|Ahead of print
Human papillomavirus and head and neck squamous cell carcinoma in a UK population: Is there an association?
Raghad Al-Dabbagh1, Nadia Al-Hazmi2, Turki Y Alhazzazi2, AW Barrett3, Paul M Speight3
1 Department of Oral and Maxillofacial Prosthodontics, King Abdulaziz University, Faculty of Dentistry, Jeddah, Saudi Arabia; Eastman Dental Institute, University College London, Oral Pathology Department, London, United Kingdom, Saudi Arabia
2 Department of Oral Biology, King Abdulaziz University, Faculty of Dentistry, Jeddah, Saudi Arabia
3 Eastman Dental Institute, University College London, Oral Pathology Department, London, United Kingdom
|Date of Submission||08-Jul-2019|
|Date of Decision||29-Jun-2020|
|Date of Acceptance||04-Jun-2020|
|Date of Web Publication||27-Jan-2021|
Department of Oral and Maxillofacial Prosthodontics, King Abdulaziz University, Faculty of Dentistry, Jeddah; Eastman Dental Institute, University College London, Oral Pathology Department, London
Source of Support: None, Conflict of Interest: None
Background: Human papillomavirus (HPV) is an evolving important risk factor for head and neck cancer (HNC), especially for individuals who do not smoke and drink alcohol. The aim of this study was to establish the prevalence of HPV infection and elucidate its association with head and neck squamous cell carcinoma (HNSCC) patients in UK population.
Methods: The presence and association of HPV was investigated in HNSCC patients in this retrospective clinical study. Samples were obtained from archived biopsies and resections. HPV screening was performed by the use of polymerase chain reaction (PCR) using the GP5+/GP6+ and the SPF1/2 consensus as primers and by immunohistochemistry (IHC). Samples of viral warts that were IHC positive for HPV and fibroepethelial polyps (FEP) were used, as positive and negative controls, respectively.
Results: The cohort included 124 patients with HNSCC with an age range of 27–97 years (median, 60 years) and a male to female ratio of 2:1. Among the 124 HNSCC, 43/124 (34.7%) were from the tongue, 74/124 (60%) presented with advanced stage III or IV disease, 112/124 (90%) had a conventional phenotype, 84/124 (68%) were moderately differentiated, and 89/124 (72%) had bands or cords at the invasive front. Of the 124 patients with HNSCC, 84/124 (68%) demonstrated the presence of HPV, 0/124 (0%) was for oral squamous cell carcinomas (OSCC). HPV16 was the associated virus type in all positive samples. However, no significant association was observed between HPV positivity and other clinico-pathological variables including age and gender of the patients, stage, and malignancy differentiation.
Conclusion: The results we provide suggest that HPV infection is low in HNSCC, in general, and absent in OSCC, specifically, in this UK population during this time period. This implies that HPV infection may not play an important role in HNSCC carcinogenesis compared to other risk factors in UK population. This information can aid in more effective treatment approaches for treating UK cases of HNSCC.
Keywords: Carcinogenesis, head and neck neoplasms, mouth neoplasms, papillomaviridae, risk factors, squamous cell carcinoma of head and neck, United KingdomKey Message Human papillomavirus infection may not play an important role in the etiology of head and neck squamous cell carcinoma in a UK population. This could aid in targeted therapy of these cancers.
|How to cite this URL:|
Al-Dabbagh R, Al-Hazmi N, Alhazzazi TY, Barrett A W, Speight PM. Human papillomavirus and head and neck squamous cell carcinoma in a UK population: Is there an association?. Indian J Cancer [Epub ahead of print] [cited 2021 Oct 22]. Available from: https://www.indianjcancer.com/preprintarticle.asp?id=308045
| » Background|| |
Head and neck cancer (HNC) is a group of cancers that afflict the diverse structures in the head and neck region. Such tumors may originate in the oral tissues, nasal cavity, throat, larynx, and the salivary glands. Globally, HNC is the ninth most common cancer and accounts for more than 90% of squamous cell carcinomas. head and neck squamous cell carcinoma (HNSCC) has been associated to environmental risk factors such as tobacco and alcohol. However, there is a small population (15–20%) of HNSCC that occur in people who do not smoke and consume alcohol, suggesting that other factors, such as human papillomavirus (HPV) may play a role.,,
The association between HNSCC and HPV was first suggested by Syrjanen et al. in 1983, when he noted microscopic histopathological changes normally associated with HPV infection (koilocytes) in HNSCC lesions. A couple of years later, Loning et al. confirmed this association when he isolated HPV DNA in both premalignant and malignant oral lesions using in situ hybridization. Since then, HPV has been implicated in HNC and oropharyngeal cancer type.,, The mechanisms by which it works differ greatly from those of alcohol and tobacco users.
HPV is a diverse group of viruses from the papillomaviridae family. There is an estimated 100 human HPV types, although a larger number is presumed to exist, but these have not been fully sequenced. HPV's role in the etiology of carcinogenesis was first identified in cervical cancer where more than 90% of these cancers can be associated to HPV infection. Clues such as the detection of HPV16 and 18 DNA sequences in cell lines derived from cervical carcinoma and their involvement in altered cell growth because of cellular mutations and altered genomic integrity, sustain the role of HPV in cervical carcinoma.,
HPV's etiologic role has been supported by epidemiologic and molecular evidence in a subset of oropharyngeal cancers (OPC) through pathways similar to those in cervical cancer. Park et al. and others have reported that oral epithelial cells become immortal when they express E6 and E7 oncoproteins from HPV16., Viral expression of E6 and E7 of HPV16 were detected more often in oropharyngeal tumors and less often in oral squamous cell carcinomas (OSCC).,, The HPV16 E6 and E7 proteins inactivate p53 and pRb, respectively, with an associated lack of p53 mutations,,, a reduction in pRb expression, and overexpression of p16 proteins. This alteration in the cell cycle regulation pathways are seen most frequently in oropharyngeal tumors, specifically in tonsillar tumors.,,
Epidemiologic case-series studies showed a large discrepancy in HPV prevalence from 0% to 100%,,,, in HNSCC cases around the world. However, HPV16 was consistently the most prevalent type associated with HNSCC.,, However, the detection of viral DNA in either biopsy or resection samples is not sufficient for the definitive implication of an etiologic role in the pathogenesis of HNSCC. HPV biological activity is needed to prove causation such as the expression of viral oncoproteins (E6 and E7), the inactivation of the p53 and pRb pathways (not through p53 and pRb mutation), the clonal association at all cancer stages (preinvasion, invasion, and metastasis), and the viral physical state and load (integrated vs. episomal and low load vs. high load). Some studies have suggested genetic subclasses of HNSCC based on HPV infection and activity within the host cell nucleus., Further studies also suggested that HPV positive HNSCC showed a 2 to 5-fold increase in mutations making HPV a serious etiologic factor. However, studies from Europe have reported ambiguous prevalence of HPV in HNC, particularly in OSCC. Therefore, the aim of this study was to establish the prevalence of HPV infection and elucidate its association with HNSCC patients in a UK population.
| » Methods|| |
Patients and archival tissue samples
This is a retrospective clinical study and ethical approval was obtained from the Joint Research and Ethics Committee at the Eastman Dental Institute (EDI) and Hospital for patients who had incisional/excisional biopsies, or surgeries, for a primary HNSCC lesion, after obtaining a written consent from all patients as per the Eastman Dental Institute and Hospital policies. The ethical approval fully abided with the World Medical Association Declaration of Helsinki. Participants who were diagnosed with HNSCC and had archival representative tissue (biopsy or resections), histologic and clinical grading were included in this study. Participants who underwent preoperative radiotherapy or chemotherapy were excluded. Samples were collected between 1986 and 2004. Participants were identified from the archived biopsies of the histo-pathological records of the EDI and the Department of Pathology of University College London, UK. All samples were fixed in a 4% dilution of 10% formalin saline concentrate and were then processed and embedded in paraffin wax. Patients who underwent preoperative radiotherapy or chemotherapy were excluded.
The following information was retrieved from the patients' notes: date of birth, gender, and site of primary tumour, date of diagnosis, differentiation, date and type of surgery, and TNM staging. Tumors were clinically classified according to the gold standard TNM system as follows: Stage I, T1N0M0; Stage II, T2N0M0; Stage III, T3N0M0, or any T with N1 M0; and Stage IV, any T with N2M0, N3M0, or any N with M1.
In addition, the mode of invasion was objectively graded according to Odell et al. as follows: Grade 1: well delineated borderline; Grade 2: infiltrating cords, bands and strands; Grade 3: small groups or cords of less than 15 cells; and Grade 4: marked extensive cellular dissociation in single cells or small groups. Two independent pathologists reviewed 10% of the hematoxylin/eosin-stained slides. Cases were divided into sample and control groups. The control group consisted of positive and negative controls (13 cases and 18 cases, respectively).
DNA extraction from paraffin embedded tissue
Measures were taken to avoid cross-contamination. To extract DNA from paraffin blocks, the Qiagen Mini kit was used following the manufacturer's instructions. Tissue sections were cut from each tissue block ~25 μm thick and put in a sterile microtube for further processing. The resulting kit DNA extraction solution was either used for polymerase chain reaction (PCR) or stored at -20°C.
HPV screening by PCR
To circumvent contamination and false-positive results, all PCR-related work was carried out in specific areas within the PCR laboratories. Viral-free DNA and negative controls consisting of PCR reagents with no DNA were used to assess and detect crossover contamination. Furthermore, the transfer of DNA to the PCR buffer was carried out with aerosol-resistant pipette tips. In addition, we used oral papillomas and viral warts that were immunohistochemically positive for HPV as our positive controls to avoid false negative results. A negative control group of fibroepethelial polyps (FEP) were analyzed to avoid false positives. The thermal profiles used had been described in previous studies.
Using the GP5+/GP6+ consensus primers
The GP5+/GP6+ L1 consensus non-degenerate primer set was used as the primary method for the detection of HPV. This detected a broad range of HPV types that generated a PCR product of approximately 150 bp. The annealing temperature used in the PCR experiment was relatively low. The PCR mixture was made of 5 μl 10× PCR buffer II, 4 μl of magnesium dichloride (MgCl2), 0.4 μl of deoxynucleoside triphosphate (dNTP), 0.2 μl of GP5+/GP6+ primers, 0.2 μl of AmpliTaq Gold and 1 μl of DNA in a ultimate volume of 50 μl. Conditions for PCR included: activation of AmpliTaq Gold for 7 minutes at 95°C, followed by 40 cycles of 45 seconds at 93°C, 45 seconds at 40°C, 90 seconds at 72°C, with a final allowance of 5 minutes at 72°C. Then the annealing temperature was changed to 46°C to improve the product yield. A separate set of positive and negative PCR controls was carried out with each experiment.
Using the SPF1/2 consensus primers
The L1 consensus primers SPF1/2 set (comprising six primers) were the universal primers that were used to detect HPV-DNA, and they formed a 65 bp product. The PCR was performed in an ultimate reaction volume of 50 μl that contained 1 μl of isolated DNA, 5 μl of 10X PCR buffer II, 5 μl MgCl2, 0.4 μl of dNTP, 0.6 μl of SPF mix primers, and 0.2 μl of AmpliTaq Gold. Conditions for PCR were as follows: activation of AmpliTaq Gold for 7 minutes at 95°C, 40 cycles of denaturation for 45 seconds at 93°C, annealing for 45 seconds at 45°C, and extension for 90 seconds at 72°C with a final allowance of 5 minutes at 72°C.
Electrophoresis of PCR products
The Mini-Protean 3 Electrophoresis was used to mix an 8% gel. The gel mixture was instantly loaded between the glass plates and well-forming combs inserted into place. After 15 minutes the combs were removed, and the sample wells rinsed with 1× TBE buffer. The apparatus was assembled following the manufacturer's instructions. The 2.5 μl of loading buffer was mixed with 5 μl of the PCR products before loading into wells. The gels were run at 200 V for 35–40 minutes. Gels were then separated from the glass plates, stained in ethidium bromide (2.5 μl/ml) for 5–10 minutes and observed under ultraviolet light and photographed.
HPV cloning and sequencing
The purification of the PCR putative major protein L1 product was carried out using the Quiaquick PCR purification kit (Qiagen) and sequenced in one direction using the T7 primer. The TOPO TA cloning kit for sequencing (Invitrogen) was used to clone the purified PCR product into vector pCR4-TOPO. Nine positive recombinant clones were randomly chosen, and sequencing of the insert DNA was done by using the CEQ DTCS Quick start kit (Beckman-Coulter). All major capsid protein L1 sequences that had at least 98% identity were considered to belong to the same species.
HPV screening by immunohistochemistry (IHC)
Sialanized slides (DakoCytomation) were used to prevent section detachment. Xylene was used to dewax the sections which were then hydrated and microwaved in Dako Target Retrieval Solution at pH 6.0, for 20 minutes on high power. They were left to stand for 10 minutes and then rinsed in tap water. Then, 1–2 drops of a biotinylated wide spectrum HPV probe (DAKOCytomation), rubber glue and coverslip were added. Sections were denatured for 6 minutes on a 90°C hot plate followed by hybridization for 60 minutes at 37°C. Slides were subsequently washed in 2 changes of 0.05% Tween 20 in TBS (TBS/Tween) on a shaker to remove the coverslips. They were then immersed in stringent wash solution (Dako stringent wash was diluted 1/50 in distilled water) at 37° for 30 minutes and rinsed in 2 changes of TBS/Tween on a shaker. The slides were placed in an incubation tray after circling the section with a hydrophobic pen. Streptavidin-AP was applied for 20 minutes and rinsed in TBS/Tween. Slides were further incubated for 60 minutes in dark after application of BCIP (5-bromo-4-chloro-3-indolyl-phosphate)/NBT (nitro blue tetrazolium). Finally, the slides were rinsed in distilled water, and counterstaining was carried out with 0.1% nuclear fast red in 5% aluminium sulphate; they were then dehydrated, mounted, and examined under Olympus BUZ light microscopy.
| » Results|| |
Patients and archival tissue samples
Our study population comprised 166 patients that presented with a histologically confirmed diagnosis of HNSCC between the year 1986 and 2004. Twenty-one of the 166 patients were excluded because the DNA was degraded and could not be analyzed. An additional 21 cases were further excluded because of lack of TNM staging data. The majority of tumor specimens (104) were obtained from resections of the primary tumor and the remaining 20 tumors were obtained from biopsies.
The final cohort (n = 124) included patients whose age ranged from 27 to 97 years (median, 60 years) with a male to female ratio of 2:1 (85:39). The primary tumors were located from the oral cavity (n = 111) including tongue (43 cases), alveolus (35 cases), floor of the mouth (19 cases), buccal mucosa (9 cases), and palate (5 cases). Five cases were from the lip and 8 from the oropharynx/tonsil (n = 8). Of these, 4 cases were in the oropharynx and 4 were in the tonsils. Ninety per cent (112/124) were conventional SCC, and 10 per cent (12/124) were verrucous carcinoma.
Among our sample, 74/124 (60%) presented with locally advanced stage III or IV disease. Sixty-eight per cent of the patients (84/124) were diagnosed as moderately differentiated, 24/124 (19%) were well differentiated, and the remainder were poorly differentiated 16/124. Seventy-two percent (89/124) had bands or cords at the invasive front, 20/124 (16%) were diffuse, and the remainder (15/124) had pushing front. Of the 16 fibroepithelial polyps (negative controls) that were suitable for analysis, 4 were men and 12 were women. Age ranged from 18–78 with a median age of 44.5 years. A summary of demographic and histopathologic parameters of all HNSCC cases are presented in [Table 1].
HPV screening, cloning, and sequencing
Of the 124 patients with HNC, 2.4% (3/124) revealed a positive presence of HPV as indicated by a 150 bp on 8% poly acrylamide gel electrophoresis of the PCR products (GP5+/GP6+, SPF1/2 set) [Figure 1]. The HPV detection rate in OSCC was 0% (0/111). Specific sequencing and cloning of the L1 major capsid protein PCR product distinctly revealed that HPV16 was the type present in all positive samples.
|Figure 1: Detection of human papillomavirus in head and neck squamous cell carcinoma specimens by polymerase chain reaction, with L1 consensus primers (GP5+/GP6 + and SPF1/2). (a) 150 bp and (b) 65 bp products were visualized on electrophoresed 8% poly acrylamide gel with ethidium bromide. Lane 1; negative HPV control, lane 2; HPV negative DNA control (FEP), lane 3; molecular weight marker, lane 4; HPV positive DNA control (HPV positive, HIV-associated warts), lanes 5,6, and 7; positive HNSCC samples. *Denotes the positive PCR product SCC: squamous cell carcinoma|
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Of the 13 positive control samples, 100% revealed a positive presence of HPV as indicated by a 150 bp and/or a 65 bp fragment on 8% polyacrylamide gel electrophoresis of the PCR products. The detection rate in the negative controls was 0%. Further analysis of the PCR HPV positive cases and 13 randomly selected negative cases by IHC confirmed the PCR analysis results—just a few HPV-infected nuclei within the tumor were detected in each HPV positive tumor [Figure 2].
|Figure 2: Detection of human papillomavirus by immunohistochemistry with a biotinylated wide spectrum HPV probe. (a) A positive HPV control (HPV positive papilloma), positive nuclei stain blue (arrows). (b) A Positive HPV tonsillar, basaloid squamous cell carcinoma. Positive nuclei are few and scattered within the tumour islands (arrows)|
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All three HPV positive HNSCC cases were moderately/poorly differentiated and were from the posterior part of the tongue and tonsils [Table 2]. Posterior tongue SCCs were of the conventional phenotype while the tonsillar cancer was basaloid [Table 2]. A summary of demographic data and clinical information of the HPV-positive HNSCC cases are presented in [Table 2].
|Table 2: Demographic and clinicopathological parameters of human papillomavirus positive head and neck squamous cell carcinoma|
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Due to the low number of the positive sample results, an association between HPV positivity and other clinico-pathological parameters such as sex and age of the patients, as well as the stage and differentiation of the malignancy, could not be verified at this moment.
| » Discussion|| |
This report is one of the few large case-series studies that included a wide age group of 27–97 years old to assess the possible prevalence and subsequent association of HNSCC, and specifically, OSCC with HPV infection in the UK. The low HPV detection rate in HNSCC (2.4%) in general, and absence in OSCC (0%) specifically, is somewhat lower than other previously reported studies.,, In this study, HPV16 infected all of the three positive tumors consistent with other studies.,,
Studies detecting HPV in HNSCC and OSCC were mostly case series that identified HPV at various rates 0%, to 100% with or without a control comparison groups.,,,[3z6] This variation in HPV prevalence might be because of geographic etiological backgrounds and methodological/technical sampling approaches or errors.
More than 100 HPV types are known to infect humans, and there is no single consensus primer that detects all types. However, both consensus primers that were used (GP5+/GP6+ and SPF1/2 set) detect HPV types that are frequently detected in lesions of the oral cavity, including HPV16. Additionally, to guarantee sensitivity and specificity of the PCR assays, we used PCR that was directed toward a DNA sequence found in all cells (4 gene rearrangements in lymphoma) to eliminate non-amplifiable samples. Then, positive and negative HPV control groups were added for comparative analysis. Furthermore, with each run of PCR, a negative DNA, a negative non-DNA, and a positive DNA sample were added. The negative controls were pivotal in detecting any possible false positive results, while the positive controls eliminated the probability of false negatives. Thus, the results reflect a true low detection of HPV in HNSCC cases in general, and absence of HPV in OSCC samples specifically, in the UK.
Formalin fixation introduces artefacts that are related to the concentration and length of fixation. Extensive cross-linking of proteins to DNA causes fixation-induced DNA degradations. This results in DNA that is often fragmented yielding relatively short PCR fragments, which might result in negative PCR. This is not the case when the amplification is less than 200 bp, which is the reason for using the GP5+/GP6+ and SPF1/2 set of primers in such samples. These L1 consensus primers form 150 bp and 65 bp products, respectively. This increases the sensitivity in HPV detection in such samples.
Additionally, the prevalence of HPV appeared to be inversely proportional to the study sample size—specifically in oral cancers. This might reflect a selection bias. Small case-series studies (n = 100) that utilized PCR-based detection methods reported a high detection rate of 11.2% (8/71) and 61.5% (16/26). Larger similar studies reported a low detection rate of HPV in OSCC (4.4%; 15/338), which is still higher than the detection rate of 0% (0/124) of OSCC cases in this study.
Moreover, this variation in prevalence might be because of diverse patient population analyzed from different geographic locations with different rates of endemic infection. Asian studies reported a high HPV detection rate in OSCC ranging from 15% (15/100) to 100% (20/20). In comparison, European studies reported a lower HPV detection rate (0%; 0/33) and (4.4%; 15/338). This is similar to the absence of HPV infection in this report (0%; 0/124).
Recent literature clearly supports the theory that oropharyngeal and tonsillar cancers are more likely to be associated with HPV than other head and neck tumors. Hence, they are phenotypically a different entity—usually poorly differentiated, basaloid SCC, and occur in patients who don't smoke and don't consume alcohol., In our study, the positive HPV samples were from the base of the tongue and tonsils and were moderately to poorly differentiated. Thus, HPV in a UK population may be associated with a particular subgroup of HNSCC—specifically oropharyngeal and tonsillar tumors. In addition, HPV-positive cases were found to be associated with more aggressive behavior, poor prognosis, and lower survival rates compared to HPV-negative cases, despite aggressive treatment approaches and radiation-based adjuvant therapy. Accordingly, early HPV screening, such as screening of oral rinse samples with Aptima HPV assay, may aid in the provision of a patient-specific treatment strategy. In other words, the HPV-negative HNSCC in the UK, may require a less aggressive treatment strategy, with less side effects and disease morbidities, as compared to the HPV-positive cases.
| » Conclusion|| |
Our study is one of a few to investigate the prevalence and association of HPV with HNSCC, specifically OSCC, in a UK population. Our results have shown low HPV infection in HNSCC, in general, and absence of HPV in OSCC specifically, in the UK population over this study period. This suggests that HPV infection may not play as important a role in HNSCC carcinogenesis relative to other risk factors in the UK. This information can aid in improving treatment modalities and treatment approaches for treating UK cases of HNSCC.
Declaration of patient consent
The authors certify that they have obtained all appropriate patient consent forms. In the form the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.
Financial support and sponsorship
This project was funded by the Deanship of Scientific Research (DSR) at King Abdulaziz University, Jeddah, Saudi Arabia and the Saudi Cultural Attaché London, UK. Thus, the authors acknowledge both funding sources for their technical and financial support.
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2]
[Table 1], [Table 2]