucosa among HPV seropositive cases was observed because buccal mucosa is histologically similar to uterine cervix and other lower genital tract mucosa.[19] Risk factors such as prolonged smoking, chewing tobacco, and tobacco-related products cause microtrauma to oral mucosa resulting in increased access for viral entry.

  Conclusion Top

Due to high morbidity and mortality in oral and OPCs, there is a need for biomarkers for early detection, diagnosis, prognosis, and monitoring of these patients. Screening for HPV status in high-risk groups of OSCC by serology might be an affordable option in the clinical settings, as knowledge of HPV serologic status may be useful in influencing further diagnostic evaluation of HPV-related cancers.

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Conflicts of interest

There are no conflicts of interest.

  References Top

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Ducatman BS. The role of human papillomavirus in oropharyngeal squamous cell carcinoma. Arch Pathol Lab Med 2018;142:715-8.  Back to cited text no. 2
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Dahlstrom KR, Anderson KS, Field MS et al. Diagnostic accuracy of serum antibodies to human papillomavirus (HPV) type 16 early antigens in detection of HPV-related oropharyngeal cancer. Cancer 2017;123:4886-94.  Back to cited text no. 5
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