Abstract
Introduction
Out of all non-communicable diseases, cancer is the leading cause of death in the 21st century. Oral cancer ranks in top ten cancers in the world and oncogenic viruses contribute to its development and are a major preventable risk factor for it.
Material and methods
In the present study we intend to find the incidence of HPV and EBV by PCR in 108 sporadic oral cancer patients and study the clinico-pathological variables in agreement to their presence or absence.
Results
We found that the incidence of EBV in oral cancer is much higher (30/108; 27.8%) than HPV 16 (14/108; 13%) and, a complete absence of HPV 18 (0/108) was reported by real time PCR. Co-infection by EBV and HPV was reported in 5.6% cases (6/108). However, on comparing this data with the corresponding clinico-pathological cofactors (age, gender, grade of tumour, tumour size, nodal status, metastasis, DOI, TIL, ENE) it was found that there was no statistical significance between the two.
Conclusion
Hence, we conclude that these oncogenic viruses are independent cofactors to oral cancer development and none of the clinico-morphologic variable is associated with the viral etiology.
Keywords: Oncogenic viruses, EBV, HPV, Real time PCR, Oral cancer
1. Introduction
Cancer is ranked as the most frequent cause of death worldwide and the most important hurdle in improving the expectancy of life. It accounts for the bulk of global mortality due to non-communicable diseases (NCDs).1
Out of these, under Head and neck tumors we categorize oral malignancy. Among this Head Neck Squamous cell carcinoma (HNSCC) is ranked between either sixth to ninth position as far as incidence is concerned.2,3
Lifestyle, oncogenic viral infections and environmental factors are known risk factors apart from genetic variables in oral carcinogenesis. It has been estimated that approximately fifty percent of oral cancers are linked to the above modifiable variables including viral infections.4
Around two thirds of the worldwide reported virus related cancers have been found to be linked to either Human papilloma virus (HPV) or Epstein Barr Virus (EBV). Viral related cancers have been estimated to be 20% of the entire oncology load, adding 2 million cases per year related to infectious pathogens, out of which 1.6 million are from developing nations.5,6
The properties of these viruses linked with cancer are determined by their capability to provoke transformation at cellular level resulting in development of tumour. For example, it is well-known that EBV and high risk HPV oncoproteins can control intra- and extra-cellular signaling pathways, aggravate genomic unsteadiness, boost the lifespan of infected cells (by inhibiting apoptosis) and subvert cellular ageing, resulting in unrestrained cell propagation.6
These fundamentals are key biological features of carcinogenesis,7 which can be provoked after oncovirus infections, including EBV and high-risk HPVs. Therefore, it is of utmost importance to emphasize that EBV and high risk HPVs can be co-present in human cancers, especially oral.8,9
Hence with an aim to recognize EBV or high risk HPV infection in oral malignancies we conducted the following study with objectives to study the incidence and infection of oncogenic viruses EBV and HPV in oral squamous cell cancers and compare their presence with other co-factors namely tumour size, depth of invasion, extranodal extension, tumour infiltrating lymphocytes and the site of tumour i.e., the pathological staging with the presence or absence of virus.
2. Materials and method
After ethical approval and informed consent 127 cases of resected specimens, pertaining to oral cancer were included in the present study during the year 2018–19. Only histologically proven cases of oral cancers were included. All cases where there was insufficient tissue for processing or no tumour/tumour <2 mm [CRS Score 3] was present post chemotherapy/radiotherapy specimen were excluded from the study sample.10
2.1. Molecular testing
Paraffin tissue blocks of cases of histologically proven oral cancers enrolled in the study were used to obtain 9-10 μm sections (10 sections per case) in eppendorf tubes for further isolation of DNA. The isolation of DNA was performed using QIAamp DNA FFPE Tissue minikit (Qiagen GmbH, Hilden, Germany). The resultant isolated DNA was eluted in 100 μL in elution buffer. The resultant was preserved at −20 °C for later experiment.
Real time polymerase chain reaction (RT-PCR) was used for viral detection. Primers from EBNA -1 (213 bp) region and BHRF-1 (208 bp) region was used for EBV detection and E6/7 for HPV 16 (96bp) and 18 (115bp) along with primer for beta globin gene (286bp) was used for housekeeping. Table 1 enlists the sequence of the primers used.
Table 1.
Synthesized primers for DNA, EBV and HPV detection.
| 1 | HPV16Fwd | 5′-GGTCGGTGGACCGGTCGATG-3′ |
|---|---|---|
| 2 | HPV16rev | 5′-GCAATGTAGGTGTATCTCCA-3′ |
| 3 | HPV18fwd | 5′-CCTTGGACGTAAATTTTTGG-3′ |
| 4 | HPV18rev | 5′-CACGCACACGCTTGGCAGGT-3′ |
| 5 | EBNA-1 QP1 Fwd | 5′-GCCGGTGTGTTCGTATATGG-3′ |
| 6 | EBNA-1 QP2 Rev | 5′-CAAAACCTCAGCAAATATATGAG-3′ |
| 7 | BHRF-1 EA1 Fwd | 5′-GGAGATACTGTTAGCCCTG-3′ |
| 8 | BHRF-1 EA2 Rev | 5′-GTGTGTTATAAATCTGTTCCAAG-3′ |
| 9 | Beta-Globin Fwd | 5′-TAGCAACCTCAAACAGACACCA-3′ |
| 10 | Beta-Globin Rev | 5′-CAGCCTAAGGGTGGGAAAAT-3′ |
2.2. PCR
Reaction mixture was prepared in PCR tubes with 10 μl of SYBR green (PowerSYBR® Green PCR Master Mix, Applied Biosystems by Thermo Fisher Scientific, Warrington, UK), 1 μl of forward and reverse primer each, 2 μl nuclease free water and 6 μl of eluted sample DNA. Thermocycler used was 48 well StepOneTM (Applied Biosystems, Singapore). 2% agarose gel electrophoresis was performed for confirmation.
2.3. Beta globin and HPV detection
TAL57 region of the beta-globin gene was used to check for DNA quality and as a control for PCR inhibitors for each sample before running the RT-PCR. Negative controls were also run by using PCR grade water and template DNA mixture. Both for beta globin and HPV the cycle run were 1) 3 min at 95 °C; 2) 40 cycles of 95 °C for 15 s; 3) 59 °C (HPV18) and 60 °C (HPV16 and Beta Globin) for 20 s; 4) 72 °C for 25 s; 5) 7 min at 72 °C.
2.4. EBV detection
To reduce false negative results and maximise detection rates, 2 primer sets were used for EBV (EBNA-1 along with BHRF-1). For EBV detection two cycles of PCR were run 1) 3 repeats of 40 s and 2) 40 repeats of 30 s. Rest experiment was similar to HPV and Beta-globin procedure as described above.11
2.5. Controls
For HPV-positive cases for HPV 16/18, cervical cancer FFPE block was used and for EBV-positive DNA, undifferentiated nasopharyngeal carcinoma FFPE block was used. DNA extracted from the blocks mentioned above was used as positive controls for RT- PCR. Negative controls as mentioned above were also run.12
2.6. Statistical analysis
Mean ± SD, median and percentage of category variables were calculated. For Qualitative variables Chi-Square test/Fisher’s exact test was used wherever found appropriate. A p value of <0.05 was considered statistically significant. Excel sheet format was used to record data and analysis was performed using SPSS version 16.0.
3. Results
In a total sample collection of 127 samples, 19 were discarded due to low DNA content in real time PCR (confirmed by beta globin housekeeping gene). The rest of the 108 samples had a CT value below 30 for beta globin PCR. Male to Female ratio was 3.9:1, mean age was 45.6 yrs and the most common age group affected being the middle age (36–50 yrs). Well differentiated carcinomas were the most common according to histological grading (71.3%). The most common T staging was T2 45.4% (tumour size ≤ 2 cm with DOI >5 mm OR tumour size 2–4 cm with DOI ≤ 10 mm), N staging was N0 68.9% (no regional lymph node metastasis), M staging was M0 100% (no distant metastasis reported), the maximum DOI reported in cases was between 5 and 10 mm (54.6%), the TIL reported was moderate TIL (50%) and absence of ENE (93.4%)
As per PCR results, HPV 16 positivity was seen in 13% samples (14/108), HPV 18 positivity was nil (0/108), EBV positivity was 27.8% (30/108) and co-infection rate was 5.6% (6/108). [Table 2]. [See Fig. 1] Melt curves analysis of the PCR tests of beta globin, EBV, HPV 16 and HPV 18 are shown in Fig. 1.
Table 2.
Occurrence of HPV16, HPV18 and EBV by PCR in cases (n = 108).
| Viral PCR (n = 108) | Positive by PCR |
|
|---|---|---|
| Number of case | Percentage (%) | |
| HPV 16 | 14 | 13 |
| HPV 18 | 0 | 0 |
| EBV | 30 | 27.8 |
| Co infection | 06 | 5.6 |
Fig. 1.
Represents the melt curve analysis and specific product formation along with controls for each real time PCR in (a) beta globin (b) EBV (c) HPV 16 and (d) HPV 18.
When the clinico-pathological parameters were correlated with presence and absence of viral co-infection, none of the results were statistically significant [Table 3].
Table 3.
Clinico-pathological parameter correlation in accordance to HPV and EBV PCR positivity and respective p values, chi square values and degree of freedom (df).
| Clinico-pathological Parameters | HPV Positive (n = 14) | HPV Negative (n = 94) | EBV Positive (n = 30) | EBV Negative (n = 78) |
|---|---|---|---|---|
| 25–35 years | 2 | 27 | 09 | 20 |
| 36–50 years | 6 | 36 | 12 | 30 |
| 51–65 years | 3 | 24 | 05 | 22 |
| >65 years | 3 | 7 | 04 | 06 |
| χ2 = 3.67 [df = 3]; p = 0.300 | χ2 = 2.07 [df = 3]; p = 0.559 | |||
| Male | 11 | 75 | 23 | 63 |
| Female | 3 | 19 | 07 | 15 |
| χ2 = 0.011 [df = 1]; p = 0.916 | χ2 = 0.225 [df = 1]; p = 0.635 | |||
| G1 | 10 | 67 | 22 | 55 |
| G2 | 4 | 25 | 07 | 22 |
| G3 | 0 | 02 | 01 | 01 |
| χ2 = 0.316 [df = 2]; p = 0.854 | χ2 = 0.708 [df = 2]; p = 0.702 | |||
| T1 | 03 | 15 | 05 | 13 |
| T2 | 06 | 43 | 15 | 34 |
| T3 | 02 | 29 | 08 | 23 |
| T4 | 03 | 07 | 02 | 08 |
| χ2 = 3.97 [df = 3]; p = 0.264 | χ2 = 0.558 [df = 3]; p = 0.906 | |||
| N0 | 08 | 65 | 21 | 52 |
| N1 | 02 | 15 | 06 | 11 |
| N2 | 03 | 11 | 02 | 12 |
| N3 | 0 | 02 | 0 | 02 |
| χ2 = 1.49 [df = 3]; p = 0.684 | χ2 = 2.569 [df = 3]; p = 0.463 | |||
| DOI | ||||
| <5 mm | 01 | 06 | 02 | 05 |
| 5–10 mm | 10 | 49 | 18 | 41 |
| >10 mm | 03 | 39 | 10 | 32 |
| χ2 = 2.103 [df = 2]; p = 0.349 | χ2 = 0.551 [df = 2]; p = 0.759 | |||
| TIL | ||||
| Mild | 04 | 27 | 12 | 19 |
| Moderate | 06 | 48 | 12 | 42 |
| Marked | 04 | 19 | 06 | 17 |
| χ2 = 0.564 [df = 2]; p = 0.754 | χ2 = 2.71 [df = 2]; p = 0.258 | |||
| ENE | ||||
| Present | 01 | 06 | 0 | 07 |
| Absent | 12 | 87 | 29 | 70 |
| χ2 = 0.028 [df = 1]; p = 0.866 | χ2 = 2.82 [df = 1]; p = 0.093 | |||
Df: degree of freedom.
4. Discussion
The incidence of HPV, EBV and co presence of both in oral squamous cell carcinoma in our study was 13%, 27.8% and 5.6% respectively. Tyan et al., in 1993 in China conducted a study using PCR and found out that OSCC harbors both EBV (44%) and HPV 16 (11%) but they found no co-infection between the two.13 This could be due to the fact that their sample size was very small. They reported maximum co-infection in nasopharyngeal carcinoma (46%) followed by hypopharyngeal carcinoma (25%) and the more common strain causing HPV infection was HPV 16. This means that HPV 16 might be of significance in initiation or propagation of cancer and also that the lymphoid tissue is more susceptible to infection.
In 2012, a meta analysis was published by Jalouli et al. with a comparison done in data of 8 different countries regarding oral SCC and virus interaction in a total of 155 patients, 4 of the countries were developed (Norway, UK, Sweden and USA) and the rest 4 were developing (India, Sri Lanka, Sudan and Yemen).14 They came to the conclusion that the most common site for oral SCC in all 8 countries was tongue (26.4%) followed by gingiva (18%) and the floor of mouth (14.8%). This correlated with our study where the most common site for oral SCC was tongue (33.3%) followed by buccal mucosa (27.7%) and gingivo-buccal sulcus (9.2%).
The studies included in meta analysis used nested and semi-nested PCR, the incidence of HPV and EBV was calculated and confirmed by seeing the gel pictures of the amplifications. They reported the overall prevalence of HPV in OSCC as 35% and the single highest incidence in Sudan (65%) followed by India (45%) and UK (45%). This was much higher as compared to our study [Table 2]. Between the HPV prevalence of Sudan and Yemen, there was a statistically significant difference whereas there was no statistical distinction between countries of Group I and II as a whole in terms of HPV prevalence. The variation in HPV prevalence in OSCC in diverse studies might be due to variable sampling techniques, PCR methods and quality of sample (frozen or fixed).
EBV on the other hand showed an overall prevalence of 55% in OSCCs. This was much higher than what we found in our study (27.8%) but due to the marked variation in the percentage, it cannot be commented upon. The span of this prevalence was huge, being 22% in Yemen to 80% in the UK.
Co-infection of EBV and HPV was seen in all eight countries with a prevalence of 21% thus inferring that these viruses play a major role in the commencement of neoplastic change of human oral epithelial cells.8 Also, the co-infection by two or more viruses might enhance the possibility of development of OSCC.15
Jiang et al.16 in 2014 indicated that EBV can be present in the healthy epithelial cells as latent virus but HPV does not infect the normal tissue. It also revealed that a considerable proportion of tonsil and base of tongue (BOT) carcinomas show co infection. The rate of co-infection being 25% in tonsil SCC, and 70% in SCC BOT as evaluated by EBER1 RT-PCR and HPV ISH by them. In the soft palate where no lymphoid tissue exists, none of their samples were infected by either of the viruses.17 However in our study the total incidence of HPV and EBV viral infection and co-infection is relatively low which was primarily due to low DNA yield quality from FFPE tissue.
Robinson et. Al18 did not find any significant correlation between the grade of tumour and HPV status of OSCC whereas Zhao et al.19 found association between HPV infection and poor histological grade by multivariate analysis (OR = 104.0, 95% CI: 11.2–962.1). Their study stated that there was better survival with HPV infection due to chemo receptiveness of these OSCC. In the study by Dorota et al.,20 a noteworthy association was found between the status of HPV and the poor histopathological grades (chi square, p < 0.05). Our results revealed no statistical significance between any of the clinico-pathological variables when compared with the EBV and HPV status. This may be due to multiple risk factors playing a role in OSCC development in our region, especially betel nut/tobacco chewing being prevalent.17
HPV DNA status in OSCC influences the therapy choices and prognosis. There has been evidence that HPV positive OSCC patients have a favorable treatment prognosis,21, 22, 23 higher survival rate and lower recurrence rate as compared to HPV-negative patients.24 Also, such patients are more sensitive to chemotherapy25 and do not require post surgery radio or chemotherapy.26
In western countries OSCC has been seen afflicting younger adults as compared to elderly recently. Golusiński et al.27 calculated that this disease affects 0.24–9.0% of people under 45 years of age in all cases. Wojciechowska et al.28 collected data showing that 19.2% oral and oropharyngeal cancers occur before the age of 50 years. Dorota et al.20 found that among this population, HPV 16 was present more often (22.9%) than in the age group of more than 50 years (12.3%) and EBV was detected in 27.3.% cases (42/154) and the most often in the age group 50–59 years (31.4%). However as stated above in our region betel nut/tobacco chewing risk factor is the major role player of the younger population being affected. Though not significant but maximum i.e., 42.9% (6/14) cases which had HPV infection were from the age group of 36–50 years. Moreover, in our study, the most common age group of EBV infection was 36–50 years (40%; 12/30) indicating that middle aged people are more prone. EBV association is seen to be likely present with higher grade of oral cancers specifically in immunocompromised individuals.29 This is of utmost importance because EBV infection is widespread in the population and about 95% adults show antibodies to EBV. EBV infection might be asymptomatic and may persist throughout life.30 But according to Kis et al.,31 in OSCC patients (73.8%), the prevalence of EBV was much higher as compared to healthy groups (19.1%).
A few authors came across an association between EBV infection (especially co-infection with papillomaviruses) and squamous cell carcinoma of the tongue and oropharyngeal sites.14,20 Studies have reported HPV-EBV coinfection up to 7.8% which is close to our observation of 5.6%. A percentage so low, cannot predict the role of coinfection in OSCC.20,32
However few authors believe that co-infection by more than one virus can lead to progression of cancer.8,16 Jiang et al.16 in their study suggested that cells infected with multiple oncogenic viruses have a superior tumorigenic potential than normal cells particularly in invasion. There has been a query with the fact that whether these are co-infection or superinfection. It is a well established fact that long term persistent infection with non-oncogenic virus can cause superinfection with oncogenic type which in turn is capable of cancer progression.
E6 and E7 oncoproteins in HPV and LMP-1 and LMP-2 expressions in EBV have oncogenic potential. TLRs sense microorganisms and sense endogenous hazardous signals and hence play an important part in the early innate immune response.33,34 Fathallah et al.34 recognized that LMP-1 of EBV is an inhibitor of TLR9 transcription which can support the superinfection theory. Hence there is still a discrepancy regarding the co-infection being superinfection or actually leading to carcinogenesis with enhanced potential. This needs to be addressed by larger cohort studies.
In our study we found that the majority of patients from the oral SCC have HPV 16 infection and it is more frequently among subjects younger than 50 years, whereas HPV 18 was not detected. EBV may play a causal role in the initiation of OSCC.
Limitation of our study is low incidence of EBV or HPV or coinfection in cases, which makes the clinicopathological correlation skewed. A larger dataset may provide better information. Hence research on the mechanisms of co-infection and/or superinfection and the part played by them in oral squamous cell carcinoma are indispensable. The information about these mechanisms may offer targets for treatment and for expansion of diagnostic methods.
Credit author statement
Madhu Mati Goel: Conceptualization, Investigation, Resources, Writing – review & editing, Supervision, Project administration, Preeti Agarwal: Conceptualization, Software, Validation, Investigation, Data curation, Writing – original draft, Writing – review & editing, Visualization. Shikha Tiwari: Methodology, Validation, Formal analysis, Sumaira Qayoom: Methodology, Shivanjali Raghuvanshi: Methodology, Vanshika Shahi: Methodology, Software, Validation, Formal analysis, Investigation, Data curation, Writing – original draft, Writing – review & editing, Visualization. Funding acquisition, Uma Shanker Singh: Resources, Supervision.
Sources of funding
Indian Council of Medical Research, India (ICMR)
Declaration of competing interest
Nil.
Acknowledgement
We are thankful to King George’s Medical University for providing the infrastructure to perform our work. We are extremely grateful to Indian Council of Medical Research, India for funding the project via letter number 493/DME/18. We also thank our Immunohistochemistry and histopathology laboratory staff for their support. The above work is cleared from Institutional Ethics committee of King George’s Medical University. .
Contributor Information
Shahi Vanshika, Email: shahivanshika@gmail.com.
Agarwal Preeti, Email: preavn@gmail.com.
Qayoom Sumaira, Email: qayoomsumaira@gmail.com.
Kumar Vijay, Email: drvkumar2007@rediff.com.
Tewari Shikha, Email: shiks01@gmail.com.
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Singh Uma Shankar, Email: ussinghjyotsna@gmail.com.
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