|Year : 2015 | Volume
| Issue : 3 | Page : 377-381
Detection of human papilloma virus (HPV) and human immunodeficiency virus (HIV) in oral squamous cell carcinoma: A polymerized chain reaction (PCR) study
Suresh Dirasantchu1, Manjula Marthala2, Sameeulla Shaik1, Raviraj Jayam1, Suman Sreeram Venkata1, Vijaykumar Bokkasam1
1 Department of Oral Medicine and Radiology, CKS Teja Dental College, Tirupati, Andhra Pradesh, India
2 Department of Oral Medicine and Radiology, Gandhi Medical College, Hyderabad, Telangana, India
|Date of Submission||19-Dec-2014|
|Date of Acceptance||30-Oct-2015|
|Date of Web Publication||25-Nov-2015|
Department of Oral Medicine and Radiology, CKS Teja Dental College, Tirupati, Andhra Pradesh
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Aims and Objectives: Certain strains of human papillomavirus (HPV) have been shown to be etiologically related to the development of uterine, cervical, and other genital cancers, but their role in the development of malignancies at other sites is less well established. Previous studies have shown HPV in tumors of the head and neck, but its prevalence has varied depending on the detection methods and the types of tumor and/or tissue examined. This study was undertaken for the detection of high-risk HPV types 16 and 18 and human immunodeficiency virus (HIV) in oral squamous cell carcinoma. Materials and Methods: Twenty-five patients histologically diagnosed with oral squamous cell carcinoma and 10 apparently normal persons as controls were selected for the present study. Two biopsy specimens were removed surgically by incision biopsy for histopathological examination and polymerized chain reaction (PCR) study. Results: Out of 25 oral squamous cell carcinoma subjects, 8 were found to be HPV positive in PCR. Out of these eight subjects, four had HPV 16 and the other four had other genotypes, and one subject was HIV positive. Conclusion: The conclusion drawn from the present study was that well-defined risk factors like HPV may play a prominent role in the development of oral squamous cell carcinomas, in addition to other risk factors. Further studies with a larger sample size are necessary to arrive at conclusions and to explore the relationship of HPV and HIV in oral squamous cell carcinoma.
Keywords: Human immunodeficiency virus, human papillomavirus, polymerized chain reaction, squamous cell carcinoma
|How to cite this article:|
Dirasantchu S, Marthala M, Shaik S, Jayam R, Venkata SS, Bokkasam V. Detection of human papilloma virus (HPV) and human immunodeficiency virus (HIV) in oral squamous cell carcinoma: A polymerized chain reaction (PCR) study. J Indian Acad Oral Med Radiol 2015;27:377-81
|How to cite this URL:|
Dirasantchu S, Marthala M, Shaik S, Jayam R, Venkata SS, Bokkasam V. Detection of human papilloma virus (HPV) and human immunodeficiency virus (HIV) in oral squamous cell carcinoma: A polymerized chain reaction (PCR) study. J Indian Acad Oral Med Radiol [serial online] 2015 [cited 2019 Mar 24];27:377-81. Available from: http://www.jiaomr.in/text.asp?2015/27/3/377/170456
| Introduction|| |
Squamous cell carcinomas (SCCs) represent the most frequent malignancy in the head and neck region. They originate from the pluristratified squamous epithelium which lines the upper aerodigestive tract, and are characterized by a multiphasic and multifactorial etiopathogenesis.  Common risk factors in head and neck SCC (HNSCC) are smoking and alcohol abuse. However, in an increasing proportion of cases, no significant smoking or drinking history has been reported. Approximately 35 years ago, the role of human papillomavirus (HPV) in cervical cancer was postulated. On the basis of epidemiological and molecular evidence, in 1995, the International Agency for Research on Cancer recognized that high-risk HPV (HR HPV) types 16 and 18 are carcinogenic in humans. 
It is widely accepted that cancer of the uterine cervix is related to HPV infection. These two HPV types are responsible for approximately 70% of cervical cancer cases.  In addition, HR HPVs are associated with other ano-genital carcinomas, including vulvar, anal, and penile cancers, and with some HNSCCs.  HPV infection was first held responsible for the development of head and neck cancer in certain individuals lacking the classical risk factors for this disease (tobacco and alcohol abuse).  The majority of HPV-related cancers contain HPV DNA integrated into the host cell genome and express only two viral genes, E6 and E7, both of which encode oncoproteins. 
The HPV involvement in head and neck carcinogenesis was first proposed in 1983 which was supported by several other authors on the basis of the following evidences: 
Epstein and Scully  reported that patients who were human immunodeficiency virus (HIV) positive had a more advanced stage of oral SCC (OSCC) and poorer survival (57% survival at 1 year and 32% at 2 years) than patients who were HIV negative (74% and 59%, respectively). The pathogenesis of OSCC in patients with HIV includes increased cell growth and proliferation caused by viral interference with tumor suppressor proteins (p53, Rb) and activity of the HIV transactivator of transcription protein and HPV. The objectives of the present study were:
- the well-assessed broad epitheliotropism of HPV;
- the morphological similarities between oropharyngeal and genital epithelia;
- the ability of immortalizing human oral keratinocytes in vitro; and
- the strongly established etiological role of HR HPV in cervical SCC.
- Detection of HPV in OSCC.
- Detection of HR HPV types 16 and 18 (HR HPV-16 and -18) in OSCC.
- Detection of HIV in OSCC.
| Materials and Methods|| |
Twenty-five patients with OSCC and 10 apparently normal people as controls were selected for the present study from the patients attending the Department of Oral Medicine at Government Dental College and Hospital, Hyderabad. The history and clinical findings of each patient were recorded in a specially prepared proforma. After obtaining written informed consent from the patients, two biopsy specimens were removed surgically by incision biopsy of the clinically diagnosed oral carcinoma lesion. One specimen was washed in saline to remove blood and was fixed in 10% buffered formalin. It was sent to the Department of Pathology, Osmania General Hospital, Hyderabad for histopathological examination to confirm the diagnosis. Another biopsy specimen was transported to the laboratory through Trizol reagent medium (for DNA stabilization) to be stored at −80°C. After the histopathological confirmation of the clinical diagnosis, the stored specimens were used for polymerized chain reaction (PCR) study to detect HPV and HIV in OSCC.
A small piece of tissue was taken in a 1.5 ml microcentrifuge tube. Tissue/pellet was dissolved in 300 μl 1X Lysis Buffer. Five microliters of 10 mg/ml proteinase K was added, mixed, and incubated at 65°C for 1-2 h, till the pellet/tissue dissolved completely. Then, 150 μl of Tris-saturated phenol and 150 μl of chloroform (CHCl3): Isoamyl alcohol (IAA) (24:1) were added. It was mixed by inversion 15-20 times and centrifuged at 13,000 rpm for 10 min at room temperature. The upper aqueous layer was transferred to a fresh 1.5 ml microcentrifuge tube and 300 μl of CHCl3:IAA (24:1) was added. It was mixed by inversion 15-20 times and centrifuged at 13,000 rpm for 10 min at room temperature. The upper aqueous layer was transferred to a fresh 1.5 ml microcentrifuge tube and equal volume of isopropanol was added. It was mixed by inversion 20-30 times and centrifuged at 13,000 rpm for 10 min at room temperature. The supernatant was drained and the pellet dried for 5-10 min. The pellet was dissolved in 50 μl of Tris-EDTA (10:1) buffer. It was incubated at 37°C for 10 min on heating block. The DNA was stored at −20°C. The PCR products were run on a 2% agarose gel mixed with DNA loading dye and analyzed along with a 100 bp DNA ladder.
| Results|| |
Twenty-five patients of histologically diagnosed OSCC were selected as subjects and 10 apparently normal persons were selected as controls for the present study. The age of 25 subjects of OSCC ranged from 26 to 70 years, with an average age of 48 years. Ten apparently normal persons were selected as controls. The age of the controls was in the range of 28-60 years [Table 1] and Graph 1]. Among 25 subjects, 18 were males and 7 were females. Among 10 controls, 6 were males and 4 were females [Table 2] and [Graph 2]. Among the 25 OSCC cases, 11 showed involvement of buccal mucosa, 5 showed involvement of tongue, 4 showed involvement of alveolus, 3 showed involvement of retromolar area, 1 showed involvement of buccal sulcus, and 1 showed involvement of the palate.
PCR for HPV was performed for all the subjects. Of the 25 OSCC subjects, 8 were HPV positive (32%) and 17 were HPV negative (68%) [Table 3] and Graph 3]. None of the 10 controls were detected as HPV positive. Of the eight HPV-positive subjects, four were detected as having HPV 16 genotype [Table 4] and the other four as having other HPV genotypes [Table 5]. None of them were detected as having HPV 18. Among the four subjects detected positive for HPV 16, two were males and the other two were females. All four subjects positive for other genotypes of HPV were males. In PCR for HIV, out of 25 OSCC subjects, 1 (4%) was detected as HIV positive and 24 subjects (94%) as HIV negative. None of the 10 controls were detected as HIV positive.
|Table 5: Other genotypes of HPV among oral carcinoma subjects and controls|
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| Discussion|| |
The etiology of malignancies, in general, is considered to be multifactorial and based on the so-called cocarcinogenic theory where intrinsic and extrinsic factors acting simultaneously or in succession are considered to be responsible for the development of malignant neoplasms. Among the intrinsic factors often mentioned are immunity, nutrition, age, heredity, and DNA composition (proto-oncogenes and oncogenes). The contributing extrinsic factors associated with OSCC include: Tobacco use, either smoked or chewed in its various forms, actinic radiation, viral infections, especially with HPV, Epstein-Barr virus, HIV, lichen planus, chronic infections such as syphilis and candidiasis, chronic irritation, and alcohol intake. ,,
In a monograph published in 2009 by the International Agency for Research on Cancer (IARC) that summarized those infectious agents for which causation has been established beyond doubt, HPV 16 was considered to be causal in a proportion of HNSCCs.  Zhang et al. reported that based on the clinical behavior of HPV infections, the viruses can be grouped into high-risk (type 16 and 18) and low-risk HPV types. In the present study, out of 25 OSCC subjects, 8 (32%) were detected as HPV positive and 17 (68%) as HPV negative. This is in accordance with the study conducted by Syrjänen  who reported evidence on HPV as an etiological factor in OSCC by analyzing the presence of HPV antigens in 40 oral carcinomas using immunohistochemistry (IHC). In the present study, among the eight HPV-positive subjects, four were detected as having HPV 16 and the other four as having other HPV genotypes. None of them showed HPV 18 genotype. It indicates that HPV 16 is more prevalent than HPV 18 in oral carcinoma.
In the present study, the age of four HPV 16 positive subjects ranged from 26 to 66 years. The age of the four subjects positive for other genotypes of HPV ranged from 30 to 70 years. Out of eight HPV-positive subjects, six were below the age of 60. It infers that HPV oral carcinogenesis is more prevalent in those aged below or equal to 60. Among the four subjects positive for HPV 16 genotype, two were males and two were females. All the four subjects positive for other genotypes of HPV were males. It infers that among oral carcinoma cases, HPV is more prevalent in males than in females.
Among the four subjects positive for HPV 16, the site of SCC was buccal mucosa in two subjects, tongue in one subject, and alveolus in the other one. Among the four subjects positive for other genotypes of HPV, the site of SCC was buccal mucosa in two subjects, palate in one subject, and the retromolar area in one subject. However, it was reported in previous studies that HPV is positive in SCCs of tonsillar pillars, base of the tongue, and oropharynx. In the present study, HPV was present in a majority of cases of carcinoma involving the buccal mucosa.
Epstein and Scully  reported that patients who were HIV positive had a more advanced stage of OSCC and poorer survival (57% survival at 1 year and 32% at 2 years) than patients who were HIV negative (74% and 59%, respectively). In the present study, out of 25 OSCC subjects, 1 (4%) subject, who was a 50-year-old male, was detected as HIV positive and the site of lesion was buccal mucosa, and 24 subjects (94%) were detected as HIV negative. It indicates that there was no association of HIV with oral carcinoma.
| Conclusion|| |
The present study results suggest that the establishment of a highly sensitive detection system for HPV, such as PCR, using an appropriate primer and probe under optimal conditions would contributes to the development of carcinogenesis research. Moreover, HPV infection could be one of the several risk factors contributing to OSCC. HPV is associated with cervix cancer and its vaccination is proved to be effective in preventing cervical cancer. Since 32% of the subjects of the present study are reported as HPV positive, the potential role of HPV prophylaxis in OSCCs should be considered. The conclusion drawn from the present study is that well-defined risk factors like HPV may play a prominent role in the development of OSCCs, in addition to other risk factors. Further studies with large sample size are necessary to arrive at a conclusion and to explore the relationship of HPV and HIV in OSCC.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]