Introduction

Along with noxious agents such as alcohol or tobacco, high-risk (HR) human papillomavirus (HPV) types of the genus alpha such as HPV16 or HPV18 play an independent and important role in the etiology of squamous cell carcinoma (SCC) of the oropharynx [1–3]. HPVs are small, non-enveloped, oncogenic DNA viruses that infect basal and stem cells of cutaneous and mucosal squamous epithelium. 40–70% of oropharyngeal cancers, mainly tonsillar and base of the tongue carcinomas, are attributed to HR-HPV infection [4, 5]. Compared to patients with HPV-negative oropharyngeal cancer, patients with HPV-associated oropharyngeal cancer are more frequently non-smokers, non-drinkers, male and of younger age [1, 4, 6]. Worldwide, approximately 400,000 new cases of oropharyngeal cancer and cancer of the oral cavity are diagnosed per year [7]. In Germany alone, roughly 5300 patients will die of oropharyngeal cancer and cancer of the oral cavity each year [8]. Depending on the stage of the tumor, treatment in Western Europe generally involves surgery and chemoradiation treatment. Both treatment response and survival rates are more favorable for HPV-positive compared to HPV-negative oropharyngeal cancer [9–12]. In recent years, the development of vaccination programs and immunotherapies has opened up new perspectives in the prevention and treatment of oropharyngeal cancer [1, 13, 14].

Similar to HPV, polyomaviruses are small, non-enveloped DNA viruses with a possible oncogenic potential. To date, 13 human polyomaviruses (HPyVs) have been discovered [15–18]. Serological studies suggest that HPyVs are widespread and that infections occur early in life [16, 19–22]. Some HPyVs such as BKPyV and JCPyV can cause severe disease in immunosuppressed patients as nephropathy (BKPyV), hemorrhagic cystitis (BKPyV) and progressive multifocal leukoencephalopathy (JCPyV) [23–26]. Merkel cell polyomavirus (MCPyV) is the only HPyV that can cause disease in non-immunosuppressed patients and is the principal etiology of Merkel cell carcinoma, a rare and aggressive neuroectodermal tumor of the skin [27, 28]. HPyV7 has recently been detected in two cases of pruritic rash in lung transplant patients [29]. TSPyV can cause the rare skin disease Trichodysplasia spinulosa in immunosuppressed patients [30]. New Jersey polyomavirus (NJPyV) was recently discovered in a pancreatic transplant recipient with retinal blindness and vasculitic myopathy [18]. To date, HPyV6, HPyV9, HPyV10 (and its variants MWPyV and MXPyV), Saint Louis PyV (STLPyV) and HPyV12 have not been associated with any pathology, and it remains to be determined whether these emerging viruses play a pathogenic role in humans [16, 17, 31–37].

We have performed a retrospective study using DNA extracted from tonsillar biopsies of patients with chronic tonsillitis/tonsillar hyperplasia or with tonsillar carcinoma. The goal of our study was to determine HPyV DNA prevalence and DNA load in benign and malignant tonsillar tissue and compare the data with those found for HR-HPV. This could help to elucidate, whether HPyV infection, besides HR-HPV infection, might contribute to oropharyngeal cancer development.

Materials and methods

Samples

We collected 80 tonsillar biopsies (palatine tonsils) of 80 patients between November 2005 and March 2014. A total of 42 patients had a histologic diagnosis of non-malignant disease (chronic tonsillitis, tonsillar hyperplasia), and 38 patients had tonsillar SCC. DNA was extracted as previously described [38], and the extracted DNA was stored in the bio-bank of Bonn University Hospital at −80 °C until virological analyses were performed. Patients’ medical files were additionally reviewed for information on smoking habits. The study was performed according to the principles of the Declaration of Helsinki. Human specimens were used after approval by the Ethics Committee of the Medical Faculty of the University Bonn (Approval Number 201/15). Two non-malignant samples had to be excluded from virological analysis due to the lack of cellular input (beta-globin gene PCR negative), leaving 40 non-malignant and 38 cancer samples from 78 patients (Table 1) for virological analysis.

Table 1 Patients’ characteristics
Full size table

HPyV and HPV detection and DNA load determination

HPyV and HPV DNA analyses were performed as recently described [39, 40]. The MCPyV polymerase chain reaction (PCR) used was established by Sihto et al. [41]. Primer and probe sequences for HPyV6, HPyV7, TSPyV, HPyV9 and HPyV10 PCRs as well as the respective reference materials were recently published [39]. Primer and locked nucleic acid (LNA) probe sequences for the detection and quantification of the remaining eight HPyVs are given in Table 2 (BKPyV, JCPyV, KIPyV, WUPyV, MCPyV, STLPyV, HPyV12, NJPyV). The analytical sensitivity of each assay was 10 copies of cloned or viral synthetic DNA (gBlocks, Integrated DNA Technologies, Leuven, Belgium), respectively. All HPyV quantitative PCRs (q-PCRs) were performed on a LightCycler 480 (Roche, Mannheim, Germany) in 20 µl reactions containing 10 µl LC 480 Probes Master (Roche), 0.2 mM each of the respective forward and reverse primer (TIB Molbiol, Berlin, Germany), 0.1 mM LNA probes (Roche) (Table 2 and [39]) and 2 µl extracted DNA. Cycling conditions were 10 min denaturation at 95 °C, followed by 45 cycles at 95 °C for 10 s, 60 °C for 30 s and 72 °C for 5 s. Negative controls contained human DNA (extracted DNA of RTS3b cells) and water instead of patient samples and never yielded fluorescence signals above the background.

Table 2 Primers and locked nucleic acid probes for the detection and quantification of BKPyV, JCPyV, KIPyV, WUPyV, MCPyV, STLPyV, HPyV12 and NJPyV
Full size table

HPV detection and typing were performed by genus alpha-specific PCR (A6/A8-PCR) and bead-based multiplex hybridization using a Luminex Lx200 (Luminex, Austin, TX, USA) as previously described [40, 42, 43]. The Luminex assay can detect 38 different HPV types, 22 high-risk (HR) or probable/possible HR-HPV types and 16 low-risk (LR) types (HR-types: HPV16, 18, 26, 30, 31, 33, 34, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 67, 68, 70, 73, 82; LR-types: HPV6, 11, 40, 42, 43, 44, 54, 55, 57, 61, 71, 72, 81, 83, 84, 89) [44].

In HPV16 or HPV18-positive samples, HPV DNA load was determined by q-PCR with type-specific primers and probes as recently described [40].

The single-copy beta-globin gene was detected and quantified as described previously [45, 46]. HPyV and HPV loads were each calculated as viral DNA copies per beta-globin gene copy [39, 40]. Cumulative HPV or HPyV DNA loads were calculated by considering all HPV or HPyV types found in the samples.

Statistical analysis

The data on quantitative variables were summarized by median (25th–75th percentile), on qualitative variables by count (percentage). Confidence intervals at level 95% for proportions were calculated according to the exact Clopper–Pearson method. Bivariable association was evaluated by cross-tabulation (calculating odds ratios) and Fisher’s exact test. Adjustment for potentially confounding variables was done by multiple logistic regression (again calculating odds ratios). Location differences in distributions (e.g., of viral load) were assessed by the non-parametric Mann–Whitney U test. p values below 5% were considered statistically significant. All analyses were done with SPSS Statistics (version 23, IBM Corp., Armonk, NY, USA).

Results

Patient characteristics

A total of 78 tonsillar biopsies comprising 40 with non-malignant disease (chronic tonsillitis, tonsillar hyperplasia) and 38 with malignant disease (squamous cell carcinoma) were included in the analysis. Patient age ranged between 2 and 81 (mean 41.4) years. Sex, age and smoking habits of patients with tonsillar cancer differed from those with non-malignant disease (Table 1). The patients with cancer were significantly older than those with non-malignant disease. 81.6% of the patients with tonsillar cancer were males, compared to 40.0% of the patients with benign disease. 47.4% of all patients were smokers. Significantly more smokers were among the patients with tonsillar cancer than among the individuals with non-malignant disease (73.7 vs. 22.5%, Table 1).

Human polyomavirus DNA prevalence in benign and in malignant tonsillar biopsies

BKPyV, KIPyV, TSPyV, HPyV9, STLPyV, HPyV12 and NJPyV were not detected in any of the samples. JCPyV (2.5%) and WUPyV (7.5%) were only found in non-malignant tonsillar tissue. HPyV7 and HPyV10 were only detected in one (2.6%) and seven (18.4%) cancer biopsies, respectively, and never in non-malignant tissue. The difference in HPyV DNA prevalence between non-malignant and malignant tissue was statistically significant only for HPyV10 (Table 3). Both MCPyV (10.0 vs. 21.1%) and HPyV6 (2.5 vs. 5.3%) were detected more frequently in cancer samples than in non-malignant tissue, but the differences were not statistically significant, neither unadjusted nor after adjustment for age, sex and smoking. The prevalence of any HPyV was higher in cancer samples compared to non-malignant tissue (42.1 vs. 20%, p = 0.034, OR 2.9 (95% CI 1.1–8.0)). However, after adjustment for age, sex and smoking, the p value was no longer significant (0.700) and the OR was lower (0.6 (95% CI 0.0–9.7)), indicating a confounding effect of these factors (Table 3). Two different polyomaviruses were found in three (12.5%) of the 24 HPyV-positive samples: JCPyV and WUPyV were found in a non-malignant sample from a 2-year-old girl, and MCPyV plus HPyV10 were detected in two HPV-negative cancer samples from a 52-year-old male and a 70-year-old female. Three or more polyomaviruses were not found concomitantly in any of the samples.

Table 3 HPyV and HPV DNA prevalence in non-malignant and malignant tonsillar tissue
Full size table

Human papillomavirus DNA prevalence in benign and in malignant tonsillar biopsies

HPV DNA was found in 44.7% of the malignant biopsies, compared to 5.0% of the non-malignant tissues (p < 0.001, OR 15.4 (95% CI 3.2–73.1)). Of the 22 HR-HPV types analyzed, only HPV16 and HPV18 were detected (Table 3). HPV16 and HPV18 were found in one non-malignant tonsillar tissue specimen each (2.5%, respectively). Sixteen cancer samples were HPV16-positive (42.1%), and one cancer biopsy (2.6%) was HPV18-positive (Table 3). With the exception of one HPV16-positive cancer that additionally carried HPV57 and HPV84 DNA, LR-HPV types were not found in any of the samples. In contrast to HPyVs, the ORs for HPV-positivity (HPV16 or any HR-HPV, non-malignant compared to cancer samples) were similar or greater after adjustment for age, sex and smoking, which argues against confounding by these variables (Table 3).

Viral DNA loads in benign and in malignant tonsillar biopsies

In all HPyV DNA-positive samples, the respective HPyV DNA loads were determined (Table 4). The HPyV loads found were very low both in non-malignant and malignant samples (Fig. 1) and did not exceed 0.008 HPyV DNA copies per beta-globin gene copy, with the exception of one cancer sample of a HPV-negative 62-year-old male heavy smoker with a HPyV6 load of close to one (0.7, Table 4). MCPyV loads were significantly higher in malignant than in non-malignant biopsies (p = 0.008), but were nonetheless very low in both groups (median 0.000004 vs. 0.000064) and also never exceeded 0.008. Median HPyV10 loads of tonsillar cancers were 0.000075, and the highest HPyV10 load found was 0.0006. Cumulative HPyV loads were not different in non-malignant compared to malignant tissue (p = 0.320; Table 4; Fig. 1).

Table 4 Viral DNA loads in non-malignant and malignant tonsillar tissue
Full size table
Fig. 1
figure 1

HPyV and high-risk HPV DNA load distribution in non-malignant tonsillar tissue and in tonsillar cancer. The four box plots show the distribution of the following viral DNA loads: cumulative HPyV DNA load of non-malignant tonsillar tissue (n = 8) (A), cumulative HPyV DNA load of oropharyngeal cancers (n = 16) (B), cumulative high-risk HPV DNA load of non-malignant tonsillar tissue (n = 2) (C) and cumulative high-risk HPV DNA load of oropharyngeal cancers (n = 17) (D). The bottom and top of the box are the first and third quartile, and the band inside the box represents the median. Whiskers are vertical lines ending in horizontal lines at the largest and smallest observed values that are not statistical outliers, i.e., values more than three IQRs from the end of a box are labeled as extreme, denoted with an asterisk (*), and values more than 1.5 IQRs but less than 3 IQRs from the end of the box are labeled as outliers (o)

Full size image

HPV16 and HPV18 DNA loads found in two non-malignant samples were similarly low as HPyV loads (Table 4; Fig. 1). In contrast, HPV16 loads and cumulative HR-HPV loads (HPV16 + 18) were higher in malignant samples compared to non-malignant tissue (median 67.469 vs. 0.000014 and 11.861 vs. 0.000007, respectively) (Table 4, Fig. 1). The difference was statistically significant for the latter (p = 0.012). Nine of the sixteen HPV16-positive cancers had a viral load above 10 copies per beta-globin gene copy and thus were most likely HPV induced. When comparing HR-HPV DNA loads of the HPV-positive cancer patients according to their smoking status, the non-smokers had significantly higher viral loads than the smokers (p = 0.047) (Fig. 2). All seven cancer patients with very low HPV16 or HPV18 loads (below 0.05 HPV copies per beta-globin gene copy) were smokers.

Fig. 2
figure 2

High-risk HPV DNA load distribution in HPV-positive tonsillar cancers of non-smokers and smokers. The box plots show the distribution of the cumulative high-risk HPV DNA loads found in tonsillar cancers of non-smokers (n = 5) (left) and smokers (n = 11) (right). Boxes represent the interquartile range and the median. Whiskers are vertical lines ending in horizontal lines at the largest and smallest observed values that are not statistical outliers, i.e., values more than three IQRs from the end of a box are labeled as extreme, denoted with an asterisk (*), and values more than 1.5 IQRs but less than 3 IQRs from the end of the box are labeled as outliers (o)

Full size image

In seven cancer biopsies (and in none of the non-malignant samples), both HPV and HPyV DNA were detected (HPV16 + MCPyV, n = 4; HPV16 + HPyV10, n = 3). In these samples, HPV16 DNA loads were significantly higher than HPyV loads (p = 0.031).

Discussion

We have analyzed HPyV and HPV DNA prevalence and viral load in 78 tonsillar biopsies comprising 40 non-malignant and 38 SCC samples. Seven of the 13 analyzed HPyVs were not found in any of the biopsies, and JCPyV and WUPyV were only found in a small percentage of the non-malignant samples. The most frequently found HPyV was MCPyV, both in non-malignant and in cancer tissue, but the difference in prevalence between the two sample groups was not significant. HPyV6 was found in 3 biopsies, one non-malignant and two tonsillar cancer biopsies, without a significant difference between the two groups. HPyV7 was only found in one cancer biopsy. HPyV10 was the only polyomavirus detected significantly more frequently in malignant compared to non-malignant tissue (18.4 vs. 0%), but HPyV10 DNA loads found in the tumor tissue were extremely low (see below). Since the patients with tonsillar cancer were significantly older than those with non-malignant disease, the difference in HPyV10 prevalence could be attributable to age. It has been shown for several HPyVs that HPyV (sero)prevalence increases with age [16]. For HPyV10, Karachaliou et al. [47] report a seroprevalence of 55.6% in 3-year-old children and of 82.3% in 4-year-old children. Gossai et al. [19] found a HPyV10 seroprevalence of 99.1% for adults from 24 to 75 years, without significant differences between age groups, between males and females, and between smokers and non-smokers.

To date, only relatively few studies on HPyVs in tonsils have been published with only one other study, Salakova et al. [48], focusing on the recently discovered HPyVs and tonsillar cancer. BKPyV and JCPyV, two HPyVs discovered 45 years ago, have been detected by PCR in 0.3–6.9% (BKPyV) and in 0–44% (JCPyV) of non-malignant tonsillar tissue in different studies [49–55]. Two earlier studies found JCPyV in 39 and 44% of non-malignant tonsillar tissue with tonsillitis or tonsillar hypertrophy using T-antigen or regulatory region targeting nested PCR protocols [53, 54]. Several more recent single-round PCR-based studies could not detect JCPyV DNA in non-malignant tonsillar tissue [49–52]. Differences in the detection rates of non-integrating polyomaviruses such as JCPyV and BKPyV should not depend on the genomic region targeted by PCR. We could confirm the low prevalence of JCPyV found in our study with a VP1-directed PCR also with a second PCR protocol [56] targeting the N-terminus of the T-antigen (data not shown). We therefore speculate that the high JCPyV prevalence found in earlier studies might be due to either a higher sensitivity or proneness to contamination of the nested PCR. Günel et al. [57] have analyzed specimens of children with recurrent tonsillitis (n = 26) or tonsillar hypertrophy (n = 25) for the presence of Epstein–Barr virus, Human Bocavirus, WUPyV and KIPyV DNA. While they found Epstein–Barr virus and Bocavirus in 12.0–53.8% of samples, they did not detect KIPyV in any of the biopsies, and WUPyV was detected in 2 of the 26 (7.7%) recurrent tonsillitis samples but in none of the tonsillar hypertrophy samples. This is almost identical to the prevalence rates found in our study for KIPyV (0%) and WUPyV (7.5%) in non-malignant samples. KIPyV and WUPyV have also been detected in previous PCR-based studies of non-malignant tonsillar tissue with prevalence rates reaching from 0 to 13.2% (KIPyV) and 0–12.0% (WUPyV) [49, 50, 52, 58–60]. Sadeghi et al. [22] found TSPyV DNA in 8 of 229 (3.5%) tonsillar tissues of children and adults with chronic tonsillitis or tonsillar hypertrophy. Two of the TSPyV-positive cases were co-infected with KIPyV or WUPyV. In contrast, we have not found TSPyV in any of our samples. However, we have analyzed fewer non-malignant biopsies than Sadeghi et al., and our sample size might have been too small to detect viruses with a low prevalence rate. Peng et al. [61] discovered MWPyV, a variant of HPyV10, and STLPyV in 2% (2/99) of swabs from the surface of palatine tonsils of Chinese children with chronic tonsillar disease. In our study, neither HPyV10 nor STLPyV was detected in non-malignant tonsils. Peng et al., however, studied more samples and did not analyze tonsillar biopsies. Recently, Salakova et al. analyzed MCPyV, HPyV6 and HPyV7 in over 200 non-malignant and malignant tonsillar tissue samples [48]. They found MCPyV, HPyV6 and HPyV7 in 10.2, 4.6 and 0.9% of non-malignant biopsies, compared to 35.7, 5.4 and 1.8% in malignant tissue, respectively [48]. Our results for these three HPyVs are closely in line with the data of Salakova et al., with both studies reporting similar HPyV DNA detection rates. In contrast to our study, the difference in MCPyV prevalence between non-malignant and malignant tissue was statistically significant in the study by Salakova et al. [48]. Kantola et al. [60] also found MCPyV DNA in non-malignant tonsillar tissues, although in only 3.5% of 229 tonsillectomy patients, which is lower than in our study and in the study of Salakova et al. [48]. Altogether, our data and these of the above-mentioned papers on HPyV prevalence suggest that lymphoid tissue such as palatine tonsils could be a possible site of persistence for several HPyVs.

Viral loads of all analyzed HPyVs were very low, below 0.01 viral copies per beta-globin gene copy for all samples, with the exception of one oropharyngeal (HPV-negative) cancer of a 62-year-old smoking male with a HyPV6 load of 0.7 copies per beta-globin gene copy. MCPyV-positive cancers had significantly higher loads than non-malignant tonsillar biopsies, but MCPyV loads were very low in both groups, and the maximum MCPyV load found in a cancer biopsy was 0.008. In contrast, viral loads of MCPyV-associated Merkel cell carcinomas were regularly above 0.1 and reached values of up to 1500 MCPyV copies per beta-globin gene copy in previous studies [62, 63]. Similar to MCPyV, HPyV10 loads were very low in all HPyV10-positive cancers (maximum 0.0006). The low HPyV DNA loads we found are in line with low tonsillar DNA loads found by others for BKPyV, MCPyV, HPyV6 or HPyV7 in non-malignant [48, 51, 55] and malignant tonsillar tissue [48]. Furthermore, Salakova et al. could not detect statistically significant viral load differences for MCPyV, HPyV6 and HPyV7 between non-malignant and malignant tonsillar tissue. It has been shown for HPV that elevated viral loads are found in HPV-induced premalignant lesions and cancers [40, 64–66]. Therefore, the very low HPyV loads found in the tonsillar cancer biopsies are in sharp contrast to both MCPyV loads of Merkel cell carcinomas and HPV loads in HPV-related lesions, thus making a causal role for MCPyV, HPyV6, HPyV7 or HPyV10 in tonsillar cancer development or maintenance unlikely. Polyomavirus Large-T-antigen is usually expressed in MCPyV-positive Merkel cell carcinomas [28, 67]. Unfortunately, material for Large-T-antigen immunohistochemistry was not available in our study, but we have shown previously that Large-T-antigen is not expressed in MCPyV-positive skin tumors or cutaneous lymphomas with low MCPyV DNA loads [63, 68, 69].

It has been shown that an increasing number of tonsillar and base of the tongue carcinomas are HPV associated [1, 3]. In our study, 44.7% of tonsillar cancers were HPV-positive, which is in line with recent studies of oropharyngeal cancer in central Europe but lower than found for northern Europe or North America [5, 11, 70, 71]. As in our study, HPV16 has consistently been the predominant type, and other HR-HPV types have only been found in a small subset of HPV-positive head and neck cancers [5, 9].

We have detected HR-HPV DNA in 5.0% of non-malignant tonsillar samples, which is in line with a previous study that found HPV16 DNA in 6.3% of 206 biopsies of patients with tonsillitis or tonsillar hypertrophy [72]. HPV loads were very low in the two HPV-positive non-malignant samples of our study. In contrast, cumulative HPV loads found in cancer biopsies were significantly higher, reaching over 3000 HPV DNA copies per beta-globin gene copy (median 12). This is in line with previous studies on HPV DNA loads in HPV-induced oropharyngeal cancers [73–75]. Interestingly, non-smoking cancer patients had significantly higher HPV loads than smokers, and all seven cancer patients with very low HPV DNA loads were smokers, strongly suggesting that these tumors were not HPV induced but rather smoking related.

Prophylactic HPV vaccination of boys is currently only recommended in a few countries (e.g., USA, Canada, Australia, Austria, Switzerland), in contrast to the HPV vaccination of girls [76]. It has not yet been shown that prophylactic HPV vaccination can reduce or prevent HPV-associated oropharyngeal cancer, but it has been demonstrated that it can significantly reduce oral HPV16 infection [77, 78]. Of the 17 HPV-positive cancers found in our study, 16 were from male patients including nine cancers with high HPV16 loads (>10) that were most likely HPV induced. This is in line with the male predominance previously described for HPV-positive oropharyngeal cancers [1]. Hopefully, gender-neutral HPV vaccination before HPV acquisition (sexual debut) will be able to prevent HPV-associated oropharyngeal cancers in the future and reverse the rising incidence described for this cancer entity.

Our study had some limitations: (1) The number of samples included in the analysis was small (<100), and HPyVs with low prevalence rates might have been missed. A larger sample also could have yielded different results concerning HPyV load distribution. (2) The age distribution, gender and smoker status were different in both groups with only a small overlap. (3) Data on lifestyle factors such as sexual preferences (oral sex) and on socioeconomic factors were not available. (4) Since only extracted DNA was available, we could neither perform in situ hybridization to localize the detected viruses within the tonsils nor immunohistochemistry to analyze Large-T-antigen expression in MCPyV DNA-positive samples. (5) We have not performed further diagnostic tests such as p16 immunohistochemistry or HPV E6/E7-mRNA determination to corroborate the attribution of HPV in the HPV DNA-positive cancers [5, 79], but the high HPV16 loads found in a subset of the samples make it likely that these cancers were HPV induced.

In summary, of the 13 HPyVs analyzed, only six have been found in tonsils and only MCPyV, HPyV6, HPyV7 and HPyV10 were detectable in cancer samples, but, with the exception of HPyV10, detection rates were similar in malignant and non-malignant tonsillar tissue. In non-malignant samples, both HPyV and HPV loads were very low with levels below 0.002 viral copies per beta-globin gene. In contrast, tonsillar HPV16 loads of non-smoking cancers patients regularly exceeded one copy per beta-globin gene copy, while HPyV loads found in cancer samples were extremely low, with a median cumulative load of 0.000071, and generally not higher than those found in non-malignant tissue. This argues against a causal role of HPyV in tonsillar carcinogenesis as it has already been discussed for other malignancies [59, 80]. While both HPV and HPyVs may persist in tonsillar tissue, only HR-HPVs seem to play a role in tonsillar carcinogenesis. It has been discussed whether HPyVs are co-factors for cancers induced by other oncoviruses such as anal, cervical, laryngeal, hepatic, gastric or skin cancer, but a link between co-infection and cancer could not yet be established [81]. Our data on HPyV DNA prevalence and load in malignant and non-malignant tonsillar tissue support the notion that HPyVs do not play a role in tonsillar carcinogenesis, neither as independent etiologic agents nor as co-infecting agents of HPV.