Abstract
The role of viruses in the etiology of oral cancer has been proposed in many studies. The aim of the present study was to analyze the prevalence of Epstein-Barr virus, Human Herpes virus type 1, Cytomegalovirus and Human Papilloma virus among patients with oral squamous cell carcinoma in a Polish population. We investigated fresh-frozen tumor tissue fragments obtained from 80 patients with OSCC using the polymerase chain reaction assay. HPV was detected in 32.5% (22.5% were HPV 16), more often in laryngeal (36%) than in oropharyngeal carcinoma (26.6%). EBV was identified in 57.5%, HHV-1 in 7.5%, and CMV in 10% of patients. Co-infection with one or more viruses was detected in 30% of cases and most frequently it was co-infection with EBV and HPV (15%). Further studies are necessary to determine the potential role of EBV and the possible importance of HHV-1 as an infection co-factor in oropharyngeal cancer.
- Oral squamous cell carcinoma
- OSCC
- larynx
- oropharynx
- Epstein-Barr virus
- EBV
- human herpes virus-1
- HHV-1
- human papilloma virus
- HPV
- cytomegalovirus
- CMV
Head and neck cancer ranks sixth among the most common malignant tumors worldwide, and is one of the major problems of public health worldwide (3). There are several types of cancers of the oral cavity, but approximately 90% are squamous cell carcinomas.
In Poland, malignant tumors are the second cause of death after cardiovascular diseases, and in 2010, they accounted for 26% of deaths in men and 23% in women (4). In 2010 in Poland, 2,709 men were diagnosed with lip, mouth or throat cancer and 1,763 died. This group of cancer accounts for 9.84% of all cancer in men and 1.4% in women. Oral cancer is second to laryngeal cancer when considering malignant tumors of the head and neck.
Many different risk factors play a role in the etiology of oral cancer. The effect of some etiological factors is well-established in the literature, such as consumption of tobacco and alcohol, poor oral hygiene and inappropriate dietary habits (5, 6). The role of oncogenic viruses in the development of oral squamous cell carcinoma (OSCC) has been proposed in several studies (7-9).
The aim of the present study was to analyze the prevalence of Epstein-Barr virus (EBV), Human Herpes virus type 1 (HHV-1), Cytomegalovirus (CMV) and Human Papilloma virus (HPV) infection among patients with OSCC in a Polish population.
Materials and Methods
Patients. The present study comprised of a group of 80 patients with a diagnosed and histopathologically-confirmed OSCC who were hospitalized at the Otolaryngology Division Hospital in Radom, Poland. The patients had neither previous radiotherapy nor chemotherapy. This research was approved by the Ethics Committee and is in accordance with the GCP regulations (no. KE-0254/181/2012).
TNM classification was carried-out according to the criteria of the Union Against Cancer (UICC) (1). Histological grading was performed according to the WHO criteria (2) (G1: well-differentiated, G2: moderately well-differentiated, G3: poorly differentiated).
The epidemiological, clinical and pathological characteristics of the studied patients are shown in Table I. Male patients dominated in the examined group (85%). The patients' ages ranged from 40 to 79 years. The most numerous group was composed of patients aged 50-69 years (55.2%), with only 15% patients being under 50 years of age. Most studied individuals (>60%) lived in urban areas. A total of 70% of the studied patients reported smoking, while 71.3% reported alcohol abuse.
Patients with stage G2 (60%), T2 (50%) and N2 (37.5%) disease prevailed. All patients had stage M0 disease (i.e. no spread to other organs). The carcinoma was located in most cases (>60%) in the larynx.
DNA extraction. The material consisted of 80 fresh frozen tumor tissue fragments taken from patients with OSCC. Fragments of fresh tissue (20 mg) were cut and homogenized in a manual homogenizer Omni TH/Omni International/Kenneswa/Georgia/USA then subjected to isolation of the genetic material using the QIAamp/ DNA/Minikit/Qiagen/Hilden/Germany, according to manufacturer's instructions. Immediately after the isolation, the efficiency and purity of the obtained eluate was checked using a spectrophotometer Epoch/Biotek Instruments/Winooski/Vermont/USA. The measurement was performed on a Take 3 plate/Biotek Instruments/ Winooski/Vermont/USA, which enables for measurement in micro-volumes (2 μl). The Microplate Reader Software Gen 5.2.0/Biotek Instruments/Winooski/Vermont/USA was used to analyze the results. The isolates were kept at −20°C until the test was conducted. To verify the quality of the obtained DNA (presence of inhibitors of Polymerase Chain Reaction), assay of β-globin was performed.
Detection of viruses. Nested PCR was carried-out for detection of CMV DNA and EBV DNA and for detection of HHV-1 DNA a single-step PCR. The region within the polymerase gene was amplified for each herpesvirus. The primer sequences are shown in Table II. All PCR reactions were carried out in a final volume of 25 μl using Taq PCR Core Kit/Qiagen/Hilden/Germany
Concentrations of PCR reaction components were prepared as follows: 2 mM MgCl2, 0.2 mM dNTPs, 0.2 μM each forward and reverse primers and 0.5 U of Taq DNA polymerase. During each run, the samples were analyzed together with one negative and positive control. Negative control were nuclease-free water, positive control were; for HHV-herpes simplex strain HF, ATCC-VR-260, for CMV-clinical sample from patients positive for CMV, for EBV-EBV-positive cell line, Namalwa, ATCC-CRL-1432. The reaction mixture containing 5 μl of extracted DNA was amplified under the following conditions: 94°C for 3 min of initial denaturation, then 35 cycles of 94°C for 30 sec, 55°C for 40 sec, 72°C for 1 min with the final extension at 72°C for 5 min. The second round amplification was performed with 1 μl of the first run product in the same conditions. HHV-1 DNA amplification consisted of 45 cycles. The final PCR products were analyzed on 3% agarose gel.
HPV detection and genotyping was performed using the INNO-LiPA HPV Genotyping Extraassay/Innogenetics/Gent/Belgium. The kit is based on the amplification of a 65 bp fragment from the L1 region of the HPV genome with SPF10 primer set. PCR products are subsequently typed with the reverse hybridization assay.
Statistical analysis. Statistical analysis was performed to investigate the relationship between EBV, CMV, HHV-1 and HPV presence and clinical and demographical parameters by means of Pearson's chi-square test. Statistical significance was defined as pp<0.05.
Results
The prevalence of viruses in laryngeal and oropharyngeal carcinomas is presented in Table III. HPV was detected in a total of 32.5% of patients, including 22.5% with HPV type 16; in four cases (10%) other types were detected, i.e. 59, 45, and 68. HPV was detected in laryngeal carcinoma (36%) more often than in oropharyngeal carcinoma (26.7%). However, this difference was not statistically significant.
EBV was identified in 57.5% of the studied samples, including 60% of laryngeal carcinomas and 53.3% of oropharyngeal carcinomas.
HHV-1 was identified in six cases (7.5%), CMV in eight cases (10%). HHV-1 and CMV were more often detected in laryngeal than in oropharyngeal cancer. This difference was not statistically significant. Co-infection with one or more viruses was detected in 30% of cases. Co-infection with EBV and HPV (15%) was detected most frequently. Co-infection between other viruses was identified: EBV with CMV in 7.5%; HPV with CMV in 5%; HPV with CMV and EBV was detected in one case.
The frequency of detection of HPV DNA increased with the age of patients, i.e. in patients under 50 years of age it was 8.3%, and in the group of patients aged over 70 years it was 83.3.3% (Table III). On the other hand, EBV was most frequently detected in the age group of those under 50 years, i.e. at 66.7%. The prevalence of HHV-1 and CMV was the highest in patients under 50 years of age and for both viruses it was 16.7%.
Discussion
The role of viruses in the development of OSCC has been proposed in many studies (7-10). The most frequently studied are HPV, EBV and HHV-1 (previously HSV-1).
Some authors divided viruses into two groups strongly associated with OSCC (HPV, HHV-1) and less frequently with OSCC (EBV, HCV) (6).
The first study to perform a comparison between different countries was published by Jalouli et al. in 2012 (11). In the present study, the overall HPV prevalence in OSCC was 35%; the highest was in Sudan (65%), followed by India (45%) and the UK (45%).
In our study among patients with oral cancer in South-eastern Poland, the overall HPV prevalence was 32.5%, primarily of HPV type 16 (22.5%). Our results are consistent with the existing literature, which estimated that 25-35% of all OSCCs are caused by HPV (9). In our previous investigation, the prevalence of HPV in patients with OSCC was 25% and 86.7% were infected with HPV type 16 (12).
An original observation that implicated HPV as a risk factor in the development of oral cancer was presented by Syrjanen et al. in 1983 (13). Since then, several studies have focused on HPV detection in oral cancer (14). A more recent study by Syrjanen and Syrjanen showed a strong association between the presence of HPV DNA, specifically HPV16, and OSCC (15). This meta-analysis showed that HPV significantly increases the risk for OSCC, compared to controls.
Data of the Centers of Disease Control and Prevention indicate that incidence rates of HPV-associated oropharyngeal cancer increased among white males (16). In our study group there existed only white males. Cases of OSCC and oral cavity squamous cell carcinomas have recently dramatically grown in number in men under 50 years old (17).
A review of the literature indicates that HPV is the most important prognostic factor in squamous cell carcinoma, while the most common genotype is HPV 16 (18). Other viruses, e.g. HHV-1 and CMV, have been identified in oral cancer (19-21).
zur Hausen drew attention as early as 1976 to the possibility of the involvement of EBV in human cancer (22). Data provided in the literature suggest that the role of EBV in OSCC depends on the geographical region (10). The prevalence of EBV in oral samples varies widely in different studies and its results are controversial (23). South-east Asian studies found a high prevalence of EBV and concluded on an etiological role of EBV in OSCC. North American studies, as well as West and North-European studies, describe a lower prevalence of EBV and conclude that the role of EBV in OSCC is questionable. Jalouli et al., studying formalin-fixed paraffin-embedded tissue samples obtained from patients with oral cancer from eight different countries, showed the highest overall EBV prevalence of 55% (ranging from 22% in Yemen to 80% in the UK) (11, 24). Sermyuta et al. are of the opinion that EBV can initiate nasopharyngeal carcinoma only in people with genetic predisposition and with concurrent environmental and dietary factors (25).
Jalouli et al. analyzed the prevalence of HHV-1 DNA in tobacco-related OSCC from Sudanese and Indian populations (24). These authors detected HHV-1 in 29% of the samples in tobacco-related OSCC and in 5% among patients with betel-related OSCC.
Several studies have shown that an individual patient may be infected by more than one viruses (21, 26, 27). Co-infection by multiple oncogenic viruses may be an important risk factor in the development of OSCC (9). Al Moustafa et al. showed that high-risk HPV and EBV co-infections play an important role in the initiation of neoplastic transformation of human oral epithelial cells (28). Data from our study and a review of literature illustrate that prevalence of HSV, HPV and EBV infections is common and may influence oral cancer development.
The prevalence of viruses in OSCC varies in many studies. In the retrospective analysis by Kruger et al. HPV was detected only in 6% of cases (29). The detection of HPV DNA depends on the method of oral specimen collection (direct swab, saliva). Different sampling techniques and different PCR methods are used to detect the genetic material. The quality of clinical samples (fresh-frozen or paraffin-embedded) may also differ. In major studies, paraffin-embedded tissues are used. In the present study, we used fresh-frozen tissue fragments.
Further studies on a larger population are necessary to determine the potential role of EBV and the possible significance of HHV-1 as an infection co-factor in oropharyngeal cancer. Early diagnosis would reduce the morbidity and mortality from oral cancer. The knowledge about predictive markers may be useful in early diagnosis, in prognostication and effective therapy.
Acknowledgements
This study was supported by a Research Grant from the Medical University, Lublin, Poland (DS 233).
Footnotes
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Conflicts of Interest
None declared
- Received November 24, 2014.
- Revision received December 10, 2014.
- Accepted December 12, 2014.
- Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved