Primary, secondary and tertiary prevention of human papillomavirus-driven head and neck cancers

European Journal of Cancer 78 (2017) 105e115

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Review

Primary, secondary and tertiary prevention of human papillomavirus-driven head and neck cancers Haı¨tham Mirghani a,*, Alain C. Jung b, Carole Fakhry c,d Department of Otolaryngology e Head and Neck Surgery, Gustave Roussy Cancer Campus, 114 rue Edouard Vaillant, Villejuif, France b Laboratoire de Biologie Tumorale, EA 3430 Universite´ de Strasbourg, CLCC Paul Strauss, Strasbourg, France c Department of Otolaryngology-Head and Neck Surgery, Johns Hopkins Medical Institutions, Baltimore, MD, USA d Department of Epidemiology, Bloomberg School of Public Health, Johns Hopkins Medical Institutions, Baltimore, MD, USA a

Received 29 December 2016; received in revised form 13 March 2017; accepted 20 March 2017 Available online 20 April 2017

KEYWORDS Oropharyngeal/ oropharynx/head and neck; Cancer/neoplasm; HPV; p16; Primary/secondary/ tertiary prevention; Screening

Abstract Human papillomavirus (HPV)-driven oropharyngeal cancers (OPCs) represent an increasing proportion of head and neck cancers that could become, in the next few decades, a public health problem in certain western countries. This significant epidemiological change strongly calls for preventive measures. Prophylactic HPV vaccination and screening programmes for early identification and treatment of premalignant lesions are currently being used to reduce the incidence of uterine cervical cancer, which is the paradigm of HPVdriven malignancy. These strategies have proven to be efficient as the incidence of cervical cancer has dramatically dropped since the 1960s in most countries where they are properly applied. The success of cervical cancer prevention encourages the development of similar approaches to prevent HPV-driven OPCs. However, a number of important limitations impede their application to HPV-driven OPCs, and the development of innovative and specific strategies dedicated to this disease are urgently needed. This article provides an overview on primary, secondary and tertiary prevention of HPVdriven OPC and discusses some directions for future research. ª 2017 Elsevier Ltd. All rights reserved.

* Corresponding author: Fax: þ33 1 42 11 70 10. E-mail address: [email protected] (H. Mirghani). http://dx.doi.org/10.1016/j.ejca.2017.03.021 0959-8049/ª 2017 Elsevier Ltd. All rights reserved.

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1. Introduction Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide, with an annual incidence rate of 600,000 cases [1]. These cancers, which are traditionally caused by excessive tobacco and alcohol consumption, are decreasing in most western countries since the 1980s due to the success of public health campaigns [2]. However, this trend is not homogenous, as cancers arising from the tongue base and the tonsils are on the rise as highlighted by recent epidemiological data from several western countries [3e5]. This increase is attributed to high-risk human papillomaviruses (HR-HPVs) and particularly to HPV16 [3e6], whose aetiological role in anogenital cancer has been clearly acknowledged for several decades [7]. Oral HPV infection is a sexually transmitted affection [8e10]. In a recent American study, the prevalences of oral HR-HPV and HPV16 infections were respectively 3,7% and 1%, which is significantly less than that at the genital level [11]. HPV-driven oropharyngeal cancers (OPCs) have specific clinical, pathological and molecular features compared to their HPV-negative counterparts [9,10,12,13]. These emerging cancers already represent the dominant form of OPCs in North America and Northern Europe, where up to 80% of OPCs are HPV-driven [3e5]. These significant epidemiological changes strongly call for preventive measures. Primary and secondary strategies are currently being used to prevent uterine cervical cancer, which is the most frequent and best-known HPV-driven malignancy [14].

These measures have proven their efficiency as the incidence of uterine cervical cancer has dropped dramatically since the 1960s in most countries where they are correctly applied [15]. From a theoretical standpoint, these good outcomes prompt the use of similar approaches to prevent HPV-driven OPCs. However, a number of important obstacles impede their application to the prevention of HPV-driven OPC, and the development of strategies specifically dedicated to this disease are urgently needed. This article provides an overview on primary, secondary and tertiary prevention of HPV-driven OPC and discusses some directions for future research (the literature search methodology is described in the Supplementary data). 2. Primary prevention Primary prevention aims to reduce the incidence of a disease within a population (Fig. 1). It involves interventions that are applied before there is any evidence of disease. As such, prophylactic vaccination against HR-HPV has proven its effectiveness. Several large randomized phase III clinical trials have demonstrated a significant reduction in the incidence of HPV 16/18 anogenital infections, genital warts and cervical and anal precancerous lesions, and have resulted in licencing and implementation of these vaccines in numerous national immunization programmes [16e21]. These very encouraging results suggest that prophylactic vaccination should decrease the incidence of anogenital cancers. However, given the considerable time lag between HR-

Fig. 1. Schematic representation of primary, secondary and tertiary strategies for the prevention of HPV-positive OPC and related morbidities. The consecutive occurrences of HPV primary infection, OPC development and post-treatment cancer relapse are represented as blue triangles. The different potential prevention methods/approaches, as well as relevant target populations are described. a e Most guidelines target adolescent girls (11e13 years of age) and some include a catch-up programme to varying degrees for older female age groups. Vaccine was also recommended for men in the USA, Canada and Australia but with different policies [16]. b e The concept of individuals at high-risk is discussed in section 3.2.1.

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HPV infection and the occurrence of cancer, the direct impact of vaccination on cervical cancer incidence will not be measurable for several decades. Although these studies have enrolled large numbers of patients, the effect of the vaccines on oral HPV infection is still poorly documented. By analogy, it is likely that these vaccines might also be effective against HPV-driven OPCs. Vaccinated individuals should be protected against HPV16/18 oral infections, which are involved in almost all HPV-driven OPCs. In animal models, vaccination was found to provide protection against oral infections and lesions of canine oral papillomavirus [22]. Studies have shown that HPV vaccination-induced high titres of type-specific antibodies in oral fluids (which include the saliva secreted by the salivary glands and the oral mucosal transudate that originates from the capillaries though the gingival mucosa) [23,24]. Moreover, these antibodies are able to neutralise HPV-pseudovirions in vitro, as recently demonstrated by Handisruya et al. [24]. To date, only one study has evaluated the preventative potential of the HPV vaccine against oral HPV infections. In this work, Herrero et al. [25] have observed that the prevalence of oral HPV16/18 infection, 4 years after vaccination, was reduced by 93% in vaccinated individuals compared with those that were not. It is however important to emphasise that this study was not originally designed to assess the efficacy of the vaccine on oral HPV infection. Consequently, several biases, including the lack of baseline oral HPV infection data and the fact that the study was carried out only on women, whereas oral HPV infections are more common in men, potentially limit the conclusion of this study. Finally, owing to the fact that oral HPV infection is mainly caused by orogenital contact (the natural reservoir of these viruses), the decrease in the prevalence of anogenital HPV infection due to vaccination might indirectly reduce the overall incidence of oral HPV infection. Unfortunately, several obstacles hamper the enthusiasm generated by vaccination. First of all, vaccine coverage remains generally insufficient, with the exception of a few countries including Australia, the United Kingdom and Denmark, where 70e80% of girls in the targeted age groups are properly vaccinated [16,26e28]. In the United States and in France, only 25e30% of females aged 16 have received the three currently recommended vaccine doses (although some studies have suggested that two doses might be sufficient) [16,29,30]. These rates are too weak to induce herd immunity that could protect unvaccinated individuals and eradicate the disease. Second, men who are up to 4 times more affected by HPV-driven OPCs [9,31] (almost 80% of all cases) are generally not eligible for vaccination. Only Australia, the United States and Canada have adopted this measure in 2011/2012, but the coverage level remains extremely low (<10e20%) [32,33]. This is probably due to the current lack of perceived benefits for

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both parents and health care providers. Thirdly, vaccination protocols that are currently recommended against cervical cancer might not be suited to OPC. Several epidemiological studies have emphasised significant differences between genital and oral HPV infection [34]. Genital infection occurs preferentially 2e5 years after the onset of sexual activity, whereas oral HPV testing peaks about a decade later [11]. To effectively prevent oral-HPV infection, vaccine-induced immunity has to be maintained for at least 2 to 3 decades, if we refer to the age that is recommended (9e12 years). However, vaccine efficacy over such a long period is unknown. Data from the study with the longest followup period has shown that vaccine-induced immunity was effective against cervical infections and precancerous lesions for at least 8.4 years [35]. Finally, it is important to highlight that even in countries with high vaccine coverage, the vast majority of the population has not been vaccinated because they were already outside of the recommended age range when anti-HPV vaccination was introduced. Most HPV-driven OPC’s that are diagnosed today and in the next decades are likely occur in this ‘pre-vaccination population’. This emphasises the need for the development of secondary prevention measures. 3. Secondary prevention The aim of secondary prevention is to detect a disease in its earliest stages of development, before symptoms appear, and to stop its progression with lighter treatment methods leading to a greater chance of recovery (Fig. 1). Cervical cancer is particularly well suited to secondary prevention, given that its natural course is characterised by the development of precancerous lesions a long time before the occurrence of cancer [14,36]. By analogy, most researchers have hypothesised that a long period of time will elapse between high-risk oral HPV infection and OPC development, making these cancers amenable to secondary prevention. However, many uncertainties regarding the natural history of tonsillar HPV infection associated to some specificities/ issues related to the anatomy of the oropharynx and the most reliable methods to detect relevant infections substantially limit the implementation of such strategies. 3.1. Screening methods Testing asymptomatic individuals to sort those who probably have the disease from those who probably do not is the first step of any screening strategy. To achieve this goal, a good screening test must be sensitive, specific, repeatable and non-invasive. 3.1.1. Cytological screening From a historical perspective, advent of the first efficient means of fighting against cervical cancer dates back

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nearly 75 years with the development of cytological tests to identify precancerous lesions or early cancer. These tests, which were designed before the aetiological role of HR-HPV was acknowledged, have led to a dramatic drop of the cervical cancer incidence in countries where screening programmes are performed [14,15,36]. Today, the vast majority of cervical cancers are being diagnosed in women without screening or with a sporadic followup. Unfortunately, such a strategy does not seem to be workable for the oropharynx. Unlike cervical cancer that develops on a flat epithelial surface (the cervical transformation zone (CTZ)) which is easy to sample, HPV-driven OPC occurs in tonsillar crypts that are deep mucosal invaginations that are technically difficult to reach. This issue was recently highlighted in a study in which tonsillar brushing to obtain liquid-based cytology specimens was performed in a cohort of 401 HIVpositive individuals, a population that is particularly exposed to HPV-induced lesions [37]. Despite the high cumulative prevalence of HPV16 oral infection (12%), no cellular change consistent with dysplasia was observed. These results contrast with the high rates of dysplastic lesions that are generally found in HIVpositive subjects with HR-HPV cervical infection [38] and suggest that superficial tonsillar brushing is inadequate to reach the tonsillar crypts. To address this issue, Franceschi et al. [39] assumed that a more ‘aggressive’ brushing would allow sampling of the crypt epithelium. However, the results were disappointing as 56% of the slides were considered to be unsatisfactory on account of too few squamous epithelial cells or of the masking effects of a large number of lymphocytes. Indeed, vigorous and deep brushing increases the number of lymphocytes to a much greater extent than the number of abnormal cells. These findings emphasise that the potential of tonsillar cytology appears limited by the difficulty of sampling the correct epithelium. 3.1.2. HPV testing-based screening New strategies to improve the efficiency of cervical cancer screening are under investigation. Despite the undeniable contribution of cytology, its lack of sensitivity (<70%) is clearly established [40]. To address this issue, cytological screening must be repeated at regular intervals from the onset of sexual activity to the age of 65 years. Therefore, identification of HR-HPV cervical infection (HPV testing) represents a promising screening strategy as demonstrated by multiple randomised trials [41e44]. HPV testing is much more sensitive than cytology for prediction of subsequent risk of CIN2, CIN3 and cancer. A negative test almost completely eliminates the risk of development of a precancerous or cancerous lesion within the next 5 years, its negative predictive value being superior to 99% [41e44]. However, its specificity is weak; a positive result can simply reflect a transient infection, which is a frequent situation with no consequence, particularly in young women [40].

This lack of specificity can be addressed by restricting HPV testing to females over 30 years old, as infection is less common at this age but with a higher risk of persistence and progression to cancer [40]. Indeed, the 10-year cumulative incidence of high-grade lesion (>CIN2) in females over 30 years with an HPV16 cervical infection is between 17 and 25%, even with a negative cytology [45]. These data prompt the use of a similar approach to identify individuals with a high-risk oral HPV infection. The largest study conducted to date has reported that the overall point prevalence of oral HPV infection in the United States of America population was 6.9% (95% CI, 5,7e8,3%) and that HR-HPVs were found in 3.7% (95% CI, 3e4.6%) of cases [11]. This evaluation was based on oral rinses and gargles that represent non-invasive methods that could be considered for large scale screening. However, these sampling methods also explore the oral cavity epithelium and consequently might overestimate the real rate of tonsillar HPV infection. Indeed, studies that have directly assessed the prevalence of HR-HPV infection in adult non-malignant tonsils have demonstrated that it is an extremely rare event, even in countries where HPV-driven OPCs are on the rise. Palmer et al. [46] have recently analysed 3377 formalin-fixed paraffin-embedded tonsil specimens, from unselected individuals aged 0e69 years who underwent tonsillectomy for benign diseases in the United Kingdom between 2004 and 2008, and found that none of these samples was HPV-infected (0%, 95% CI 0e0.089%). They further focussed on a population that is supposed to be at higher risk for HPV infection (i.e. men aged 25e34 years and >44 years). This analysis was carried out on homogenised fresh-frozen tonsil tissue (511 cases) using two different very sensitive polymerase chain reaction (PCR) approaches. However, no sample was found to be HPV-positive (0%, 95% CI 0e0.58%). These findings corroborate two previous smaller cohort studies [47,48] and highlight the discrepancies between oral rinses or gargles and the direct analyses of tonsillar tissue in terms of HPV detection. Although technical issues and geographical variations in oral HPV prevalence cannot be excluded, it is likely that the HPV detected by oral rinses and gargles comes from the oral cavity or non-tonsillar oropharyngeal anatomical subsites, and consequently raise concerns about the outcomes’ relevancy. This limitation, combined with the lack of knowledge with regard to the natural history of oral HPV infection (particularly the likelihood of oral HPV16 infection to persist and to progress towards precancerous and cancerous lesion) are not in favour of screening strategies based on the detection of HPV DNA identified by oral rinses or gargles. 3.1.3. Serological assay Serological assays that identify an immune response against several HPV antigens are currently available. In

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contrast with antibodies against HPV-L1 that represent lifetime cumulative exposure to an HPV infection at any anatomic site (genital, anal or oral) and that do not imply the presence of an HPV-related tumour, antibodies against HR-HPV E6 and E7 oncoproteins occur in response to an underlying HPV-driven neoplastic process [49]. Kreimer et al. [50] have shown that HPV16 E6 antibodies were detectable in prediagnostic sera (serum that was collected before the diagnosis) of patients with HPV-driven OPC. They analysed 638 patients with aero-digestive tract malignancies (135 OPSCC irrespective of HPV status and 503 patients with laryngeal, oesophageal or oral cancers) and 1599 controls (healthy individuals without cancer). HPV16 E6 seropositivity was observed in prediagnostic samples for 47 (34.8%) patients with OPSCC (the HPV status was unknown during the trial), 6 (1.2%) patients with other cancer type and 9 (0.6%) controls. Seropositivity against HPV16 E6 was significantly associated with the risk of OPC (OR: 274; 95% CI: 110 to 681) but not with cancer affecting other anatomical sites. Interestingly, the increased risk of OPSCC among HPV16 E6 seropositive participants was independent of time between blood collection and diagnosis and was observed more than 10 years before diagnosis. This finding suggests that a specific humoural immune response against HPV-driven malignancies can be detected even at a subclinical disease stage and supports the assumption that precursor lesions precede HPV-driven OPCs. Therefore serology might be a helpful and non-invasive screening tool that warrants further investigation [51]. 3.2. Screening-related issues 3.2.1. Large scale or selected population screening Despite a significant increase over the last few decades, the incidence of HPV-driven OPC remains limited and probably insufficient (<10 per 100,000 individuals) [52] to conceive screening strategies at the general population level. Even if highly specific screening tests were available, their predictive positive value (PPV) would be insufficient to accurately identify individuals at risk, as recently highlighted by Castle PE [53]. To address this issue, identification of a group of subjects at higher risk is necessary. Studies describing the clinical features of HPV-driven cancer patients and healthy individuals with oral HPV infection have indicated that males were more affected than women and that some sexual behaviours (high number of partners.) were risk factors [9e11]. Therefore, an interesting target population to screen might be men aged 55 to 65 (in which the prevalence of HPV infection was shown to peak) with given behavioural features (i.e. high number of lifetime sexual partners). However, it might prove challenging to define accurate and reliable threshold to define this potentially enriched population.

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Partners of patients with an HPV-driven malignancy might also represent a relevant target population. Indeed, we can assume that these individuals were more exposed than the general population to high-risk HPVs. Although it is obvious that the vast majority of partners spontaneously clear their infection, we cannot rule out that some of these infections persist and might progress to cancer. A dozen case reports have described cases of HPV-driven cancers in husbands and wives, and in some studies the same viral strain was found in both partners, suggesting that the transmission occurred from one spouse to the other [54]. Additionally, five larger studies based on population registries have reported an increased risk of HPV-driven cancers in spouses of patients with genital malignancies [54]. 3.2.2. Identification of lesions in individuals with a positive screening test Identification and treatment of precancerous and early cancerous lesions, in patients with a positive screening test, are the ultimate goals of any secondary prevention strategy. In the cervix, patients with significant cytological abnormalities are referred for colposcopy, and those with histologically proven alterations will receive a treatment to destroy or remove the CTZ, which is a well delimited/defined anatomic region. Unfortunately, the situation is much more complex in the oropharynx. Indeed, even if a screening test for HPV-driven OPC was identified with acceptable clinical performance, significant limitations remain for the implementation of a screening programme for HPV-driven OPC. First, the oropharyngeal lining represents a wide surface/area composed of lymphoid tissue and mucosal invaginations whose clinical and endoscopic exploration is much more difficult than that of the CTZ. This difficulty is compounded by the fact that HPV-driven OPCs arise from palatine and lingual tonsils crypts [55] which are hard to reach with conventional visual and tactile examination. The use of adjunctive techniques such as acetic acid or lugol’s iodine might be of interest but was not assessed thoroughly in this indication. Second, whereas oral premalignant lesions (e.g. leukoplakia, erythroplakia) induced by tobacco and alcohol are well described, the clinical appearance of HPV-related premalignant oropharyngeal lesions has not yet been adequately described, and this represents an obvious limitation. To address these issues, some teams have recently evaluated innovative diagnostic approaches, with encouraging results that prompt further validation on larger cohorts of patients. Ebisumoto et al. [56] have reported their successful experience to detect minute p16-positive tonsillar cancer lesions on transoral examination with narrow band imaging (NBI). This imaging technique works by filtering white light into specific light wavelengths that are absorbed by haemoglobin and consequently

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enhances the visibility of the mucosal superficial vasculature [57]. Thus, superficial lesions are identified, in view of their neoangiogenetic pattern of vasculature. Using a flexible videolaryngoscope with NBI, Ebisumoto et al. showed that it enhances minor mucosal defects on the surface of the tonsils, thus allowing the identification of tumours that cannot be seen upon macroscopic examination with conventional white light because they are almost completely covered with normal mucosa. This method is particularly interesting as it is non-invasive and can be used in an outpatient setting. However, it is unclear whether transoral examination with NBI is beneficial if the lesion is completely surrounded with normal mucosa. Moreover, the literature on the use of NBI in the head and neck region is still very sparse. Another potential alternative approach for the detection of early tumours of the oropharynx is transcervical and intraoral ultrasonography. This imaging technique is particularly adapted to visualise the base of the tongue, the examination of which is challenging for anatomical reasons. Recent articles have described the sonographic appearance of the oropharynx [58] and have shown that with this technique, it is feasible to visualise and evaluate base of tongue cancers [59]. Fakhry et al. [60] have assessed the benefit of ultrasonography to identify the primary site in a series of ten patients with head and neck cancers of unknown primary (HNCUPs). On ultrasound examination, a candidate oropharyngeal lesion with characteristics consistent with a primary tumour was identified in all cases. During endoscopy under general anaesthesia, five of ten cases had clinically discernible masses which were histopathologically confirmed. The primary site of two additional cases was confirmed with biopsies ‘directed’ by the ultrasonography data, whereas three patients remained with unknown primary. This proof of concept study demonstrates that ultrasound helps to identify certain oropharyngeal lesions as small as 5 mm. These findings, also preliminary, have exciting implications for future screening strategies. Indeed, ultrasonography could provide a potential means of visualising and sampling suspicious lesions in high-risk populations and therefore warrants further study. 4. Tertiary prevention Tertiary prevention primarily aims to prevent or control the morbidity caused by cancer therapy, but it also encompasses the prevention of cancer recurrence (Fig. 1). Although the prognosis of HPV-driven OPSCC is significantly better than that of their HPV-negative counterparts, up to 25% of patients will recur within 24 months of treatment [61,62]. Traditionally, posttreatment follow-up of patients with HNSCC is based on clinical examinations and medical imaging.

Recurrences are frequently diagnosed at a late stage once the symptoms appear and when treatment options are limited, with dismal outcomes. These delays are mainly caused by treatment-related aftereffects (anatomical changes, fibrosis.) preventing the early detection of disease relapse. These difficulties make biomarkers attractive for optimising patient monitoring, as they might be detected before the development of any clinical or radiological evidence of disease recurrence. Comparable follow-up strategies have already demonstrated their effectiveness in several types of malignancies, including prostate, breast and thyroid cancers. Unfortunately, to date no validated biomarkers exist for monitoring HNSCC during follow-up. The emergence of HPV-driven OPSCC has opened up new possibilities. As these cancers are a long-term consequence of a viral infection, their molecular profile is clearly distinct from that of other HNSCC that are traditionally induced by tobacco and alcohol abuse [12,13]. HPV-driven malignant cells express viral DNA sequences, and particularly E6 and E7 viral oncogenes, to which they are addicted [63,64]. This induces an adaptive immune response directed against the viral antigens that are displayed on their surface [65e67]. These features, specific to HPV-driven cancers, might be used as tumour biomarkers. Indeed, HPV DNA is released by tumour cells (cell free DNA and/or DNA in exfoliated cells) in body fluids, and antibodies against the viral oncoproteins E6/E7 are frequently detected in patients with HPV-driven cancers, particularly at the oropharyngeal and anal level, whereas they are rarely found in healthy individuals [50,51,68]. Consequently it might be relevant to use these molecular biomarkers to improve post-treatment follow-up. 4.1. Oral fluid biomarkers Rettig et al. [69] examined whether HR-HPV DNA detection in oral rinses after treatment of HPV-driven OPSCC is associated with recurrence and survival. Oral rinse samples were collected at diagnosis and after treatment (9, 12, 18, and 24 months after diagnosis). Persistent HPV16 DNA was found in 5/124 patients, all of which recurred (100%) within 3e11 months after oral rinse collection. In contrast, only 9/119 (8%) patients without HPV16 DNA persistence relapsed within the year after the last oral rinse sampling. Persistent oral HPV16 DNA was associated with worse disease-free survival (DFS) (hazard ratio, 29.7 [95% CI, 9.0e98.2]) and overall survival (OS) (hazard ratio, 23.5 [95% CI, 4.7e116.9]). This study indicates that patients with HPV-16 DNA in surveillance salivary rinses are at significant risk for recurrence, and is consistent with the findings of two previous smaller cohort studies. Chuang et al. [70] studied 20 patients with HPV-driven OPSCC, and they noted four disease recurrences, of which two

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had positive surveillance salivary rinses (sensitivity Z 50%). By contrast, none of the 16 patients without recurrences tested positive (specificity Z 100%). Ahn et al. [71] made the same observation after performing a comparable study in a 38-patient cohort with HPV-driven OPSCCs for which HPV DNA was detectable in oral rinses before treatment. They noted three disease recurrences among the four patients with HPV16 DNA in their post therapeutic oral rinses, and only two among the 34 patients without HPV16 DNA persistence. 4.2. Blood biomarkers HPV16 E6 serology and detection of circulating HPV16 DNA are also promising approaches, as blood offers an attractive and non-invasive means for cancer surveillance. To consider HPV antibody levels as a candidate biomarker for surveillance after treatment, it is important to investigate whether antibody levels change after treatment. Cervical cancer research has suggested that a decrease in antibody levels between pre- and posttreatment were associated with remission, whereas persistent or increasing antibody levels showed evidence of residual or recurrent disease [72,73]. However a substantial proportion of women with cervical cancer have no immunological response to HPV-specific antigens. Indeed, humoural immunity seems more difficult to induce in the cervix than in the oropharynx or the anal canal (which is also caused by HR-HPV), which contains significantly more lymphoid tissue [74,75]. Moreover, the broad distribution of HPV genotypes responsible for cervical cancer precludes the examination of serological response to one antibody type [76] (HPV16 is responsible for almost 90% of HPV-driven OPSCC). To date, few studies have focussed on HPVdriven OPSCC, but in general they all confirmed that antibody titres drop significantly after treatment and might have also a prognostic value [77e79]. Koslabova et al. [78] reported that this decrease was significantly more frequent in patients without recurrences, compared to those who relapse. Of the 16 patients who recurred at one-year follow-up, 6 were seropositive at enrolment, of which 5 had no decrease in antibody levels during follow-up. Fakhry et al. [79] have studied 60 patients with HPV-driven OPSCC, and they have reported a significant decline in the levels of antibodies against HPV16-specific oncoproteins after treatment. Notably, higher levels of E6 antibody at diagnosis were associated with a significant increased risk of disease recurrence (HR 7.1, 95% CI 1.2e43.2, p Z 0.04). Furthermore, to determine whether the observed declines in antibody titres were specific to HPV or workable for antibody levels in general, BK polyomavirus (BKPyV) antibody titres were evaluated over time. BKPyV enzyme-linked immunosorbent assay (ELISA)

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was chosen as a majority of individuals are expected to be seropositive, and thus changes in antibody level could be easily measured. The authors found that in contrast to declines in HPV16 early oncoprotein antibodies, BKV antibody titres remained stable over time. Detection of HPV circulating DNA (cDNA) is also a promising approach. Normal HPV life cycle is strictly epithelial and there is no viraemia [80]. Several studies have shown that HPV cDNA is absent in the blood of women with cervical HPV infection, whereas it is detectable in patients with cervical invasive lesions [81,82]. This DNA is released by cancer cells, as confirmed by sequencing studies that have demonstrated that the HPV type detected in the plasma was identical to that found in the cervical tumour from the same patients [83,84]. Capone et al. [85] were the first to demonstrate that HPV cDNA was also present in plasma of patients with HPV-driven OPSCC, 6 of the 13 (46%) patients they studied had HPV cDNA in their sera. However, as samples were collected at a single time point during patient care, it was not possible to assess whether HPV cDNA might be a useful biomarker for monitoring treatment response and early identification of recurrences. Indeed, in nasopharyngeal carcinoma post-treatment EpsteineBarr virus (EBV) cDNA level is highly indicative of disease persistence or recurrence [86,87] and is currently being tested in a prospective multi-institutional phase III clinical trial. To answer this question, Cao et al. [88] performed serial HPV cDNA measurements during and after therapy in 14 patients with HPV-driven OPSCC. They noted a rapid decline in HPV cDNA that became undetectable at treatment completion in all patients. Interestingly, in three patients HPV cDNA rose to discernable levels at the time of metastasis. More recently, Dahlstrom et al. [89] have retrospectively studied HPV cDNA in a large cohort of HNSCC including 114 assumed HPV-related tumours. They found that patients in advanced disease stages have higher rates of pre-treatment HPV cDNA. Progressionfree survival and disease recurrence were not significantly correlated to pre-treatment HPV cDNA levels. However, this study was not properly designed to assess HPV cDNA as a monitoring biomarker. Moreover, tumour HPV status was defined exclusively on the basis of HPV DNA identification by PCR, which is clearly not the most reliable method to identify accurately HPV-driven OPSCCs [90]. These three strategies seem relevant to the optimisation of patient follow-up. They are simple, non-invasive and potentially cost-effective. However, they need to be validated prospectively in a larger and more rigorous study. Indeed, most of the existing data derives from retrospective studies with obvious limitations, including small cohorts, lack of post-treatment surveillance standardisation, and in some cases inadequate tumour HPV status definition. It is also important to highlight

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that each of these tests is negative in 10e40% of patients, even though the cancer is HPV-induced. This lack of sensitivity is probably related to technical issues. For instance, Jeannot et al. [91] have recently demonstrated that droplet digital PCR (ddPCR) was more effective in detecting HPV cDNA than classical real-time quantitative polymerase chain reaction (qPCR) methods. Indeed, they were able to identify HPV cDNA in 61/70 (87%) serum samples from patients with different types of HPV-related carcinomas (uterine cervix, anal canal and oropharynx), whereas the sensitivity of classical qPCR methods ranges from 6.9 to 65% [92,93]. Alternatively, a biomarker combination (concomitant use of several markers) might overcome this limitation and consequently increase the overall sensitivity. Moreover, confirmation that these biomarkers predict disease recurrence could open new domains of research. New imaging methods to visualise early disease recurrence, such as the use of a labelled tracer that specifically binds to cells expressing HR-HPV’s E6/E7 oncoproteins or p16 proteins, could be developed [94]. It is also conceivable that new treatment options might be elaborated in the future (such as therapeutic vaccination) for patients with subclinical disease (individuals with positive biomarkers but no anatomically detectable lesion).

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5. Conclusion Prevention of HPV-driven OPC is an important issue considering its growing incidence. Prophylactic HPV vaccination should be recommended and expanded. However, several decades will be necessary to obtain sufficient coverage at the global level. For this reason, particular efforts should be deployed to develop secondary and tertiary preventive measures.

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Conflict of interest statement [16]

The authors have nothing to declare. Appendix A. Supplementary data

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Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.ejca.2017.03.021. [18]

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