1Department of Virology, Medical University, Lublin, Poland
2Chair and Department of Otolaryngology and Laryngological Oncology, Medical University Lublin, Poland
*Corresponding author: D. Polz-Gruszka, Department of Virology, Medical University of Lublin, Lublin, Poland; e-mail: firstname.lastname@example.org
Submitted 14 February 2015, revised 11 May 2015, accepted 7 July 2015
The aim of this study was to analyze the prevalence of BK virus, Human Papillomavirus and Epstein-Barr virus in oropharyngeal cancer, and to test our hypothesis that BKV/HPV/EBV co-infection plays a role in oropharyngeal squamous cell carcinoma. The correlation between viral infection, OSCC, anatomic location, pre-treatment staging, evidence of metastases to lymph nodes, and grading was also investigated. The examination samples were collected from 62 patients from paraffin tissue blocks. Males (90.3%) with, smoking (83.9%) and alcohol abuse (67.7%) problems prevailed in the studied group. G2 histological type was recognized in 80.6% cases. T4 (77.4%) and N2 (56.5%) traits occurred in the majority of patients. No cases of metastasis were observed (M0 100%). HPV – 24.2%, EBV – 27.4% and BKV 17.7% were detected in the studied samples. We observed co-infection EBV/BKV in 8% of cases, HPV/BKV in 4.8%, and HPV/EBV in 9% cases. Only in two cases co-infection of all three viruses was found.
Key words: BKPyV, Epstein-Barr virus (EBV), Human Papilloma virus (HPV), oral squamous cell carcinoma (OSCC), oropharynx
Head and neck cancer is a very important problem in public health worldwide and as well as in Poland. In Poland oral and oropharyngeal cancer constitutes 3.8% cancers among men and 1.3% cancers among women (Didkowska et al., 2013). Squamous cell carcinoma (SCC) constitutes 90% of all cases of cancers localised in the oral cavity and the oropharynx. The etiology of OSCC is considered to be multifactorial. Oncogenic viruses have also been involved in OSCC development (Hillbertz et al., 2012; Scully, 2011; Alibek et al., 2013). The cancerogenicity of HPV in humans was conducted by the International Agency for Research on Cancer (IARC) in 2007 and 2012. It was concluded that there is evidence indicating that HPV 16 can cause oral cancer (IARC, 2007; IARC, 2012). Ten to thirty percent of oral cancers are associated with HPV.
A number of studies also suggest the participation of Epstein-Barr virus (EBV) in oral cancer (Acharya et al., 2014; Kis et al., 2009; Jalouli et al., 2010; Jalouli et al., 2012). It is one of the most common viruses in humans (Alipov et al., 2005). EBV was the first human virus to which oncogenic potential was attributed, and it is classified as group 1 carcinogen by the World Health Organization’s International Agency for Research on Cancer (IARC, 1997). EBV is correlated with nasopharyngeal and gastric carcinoma, squamous cell carcinoma, Hodgkin’s lymphoma and Burkitt’s lymphoma (Peh et al., 2003; Slots, 2005; Slots et al., 2006; Tiwawech et al., 2008; Perera et al., 2010; Chen, 2011).
The BK polyomavirus (BKPyV,) is a small, non-enveloped, circular double-stranded DNA virus, belonging to the Polyomaviridae family, genus Orthopolyomavirus. The Orthopolyomavirus genus includes the JC virus (JCPyV), BK virus (BKPyV), the Simian virus SV40, Merkel cell polyomavirus (MCV, MCPyV) and trichodysplasiaspinulosa-associated polyomavirus (Benett et al., 2012). All polyomaviruses encode regulatory proteins, named large and small tumor antigens (LT-ag and ST-ag). The LT-ag has oncogenic potential in cell culture and animal models. It is estimated that up to 90% of the general population may be BKV infected, mainly during childhood (between 5 and 10 years of age). The transmission route is not clearly defined; however, the orofecal and respiratory route transmission is most possible as the upper respiratory tissues are more susceptible to infections. Additionally, BKV-induced nephropathy is a well-known problem among kidney transplant recipients. What is more, the BKV DNA was detected in human brain tumors, in neuroblastoma, in urinary tract tumors, in carcinomas of the uterine cervix, vulva, lips and tongue, and in Kaposi’s sarcoma. (Tognon et al., 2003; Neirynck et al., 2012; Schowalter et al., 2012; Raeesi et al., 2012; Konietzny et al., 2012). The correlation between BKV and metastatic bladder carcinoma among immunosuppressed transplant recipients, and among BKV and prostate and bladder carcinoma was analyzed. It is probably consequence of the kidneys being the main site of BKV latency (Tognon et al., 2003). On the other hand, salivary glands are described as a potential location of the virus as the presence of BKV genetic material was detected in saliva (Jeffers et al., 2009). The BKV virus is detected in the oral cavity, and probably has tropism to squamous cells.
The aim of this study was to analyze the prevalence of BK virus, Human Papillomavirus and Epstein-Barr virus in oropharyngeal cancer, and to test our hypothesis that BKV/HPV/EBV co-infection plays a role in oropharyngeal squamous cell carcinoma.
Materials and Methods
The present study comprised a group of 62 patients with a diagnosed and histopathologically confirmed OSCC who were hospitalized between 1995-2005 at the Chair and Department of Otolaryngology and Laryngological Oncology of the Medical University of Lublin, Poland. The patients had not received radiotherapy or chemotherapy before. TNM classification was done according to the criteria of the Union Against Cancer (UICC) (Sobin et al., 2009). Histological grading was performed according to the World Health Organization criteria, which divide tumors into three types: well differentiated (G1), moderately differentiated (G2), and poorly differentiated (G3) (Cardesa et al., 2010).
DNA extraction. DNA was extracted from three, 10-µm thick sections of formalin-fixed and paraffin-embedded tissue. The samples were transferred to Eppendorf tubes after cutting deep into the block. The microtome blade was changed after each time. One milliliter of xylene was added into test-tubes, mixed using puls-vortexing, and later centrifuged (3 min, 8000 rpm). Then the supernatant was removed. One milliliter of 96% ethanol was added to remove the residual xylene. The samples were vortexed and then centrifuged at 800 rpm for 3 minutes. The supernatant was carefully removed. Opened tubes were incubated at 37 °C until residual ethanol evaporated.
DNA measuring. Measurement of DNA concentration was made using spectrophotometry, and β-globin gene was amplified to evaluate the DNA extraction process and the presence of amplification reaction inhibitors.
Detection of BKV. The polymerase chain reaction (PCR) method was used to detect the BK/JC virus in the specimens. With the aim of detecting the genetic material of the BK/JC virus, the primers described for the first time by Arthur et al. (1989) were used (Table I). The oligonucleotides attach to a highly conservative region of early coding T-Ag. Because of a high homology of BKV and JCV genomes (75%), polymers are complimentary to the DNA of both viruses. The described primer pair can therefore be used for detecting both BK and JC viruses. The PCR product sequence is specific for a given virus. Primers amplify a 176 bp fragment of BKV genetic material, and 173 bp fragment of JCV genetic material. The final concentrations of the PCR reaction mixture were as follows: 2.0 mM MgCl2, 200 µM dNTPs, 0.25 µM of each primer, 0.5 U Hot Start Taq DNA polymerase (Qiagen/Hilden/Germany). Amplification was performed under the following conditions: initial denaturation 94 °C 15 min., followed by 40 cycles: 94 °C 1 min., 55 °C 1 min., 72 °C 1 min.; final extension: 72 °C 10 min. During each PCR run, the samples were tested, together with one negative and one positive control. DNA from urine of a kidney transplant patient was used as a positive PCR control to assess the success of amplification (ATCC VR-837). PCR reagents without template DNA served as a negative control. The PCR products were analyzed using electrophoresis in 2% agarose gel.
Epidemiological and clinical characteristics of patients.
|N = 62||%|
|age||40 – 49||12||19.4|
|place of residence||urban||37||59.7|
|histology stage G||1||2||3.2|
Detection of EBV. Nested PCR was carried out for detection of EBV DNA (54 bp). The primer sequences are shown in Table II.
The primer sequences used for the studied viruses.
|Virus||Primer sequences||Product size|
5’–GTC ATC TAC GGG GAC ACG GA–3’
5’–AAG AAG AGA TAT GTG GGG GT–3’
5’–ACC CGG AGC CTG TTT GTG GC–3’
5’–GGA GAA GGT CTT CTC GGC CTC–3’
All PCR reactions were carried out in the final volume of 25 µl using Taq PCR Core Kit (Qiagen/Hilden/Germany). The concentrations of PCR reaction components were prepared as follows: 2 mM MgCl2, 0.2 mM dNTPs, 0.2 µM of each forward and reverse primer and 0.5 U of Taq DNA polymerase. During each run, the samples were analyzed together with one negative and positive control. The negative control consisted of nuclease-free water, while the positive control for EBV was 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 minutes of initial denaturation, then 35 cycles of 94 °C for 30 seconds, 55 °C for 40 seconds, 72 °C for 1 minute with the final extension at 72 °C for 5 minutes. The second round amplification was performed with 1 µl of the first run product in the same conditions. The final PCR products were analyzed on 3% agarose gel.
HPV detection and genotyping. 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. PCR products are subsequently typed with the reverse hybridization assay. This kit identifies 28 HPV genotypes: HPV 6, 11, 16, 18, 26, 31, 33, 35, 39, 40, 43, 44, 45, 52, 52, 53, 54, 56, 58, 59, 66, 68, 69, 70,71, 73, 74, and 82.
Statistical analysis. Statistical analysis was performed to investigate the relationship between EBV and HPV presence and clinical and demographical characteristics of patients were determined by means of Pearson’s chi-square test and with Kruskal-Wallis test for small groups. Statistical significance was defined as p < 0.05.
This research was approved by the Ethics Committee and is in accordance with the GCP regulations (no. KE-0254/133/2013).
Males (90.3%) with, smoking (83.9%) and alcohol abuse (67.7%) problems prevailed in the studied group. G2 histological type was recognized in 80.6% of cases. T4 (77.4%) and N2 (56.5%) traits occurred in the majority of patients. No cases of metastasis were observed (M0 100%). Characteristics of patients with oropharyngeal carcinoma are shown in Table I.
In the studied samples HPV was detected in 27.4%, EBV in 29%, and BKV in 17.7%. We observed co-infection EBV/BKV in 8% of cases, HPV/BKV in 4.8%, and HPV/EBV in 9.7%. Only in two cases co-infection all three viruses was detected (Fig. 1). The presence of JCPyV was observed in none of the studied samples. There isn’t statistical significance between HPV, EBV, BKV infection and sex, age, place of residence, tobacco smoking, alcohol use, G, T, N features. Detailed results are shown in Table III.
Correlation between HPV, EBV, BKV and epidemiological and histological grades and TN classification
|HPV+ (N=17)||EBV+ (N=18)||BKV+ (N=11)|
|place of residence||urban||9||52.9||0.4725||12||66.7||7||63.6||0.7679|
Literature on the role of HPV virus, and especially HPV 16 in oral squamous cell carcinoma is abundant (Syrjanen et al., 1983; Szostek et al., 2009; Jalouli et al., 2010; Jalouli et al., 2012; Syrjanen and Syrjanen, 2013; Metgud et al., 2014; Sand and Jalouli, 2014; Sathish et al., 2014). The frequency of HPV occurrence varies in different populations and is dependent on the kind of the clinical material and the applied research methods. According to the available data, the prevalence of HPV in squamous cell carcinoma of the oral cavity and oropharynx varies and ranges from 8% to 74% (Castro and Busolotti, 2006; Ritchie et al., 2003; Sathish et al., 2014). In the present study, HPV DNA was detected in formalin-fixed and paraffin-embedded tissue in 24.2% cases. In our earlier studies from fresh frozen tissue, HPV was detected in 32.5% (HPV 16 composed 22.5%) (Polz-Gruszka et al., 2015). The presence of EBV was found in 27.4% of the studied patients. The role of EBV virus in OSCC was first observed by zur Hausen (1976), while in Poland by Szkaradkiewicz et al. (2002). Other authors also emphasize the role of EBV in the development of OSCC (Kis et al., 2009; Jaloluli et al., 2010; Jaloluli et al., 2011; Senyuta et al., 2013). Jaloluli et al. (2012) detected the presence of EBV In 55% of samples from 8 different countries. A number of studies point to the co-infection by HPV and EBV in cervix cancer (Szkaradkiewicz et al., 2004) and in oral squamous cell carcinoma (Al Mustafa et al., 2009; Achayra et al., 2014; Jiang et al., 2015). Several articles indicate that co-infection by multiple oncogenic viruses may be an important risk factor in the development of OSCC (Jalouli et al., 2011; Achayra et al., 2014; Jiang et al., 2014; Sand et al., 2014). Al Moustafa et al. (2009) showed that high-risk HPV and EBV co-infections play an important role in initiating neoplastic transformation of human oral epithelial cells.
In our study co-infection HPV/EBV was identified in 9.7%. Jiang et al. (2014) suggest that co-infected cells can have a higher tumorigenic potential than normal cells.
BKV DNA was also detected (17.7%) in the studied material from oropharyngal cancer. In our earlier studies of the clinical material from oral cavity carcinoma BKV DNA was detected in 18.5% cases (Polz et al., 2015). Recent data suggested a correlation between BK virus and various types of human cancers, Kaposi’s sarcoma, brain tumors, and tumors of the urinary tract (Hachana et al., 2012). The role of BKV in oral squamous cell carcinoma is controversial. What is more, BKV DNA presence was confirmed in high-grade squamous intraepithelial cervical lesions (precancerous lesions) (Comar et al., 2011). Some authors suggest that BK virus may be a potential co-factor for HPV in the development of cervical neoplasia (Fraase et al., 2011), especially together with the HPV genotype 16 (Comar et al., 2011). Burger-Calderon et al. (2014) suggests a connection between BKPyV and the oral compartment. BKV DNA was detected in tonsilar biopsy specimens and nasopharyngeal aspirates. Besides, Moens et al. (2014) suggest that polyomaviruses, including those induced by other oncogenic viruses, may be a co-factor in the development of cancer. In our studies HPV/BKV co-infection was detected In 4.8% cases, while EBV/BKV in 8%. In the light of our research results and the opinions of other researchers, we cannot exclude the role of BKV in oral SCC, taking into account the fact that the genetic material of BKV was detected in saliva (Jeffers et al., 2009). Double or mixed infection with other oncogenic viruses may induce transformation. The question remains open whether BKPyV play a role in oral squamous cell carcinoma or it is a co-factor for cancers induced by other oncoviruses. It is well-known that chronic infection affects the immunological response of the host. Primary infection with a nononcogenic virus may promote superinfection with oncogenic virus capable of neoplastic transformation. The oncogenic potential of HPV is related to the expression of E6 and E7, whereas the oncogenic potential of EBV – to the expression of LMP-1 and LMP-2 and of BKV – LTag. Toll-like receptors (TLRs) play a critical role in the early innate immune response to invading pathogens by sensing microorganism and they are involved in sensing endogenous danger signals. LTag of virus BKPyV as well as the protein of LMP-1 of virus EBV lowers the expression of TLR9, which can favour the aforementioned superinfection (Moens et al., 2014). According to Siennicka et al. (2013), TRLs simulation with microbial ligands influences EBV replication.
These observations provide insights for future studies of EBV and BKPyV pathogenesis and the association with oral squamous cell carcinoma. Future studies on the mechanisms of co-infection and/or superinfection and their role in oral squamous cell carcinoma are necessary. The knowledge about these viruses may provide targets for therapy and for diagnostic methods.
This study was supported by a Research Grant from the Medical University, Lublin, Poland (DS 233). We are thankful to prof. Wiesław Gołąbek (Chair and Department of Otolaryngology and Laryngological Oncology in Lublin 1998-2010) for his help in clinical material collection.
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