HPV-16 E6 L83V variant in squamous cell carcinomas of the upper aerodigestive tract

Abstract

Purpose

The aim of this prospective case series study was to determine the prevalence of HPV-DNA, analyze the E6 mRNA expression, identify intra-type variation in the E6 oncogene in upper aerodigestive tract (UADT) squamous cell carcinoma (SCC), and correlate the presence of HPV-DNA with several clinical parameters and outcome.

Methods

Frozen samples of UADT-SCC were analyzed for the presence and characterization of HPV-DNA and RNA sequences by means of polymerase chain reaction (PCR), reverse transcriptase-PCR, and direct sequencing of amplified products.

Results

HPV-DNA sequences were detected in 10% of the tumors, all of which were typed as HPV-16. Positivity for HPV-16 E6/E7 mRNA was observed in five of the eight HPV-positive tumors (62.5%). The HPV-16 E6 L83V variant was present in five cases. Multivariate analysis identified a history of absence of smoking (P = 0.009) as a predictor of HPV-positive tumor. No significant differences in overall and disease free survival curves were observed between patients with HPV-positive tumors and patients with tumors without detectable HPV-DNA.

Conclusion

Our findings support the etiological participation of HPV-16 in a subset of UADT-SCCs from patients lacking traditional risk factors. The potential prognostic significance of HPV-16 E6 L83V variant in HPV-16 positive UADT-SCCs should be more extensively investigated.

Introduction

In 2006, upper aerodigestive tract cancer (oral cavity, pharynx and larynx) was estimated to be the seventh most common cancer in Europe (Ferlay et al. 2007). By far, squamous cell carcinoma (SCC) is the most common malignancy of the upper aerodigestive tract (UADT) typically occurring in older adults in their sixth and seventh decades, and is twice as common in men than in women (Muir and Weiland 1995).

Chronic exposure of UADT epithelium to tobacco and alcohol, alone or in combination, are well-established risk factors. The effect of combined exposure to alcohol and tobacco on risk of head and neck cancer appears to be multiplicative (Pelucchi et al. 2006). In terms of population attributable risks, it has been estimated that approximately 80% of oral and pharyngeal cancer cases in men and about 65% of cases in women may be prevented by avoiding alcohol and tobacco exposure (Pelucchi et al. 2006).

However, certain individuals lack these typical risk factors. Furthermore, although public health efforts intended to reduce tobacco use have successfully reduced the prevalence of cigarette smoking, a significant increased incidence of oral and oropharyngeal SCC in younger population has been observed in Europe and in the United States (Hammarstedt et al. 2007; Sturgis and Cinciripini 2007; Chaturvedi et al. 2008). Human papillomavirus (HPV) is a necessary cause of virtually all invasive cervical cancers (Bosch and de Sanjosé 2002). Studies on the mucosal oncogenic types of HPV have demonstrated that the products of two early genes, E6 and E7, play a key role in malignant transformation. E6 oncoprotein is able to induce deprivation of the tumor suppressor protein p53 via the ubiquitin pathway. On the other hand, E7 oncoprotein has high affinity for pRb and promotes its degradation, that results in the release of active E2Fs which in turn activate transcription of a group of genes encoding proteins essential for cell cycle progression, such as cyclin E and cyclin A (Tommasino 1997). If it persists, then oncogenic type HPV infection results in unscheduled proliferation and genome instability, turning cells malignant.

Substantial molecular and epidemiological evidence suggest that HPV plays an etiologic or a cofactor role in the pathogenesis of a subset of UADT-SCC (D’Souza et al. 2007; Charfi et al. 2008). Particularly, several data suggest that an increasing amount of oropharyngeal carcinomas may be attributed to HPV infection, mainly type 16 (Sturgis and Cinciripini 2007; Chaturvedi et al. 2008). HPV oral infection is mainly sexually acquired and the recent increase in the incidence of oropharyngeal carcinoma has been considered the result of changing sexual behaviors (Chaturvedi et al. 2008).

The aim of this study was to determine the prevalence of HPV DNA, investigate the E6/E7 mRNA expression, identify intra-type variation in the E6 oncogene in UADT-SCC, and correlate the presence of HPV-DNA with several clinical parameters and outcome.

Patients and methods

Patients with a diagnosis of UADT-SCC seen at Otolaryngology Clinic II-Regional Center for Head and Neck Cancer, University of Padua, Treviso Regional Hospital, between May 2003 and July 2006 were considered for inclusion in this prospective case series study.

Criteria for inclusion in the study

The criteria for inclusion in the study were: previously untreated, histologically proven, SCC of the UADT (oral cavity, oropharynx, hypopharynx, and larynx).

Histological analysis

The diagnosis of SCC and the score of histological grading was determined by an experienced head and neck pathologist. The World Health Organization criteria were used as follows: grade 1 (G1), well differentiated; grade 2 (G2), moderately differentiated; and grade 3 (G3), poorly differentiated. The presence of basaloid features (small crowded cells with hyperchromatic nuclei, scant cytoplasm, small cystic spaces) were also evaluated and described (Wain et al. 1986).

Staging

All patients were evaluated by physical examination, fiberoptic endoscopy of the upper respiratory tract, tracheobronchoscopy, esophagoscopy, chest X-ray, head and neck contrast-enhanced computed tomography ± magnetic resonance imaging. The subsites of the neoplasms were defined according to International Union Against Cancer (UICC) criteria. When a tumor involved more than one subsite, its original subsite was considered the one in which the largest neoplastic mass was present. Tumors were staged according to the 2002 UICC’s TNM classification (Sobin and Wittekind 2002).

Surgical treatment

Surgical resection was performed using either a transoral, a transcervical, or a combined approach including an ipsilateral or bilateral neck dissection. Reconstruction, when indicated, was performed with either a pectoralis major musculocutaneous flap or a microvascular free flap. Neck dissection was performed before resection of the primary tumor. Postoperative radiotherapy was used in patients with T3-T4 tumors, perineural tumor spread, positive or close surgical margins, multiple metastatic nodes, or nodes with extracapsular extension. The planning target volume was irradiated with a dose 50 Gy of 4–6 MV photons of linear accelerator administrated in 2 Gy daily fractions applied five times weekly. The spinal cord was limited to a maximum of 44 Gy. An electron beam boost of 10 Gy up to cumulative dose of 60 Gy was given to the involved neck region in patients with multiple metastatic nodes or with evidence of extracapsular extension and to the pathologic-positive surgical bed.

Non-surgical treatment

Patients who underwent radiotherapy alone were prescribed to receive a dose of 66–70 Gy in 33–35 fractions over a period of 7 weeks. Patients who underwent concurrent chemoradiotherapy received at least 2 cycles of chemotherapy concurrently with radiotherapy. The chemotherapeutic regimen included cis-platinum 100 mg/m² on day 1, 5-fluorouracil 1,000 mg/m² as a continuous infusion on days 1–5.

Collection of specimens

Two tumor samples were collected from each patient from a non-necrotic area of the suspected tumor before any cancer treatment; one was fixed in 10% buffered neutral formalin for histological examination in order to confirm the diagnosis, and the other was snap-frozen in liquid nitrogen and kept at −80°C for molecular analyses.

Detection of HPV DNA and RNA sequences

DNA was extracted by using the proteinase K/phenol/chloroform procedure, and total RNA by means of the RNeasy Mini kit (Qiagen GmbH, Germany) according to the manufacturer’s instructions. Amplification of a 268-bp fragment of the beta-globin gene was performed to verify DNA quality using PC04/GH20 primers. HPV detection was conducted using MY09/MY11 general consensus primers (which amplify a fragment of approximately 450 bp), as previously described (Del Mistro et al. 2001). HPV types were identified by restriction fragment length polymorphism (RFLP) analysis of the amplified products. Amplification of a 323-bp fragment in the HPV 16 E6 region by type-specific primers H16L1/H16R3 was also performed on all samples (Del Mistro et al. 2006); type-specific primers 16E2-1/16E2-2 that amplify a 1,192-bp fragment in the E2 region were used on HPV 16 positive samples (Das et al. 1992). DNA from SiHa (HPV 16 positive) and HeLa (HPV 18 positive) cell lines were used as positive controls.

Detection of E6/E7 mRNA full-length transcripts was performed with the PreTect HPV Proofer Norchip assay (Alfa Wassermann, Milano, Italy), as described by the manufacturer’s instructions; the test is a multiplex nucleic acid sequence based amplification (NASBA) assay, and is capable of specifically identifying types 16, 18, 31, 33, and 45 (Molden et al. 2007).

Sequence analysis of HPV

Viral variants were characterized by direct sequencing of the E6 fragment; PCR products were purified by ExoSAP-IT (USB Corporation, OH, USA), subjected to cycle sequencing by ABI PRISM Big Dye Terminator Cycle Sequencing kit (Applera, Foster City, CA, USA), and sequencing reactions were run on the ABI PRISM 310 Genetic Analyzer (Applied Biosystems). The BLAST server was used to match all sequences available in GenBank [http://www.ncbi.nlm.nih.gov/]; variants were defined by comparison to HPV 16 prototype sequences.

Follow-up

The routine follow-up program consisted of locoregional examination at 2-months intervals during the first year, 3-months intervals in the second year, 4-months intervals between the third and fifth years and every 6 months thereafter.

Statistical analysis

The following parameters were recorded for each patient and analyzed for potential correlation with HPV status: gender, age, history of smoking, alcohol abuse (≥0.5 L), tumor site, TNM stage, tumor grading, basaloid features, and presence of comorbidity as evaluated by “The Adult Comorbidity Evaluation index 27” (ACE-27) (Hollenbeak et al. 2007).

Fisher exact test was used to assess group difference on categorical data. A multivariate logistic regression analysis was undertaken using a forward stepwise technique, in which including significant variables and those with a trend toward significance as determined by univariate analysis, in order to identify independent predictors for HPV-positive tumors.

Survival analysis was calculated using the standard Kaplan–Meier method. Overall survival was defined as the interval between the date of the end of treatment and death. Disease free survival was defined as the interval between the date of the end of treatment and the date of objective disease recurrence or death from any cause. Tests of significance were based on log rank statistic.

Tests were 2-tailed, and levels of statistical significance have been calculated at the 5% level of probability. Statistical analysis was performed using the SPSS/PC software package (SPSS Inc., Chicago, IL).

Ethics

The local institutional review board approved the study protocol and informed consent was obtained from the patients.

Results

Patients characteristics and treatment

A total of 77 previously untreated consecutive patients with UADT-SCC were recruited at Otolaryngology Clinic II-Regional Center for Head and Neck Cancer, University of Padua, Treviso Regional Hospital.

Table 1 summarizes the clinical and behavioral patients characteristics. The 77 patients in the study consisted of 66 males (85.7%) and 11 females (14.3%) ranging in age from 46 to 89 years (median 64). Overall, exposure to tobacco and/or alcohol was recorded for 61 patients (79.2%).

Table 1.

Anatomo-clinical and behavioral patients characteristics

Characteristic	N (%)	Median (range)
Gender
Female	11 (14.3)
Male	66 (85.7)
Age (years)		64 (46–89)
Tobacco
Yes	54 (70.1)
No	23 (29.9)
Alcohol
Yes	46 (40.3)
No	31 (59.7)
T category
T1	19 (24.7)
T2	21 (27.3)
T3	23 (29.9)
T4	14 (18.1)
N category
N0	40 (51.9)
N1	14 (18.2)
N2	21 (27.3)
N3	2 (2.6)
Stage
I	13 (16.9)
II	8 (10.4)
III	22 (28.6)
IV	34 (44.2)
Grading
G1	17 (22.1)
G2	33 (42.9)
G3	27 (35.1)
Basaloid features
Yes	3 (3.9)
No	74 (96.1)
ACE 27
0–1	46 (59.7)
2–3	31 (40.3)
Site
Oral cavity	10 (13.0)
Oropharynx	22 (28.6)
Larynx	38 (49.4)
Hypopharynx	7 (9.1)
Treatment
Surgery	30 (39.0)
Surgery + RT	39 (50.6)
RT	3 (3.9)
CTRT	5 (6.5)
HPV status
Positive	8 (10.4)
Negative	69 (89.6)
Follow-up (months)		31 (12–58)

RT radiotherapy, CTRT concurrent chemoradiotherapy

Among the 77 UADT-SCC, 58% were in the larynx-hypopharynx, 29% in the oropharynx, 13% in the oral cavity. A surgical treatment (± radiotherapy) was carried out in 90% of patients.

HPV status

High risk HPV-DNA sequences were detected in 10% of the tumors, all of which were positive for type 16, as indicted by both RFLP analysis on MY09/MY11 amplified products and E6 type-specific amplification. Data concerning the HPV-positive tumors are shown in Table 2. In reference to the tumor site, HPV-16 was present in 18.2% of oropharyngeal SCCs, 20.0% of oral SCCs, and 4.4% of laryngeal-hypopharyngeal SCCs. Positivity for HPV-16 E6/E7 mRNA was observed in five of the eight HPV-DNA positive tumors; the negative result for 2/3 negative cases was confirmed on a second RNA preparation. The HPV16 E6 L83V variant was present in five cases, and the prototype in the remaining three. Amplification of the E2 region was obtained in six of the eight HPV-positive cases (a weak band was observed in two cases), while the remaining two were negative, a result indicative of viral integration.

Table 2.

Anatomo-clinical and virological data of HPV-positive cases

Patients	Gender	Age	Site	TN	Treatment	HPV	mRNA E6/E7	E6 variant	E2
1	M	74	OC	T1N2b	Surg + PORT	HPV16	Positive	L83V	Positiveb
2	M	58	OC	T1N0	Surg	HPV16	Positive	L83V	Positive
3	M	75	OPh	T1N2b	Surg + PORT	HPV16	Negative	L83V	Positive
4	M	72	OPh	T1N0	Surg	HPV16	Positive	L83V	Negative
5	M	68	OPh	T2N0	Surg	HPV16	Positive	Prototype	Positive
6	M	53	OPh	T2N2b	Surg + PORT	HPV16	Positive	L83V	Negative
7	M	62	L	T4N1	Surg + PORT	HPV16a	Negative	Prototype	Positiveb
8	M	67	Hph	T4N3	CTRT + Surg	HPV16a	Negative	Prototype	Positive

M male, OC oral cavity, OPh oropharynx, L larynx, Hph hypopharynx, Surg surgery, PORT postoperative radiotherapy, CTRT concurrent chemoradiotherapy

^aCases in which the results were indicative of a lower viral load

^bCases showing a weak amplification band

HPV status and clinical parameters

Univariate analysis (Table 3) revealed a significant negative correlation between history of smoking [odd ratio 0.11 (95% CI.0.02–0.59); P = 0.008], alcohol abuse [odd ratio 0.08 (95% CI 0.01–0.66); P = 0.006] and HPV-positive tumors. A trend toward significance was observed for the relationship of HPV-positivity to oral-oropharyngeal site of the tumor [odd ratio 4.9 (95% CI 0.93–26.4); P = 0.061]. Multivariate analysis identified only a history of absence of smoking [hazard ratio = 10.3 (95% CI 1.7–59.8), P = 0.009] as a predictor of HPV-positive tumor.

Table 3.

Univariate analysis of several parameters in relation to tumor’s HPV status

Characteristic	HPV negative	HPV positive	P value *
Gender
Female	11	0	0.593
Male	58	8
Age (years)
≥65	33	6	0.263
<65	36	2
Tobacco
Yes	52	2	0.008
No	17	6
Alcohol
Yes	45	1	0.006
No	24	7
T category
T1-2	34	6	0.266
T3-4	35	2
N category
N0	37	3	0.470
N1-3	32	5
Stage
I–II	18	3	0.676
III–IV	51	5
Grading
G1–2	46	4	0.440
G3	23	4
Basaloid features
Yes	3	0	1.000
No	66	8
ACE 27
0–1	40	6	0.463
2–3	29	2
Site
Oral cavity–oropharynx	26	6	0.061
Larynx–hypopharynx	43	2

RT radiotherapy, CTRT concurrent chemoradiotherapy

* Fisher exact test

HPV status and outcome

The median follow-up for surviving patients was 31 months (range, 12–58). Actuarial overall survival and disease free survival of all patients 3 years after treatment was 71.8% (95% CI, 53.4–90.2%) and 56.9% (95% CI, 36.2–77.6%), respectively. Taking into account the whole follow-up period, no significant differences in overall survival (χ² logrank = 0.041; P = 0.840) and disease free survival curves (χ² logrank = 0.006; P = 0.937) were observed between patients with HPV-positive tumors and patients with tumors without detectable HPV-DNA.

Discussion

Prevalence of HPV-positive tumors showed a trend toward correlation with the site of origin, in accordance with published data (Kreimer et al. 2005). Carcinomas located in the oral cavity and oropharynx contained type 16 DNA sequences much more frequently [18.7% (6/32)] than those located in the larynx and hypopharynx [4.4% (2/45)] (P = 0.061). More importantly, while 83.3% (5/6) of the former expressed E6/E7 mRNA transcripts, none of the latter two did so. As suggested by Braakhuis and colleagues, low levels of HPV DNA and absence of viral transcriptional activity are likely to have no or limited biologic significance, and could indicate that in these tumors the virus does not play a pathogenetic role (Braakhuis et al. 2004). Indeed, HPV-DNA detection per se in a HNSCC does not prove a causal association (Psyrri and DiMaio 2008); causality is linked to viral transcriptional activity and cellular deregulation, as demonstrated in cervical carcinogenesis with detection of E6/E7 m-RNA in more than 99% of HPV16-positive cases (Kraus et al. 2006).

A recent systematic review has shown that the prevalence of HPV infection in patients with oropharyngeal carcinoma is about 35%, with a higher rate in North America (47%) compared to Europe (28.2%). When considering specifically HPV-16, the prevalence of positive oropharyngeal tumors was 42.1% in North America and 23.8% in Europe (Kreimer et al. 2005). This is in contrast with our series and another Italian study (Badaracco et al. 2007) where only 18.7 and 17.9%, respectively, of patients with oropharyngeal carcinoma harbored HPV-DNA. This divergence in prevalence may result from differences in life style and risk-factor exposures. In an area, like Northeast of Italy, with high wine consumption, a higher rate of head and neck cancers is probably attributable to smoking and alcohol abuse (Barra et al. 1990); alcohol exposure has been recorded for ≥40% of the patients in Italy (Present study; Badaracco et al. 2007) and for ≤30% of the patients in USA (Gillison et al. 2000). On the other hand, herpes simplex-2 seroprevalence, a surrogate marker of sexual habits, is about 5.5% in Italy against 17.2–25.6% in the US (Suligoi et al. 2000; Leone et al. 2004; Xu et al. 2006).

On the other hand, the divergence in prevalence of HPV-related UADT-SCCs between this study and previous studies is most likely not due to the different methodology used. Detection and typing of HPV-DNA sequences can be performed by using different molecular strategies. The most widely used and validated consensus primers (MY09/11, GP5+/6+, SPF1/2) target a conserved region in the L1 gene, and give rise to amplicons of different lengths; these can be typed by sequencing, or by hybridization with type-specific probes, or by restriction fragment length polymorphism analysis (RFLP). The different strategies (consensus PCR and typing method) differ in sensitivity for specific types and for detection of multiple infections, and in performance with different samples. The use of frozen tissues and analysis by MY-PCR followed by RFLP with RsaI, HaeIII, DdeI (and PstI when necessary) restriction enzymes, associated with HPV 16 type-specific PCR in our study were chosen in order to have a high sensitivity in the detection of HPV sequences, particularly type 16, the most frequently found in HNSCC, and differences in the prevalence of HPV-positive cases cannot be attributable to methodological issues. The reproducibility and high sensitivity of the MY09/11 amplification assay was recently reported by other authors (Nobre et al. 2008).

Our results seem to support the hypothesis that risk factors for HPV-positive and HPV-negative UADT-SCC are distinct. A significant correlation between absence of smoking and drinking and HPV-positive tumors was observed. Recently, Gillison et al. found markedly different risk factors for HPV-16 positive and HPV-16 negative head and neck SCC in a hospital-based case-control study, with several measures of sexual behavior being associated with HPV-16 positive tumors and, on the other hand, tobacco and alcohol use and poor oral hygiene being the dominant risk factors with HPV-16 negative tumors (Gillison et al. 2008).

These data along with several molecular evidences support the existence of two different etiologic pathways of head and neck cancer development: one caused by tobacco and alcohol-mediated oncogenic insult and characterized by downregulation of p16 protein and TP53 gene mutation, and the other caused by high risk HPV-mediated carcinogenesis and mainly characterized by wild-type TP53 gene and upregulation of p16 protein (Gillison et al. 2008; Psyrri and DiMaio 2008).

In our series, no significant differences in overall survival and disease free survival were observed between patients with HPV-positive tumors and patients with HPV-negative tumors. Most retrospective studies have shown a better outcome in patients with HPV-positive head and neck SCC (Gillison et al. 2000; Schwartz et al. 2001; Lindel et al. 2001; Weinberger et al. 2006). However, not all the studies show consistent results. Several authors reported no correlation between HPV infection in head and neck cancer and survival (Pintos et al. 1999; Riethdorf et al. 1997; Hoffmann et al. 2005). Recently, Fakhry and colleagues confirmed the prognostic significance of HPV infections in a prospective randomized phase II trial of 96 patients with oropharyngeal and laryngeal carcinomas; in this study, the hazard ratio for overall and progression free survival in HPV-positive patients was 0.36 (P = 0.02) and 0.27 (P = 0.01), respectively. However, when the analysis was restricted only to the patients who had oropharyngeal cancer, hazard ratio (95% confidence interval) for both overall and progression free survival encompasses 1.0 (Fakhry et al. 2008). Conversely, a recent meta-analysis by Ragin and Taioli suggested an oropharyngeal restricted prognostic significance of HPV-positivity with no difference in overall survival between HPV-positive and HPV-negative non-oropharyngeal patients (Ragin and Taioli 2007). A statistically significant association with 2-year overall survival was recently demonstrated in a group of Italian patients (Badaracco et al. 2007). The better outcome in patients with HPV-positive tumors has been attributed to an enhanced radiation and chemotherapy sensitivity due to an intact p53-mediated apoptotic response (Ferris et al. 2005; Butz et al. 1996). Therefore, the lacking evidence for an association between HPV status and prognosis in this series may partially be related to the fact that only few patients received irradiation with or without concomitant chemotherapy as elective treatment.

Data on HPV 16 viral variants have been reported so far in a limited number of studies; Gillison and colleagues (2000) investigated 52 HNSCC cases, while Badaracco et al. examined intra-type variation of a subset of their HNSCC tumors by sequencing a fragment of the L1 region (Badaracco et al. 2007). HPV-16 has been shown to display intratypic sequence variants that are known to differ in oncogenic potential, antigenic properties and geographic distribution; several mutations have been described within the HPV 16 E6 region. Sequence analysis of the E6 gene of our HPV16-positive UADT-SCCs showed the presence of the T350G mutation in 5/8 cases, all of which located in the oral cavity and oropharynx [5/6 (83.33%)], while the two cases in the larynx and hypopharynx contained HPV 16 prototype sequences. Of the naturally occurring HPV 16 viral variants, the T350G (L83V) is the most frequently found among invasive cervical cancers (Del Mistro et al. 2006; Tornesello et al. 2004) and has been linked to a higher oncogenic potential than the prototype (Lichtig et al. 2006). The E6 variant distribution found among our UADT cases (5/8) reflects the frequency observed in this area by sequence analysis in cervical samples; the L83V variant predominates over the prototype, with a higher prevalence among women with invasive cervical cancer and high grade lesions than among women with no or low grade lesions (Tornesello et al. 2004, data not shown). The L83V variant was the most frequently found (although to a lesser extent; 20/52) also among the cases examined by Gillison and colleagues. Since no data are yet available, we can only speculate whether HPV-16 E6 L83V variant confers UADT-SCC a more aggressive clinical behavior that might have neutralized, in the present series, the reported more favorable outcome of patients with HPV-positive UADT-SCC. Additional studies are necessary in order to establish the prevalence of this variant and its potential prognostic significance in HPV-16 positive UADT-SCC.

Conclusion

Our findings support the etiological participation of HPV-16 in a subset of UADT-SCCs from patients lacking traditional risk factors, particularly tobacco smoking. The “no drinker and no smoker population” showed a relative higher risk of HPV-associated UADT-SCC; the different risks probably imply two different pathogenic mechanisms as outlined by Psyrri and DiMaio in a recent review (Psyrri and DiMaio 2008) and may explain the increase of oral and oropharyngeal cancers in the last decades. More attention should be probably paid in collecting the history of the patients, and counseling for oral HPV infection risk factors should be implemented. HPV-related UADT-SCC seem to display an enhanced radiation sensitivity. Therefore, the detection of HPV infection in UADT-SCC may be of significance in planning the treatment and consequently in improving the prognosis. The currently available HPV vaccines preventing persistent cervical infection from HPV-16 may have an impact in the incidence of HPV-related head and neck cancers by reducing virus circulation in the population and by protecting the mucosa of the upper aerodigestive tract from HPV infection.

In contrast with most previous reports, no association between HPV status and prognosis has been found. The type of treatment administered and the virulence of HPV-16 E6 L83V variant may be responsible for these findings. Finally, the potential prognostic significance of HPV-16 E6 L83V variant in HPV-16 positive UADT-SCCs should be more extensively investigated.