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HPV DNA/RNA detection in various oral and oropharyngeal biomaterials identifies active HPV infections also in non-neoplastic tonsils

Highlights • Fist study analysing tissue, brushes and sputum for all patients also for viral mRNA.• HPV detection performed in brushes/sputum inconsistently reflect tissue HPV status.• In terms of virus activity, p16 seems insufficient to predict tissue HPV status.• E6-mRNA analysis detects active HPV infection in non-neoplastic tonsillar tissues.• Data gives rise to the question whether tonsillectomy might prevent TSCC.


Introduction
Infection with human papillomaviruses (HPV) are causing anogenital, specifically cervical cancers [1][2][3] and a subset of head and neck squamous cell carcinoma (HNSCC). In the head and neck, specifically SCC of the tonsils (TSCC), located in the anatomical region of the oropharynx, are often characterized by infection with predominantly HPV16 [4][5][6][7][8] . TSCC harboring active HPV infection, characterized by HPV-DNA and -RNA expression, are considered to be truly HPV-driven [9] with HPV prevalence rates in this tumor entity between 30% and 90% of cases dependent on the geographical region the patients live in [10][11][12] .
to be infected before clinically evident lesions can be detected. Thus, HPV initially infects neoplasia-free, clinically healthy mucosa, in the area of the upper aerodigestive tract as well as other anatomical sites. Therefore, clinically normal tissue of the tonsils is of particular interest because its HPV status provides information on the epidemiology of HPV in neoplasia-free tissues of the oral cavity and oropharynx. Moreover, precise HPV prevalence rates of clinically normal mucosa or tonsils can help to better understand HPV's natural history and the progression from HPV presence to carcinogenesis. HPV prevalence rates in non-neoplastic tonsils are reported to be between 0% and 12.5%. Negative HPV detection studies have been reported from non-neoplastic tonsils in children [17] , children and adults [18][19][20] and adults only [21] , while positive HPV detection studies come predominantly from children and adults [22][23][24] .
Gargle and sputum samples are currently evaluated to determine whether HPV diagnostics on these minimally-or non-invasively obtained materials can provide reliable information regarding the HPV status of the tonsillar tissue [ 25 , 26 ]. Such a non-invasive surrogate method for detection of HPV infection could be suitable for developing screening programs, possibly to identify individuals who could particularly benefit from HPV vaccination or should be monitored more closely to prevent carcinogenesis. However, data obtained from tonsillar tissue, tonsillar brushes, gargle or sputum samples from the same subjects are currently not promising [24][25][26] , regardless of whether neoplastic or non-neoplastic tissues have been examined. Franceschi and colleagues [24] conclude that brushes taken from non-malignant tonsillar tissues are not suitable for detecting precancerous lesions. Concordance rates in mouthwashes and tissue samples from the same patients with TSCC of only 58% were reported [24] . In addition, the same authors analyzed tonsillar swabs as well and found concordance rates between swabs and tissue samples of 65% only.
All these studies have the disadvantage of analyzing material of patients having either benign [24] or malignant lesions [27] or have enrolled only small numbers of patients with benign lesions with limited information about the HPV-status of these benign tissues [27] . Moreover, the vast majority of studies reviewed were performed in a retrospective setting while data from prospective studies are scarce.
Therefore, the aim of the present prospective study was to analyze the HPV-status of intraoperatively obtained tonsillar tissue specimens and swabs, and sputum samples of patients with either TSCC, tonsillar hyperplasia, or chronic or recurrent tonsillitis. The non-neoplastic and malignant tonsillar entities have been tested for HPV-DNA-and -RNA in order to compare their HPV prevalence rates and to possibly draw conclusions on aspects of their natural history. Tonsillar swabs and sputum samples from the same patients were analyzed to determine the validity of these biomaterials as surrogate markers for tissue HPV status. In addition, p16 INK4A -expression was determined in all tissue samples to further investigate the validity of this protein as a surrogate marker for HPV infection, a still highly controversial topic.

Study design
In a prospective setting, patients with tonsillar squamous cell carcinoma (TSCC) or non-neoplastic tonsillar lesions, namely tonsillar hyperplasia (H) or chronic or recurrent tonsillitis (CRT), were enrolled when treated at the ENT Departments of the University Hospitals of Kiel and Rostock and the ENT Department of the Asklepios Klinik Hamburg-Harburg and Klinikum Oldenburg (all Germany). Smoking habit and age were documented. Each patient was asked to deliver 2 sputum samples directly preoperatively, which were immediately taken to the laboratories of the respective institution. For subsequent DNA and RNA isolation from the sputum, the first sputum sample was supplemented with 2 ml nucleic acid stabilizer (AmpTec, Hamburg, Germany). The second sample was centrifuged for 10 min (Heraeus Megafuge 1.0 or equivalent, at 600 g) to separate the cellular fraction. The resulting supernatant was supplemented with 2 ml of nucleic acid stabilizer and the cellular fraction was dissolved in 1.5 ml of the stabilizer. All samples were stored at room temperature until nucleic acid extraction, which was performed in laboratories in Kiel. Intraoperatively, patient material was extracted in different ways depending on the diagnosis: In CRT-and H-patients palatine tonsils of both sides were swabbed and resected; in TSCC-patients only the affected tonsil was swabbed and resected. From each tonsil eligible for analysis, 2 tonsillar swabs were taken with the Buccalyse swabs (Isohelix, Harrietsham, United Kingdom). One swab was used to extract DNA, the other swab was transferred into 700 μl of the abovementioned stabilizer (AmpTec, Hamburg, Germany) to extract RNA. All swabs were stored at room temperature until nucleic acid extraction in Kiel. Tissue specimens (approx. 1 cm 3 ) of each tonsil was shock frozen in liquid nitrogen and stored at − 80 °C until nucleic acid extraction in Kiel. A second, adjacent piece of tissue was kept in formaldehyde for immunohistochemistry. cDNA synthesis RNA (200 ng) was transcribed into cDNA (TR-cDNA synthesis kit; AmpTec, Hamburg, Germany) under the following reaction conditions: 30 min at 16 °C, 30 min at 42 °C, 5 min at 85 °C followed by 5 min storage on ice. To analyze cDNA integrity a PCR from each sample using 18S RNA primers (Promolgene; Berlin, Germany) according to the manufacturer's protocol was performed.

HPV detection
HPV-DNA detection was performed by PCR amplifying 50 ng per sample using the primers GP5 + /GP6 + , as described previously [28] . For all (RT)-PCR reactions the Quanta Biosciences Perfecta PCR mix, utilizing the AccuStart TM Taq DNA-Polymerase, was used. PCR conditions were as follows: initial denaturation: 15 min 95 °C, followed by 40 cycles of 1 min 95 °C, 1 min 40 °C and 1 min 72 °C, and a final elongation: 5 min 72 °C. DNA integrity was analyzed using genomic primers for the housekeeping gene ß2-microglobin (B2M; Promolgene) according to the manufacturer's protocol; in brief, initial denaturation 10 min 95 °C; followed by 40 cycles:20 s 95 °C, 20 s 60 °C and 20 s 72 °C. Additionally, a positive control (a synthetic oligonucleotide of the HPV L1 gene, covered by the GP5 + /GP6 + primers; Eurofins; Ebersberg Germany) was amplified in the GP5 + /GP6 + PCRs. Amplification products were sequenced by Sanger sequencing and alignments were obtained from the GenBank online BLAST server ( http://blast.ncbi.nlm.nih.gov/Blast.cgi ). HPV-RNA std: standard deviation, N/A no information available, numbers in parenthesis are percentage among disease type. The entire patient population ( n = 215 patients) was comprised of patients with non-neoplastic chronic or recurrent tonsillitis (CRT; n = 107), tonsillar hyperplasia (H; n = 56) and patients with tonsillar squamous cell carcinoma (TSCC; n = 52).

Smoking history of the patients
Smoking habit was classified as never smoker, active smoker and former smoker, with the latter having stopped tobacco consumption at least 2 years prior to diagnosis. This classification has led to significant results published by our group [ 10 , 13 ]. In addition, information provided by patients regarding their substance abuse must be interpreted with caution and the meaning of pack years has become variable as cigarette packs, at least in Germany, contain between 10 and 40 cigarettes per pack.

Statistical analyses
Fisher's exact test (SPSS 20.0 software) followed by Bonferroni posttest, where appropriate was performed to correlate the patients' smoking habit and the tonsillar HPV-DNA-status, smoking habit and tonsillar p16 INK4A -expression as well as the correlation between tonsillar HPV-DNA-status and p16 INK4A -expression. Student's t -test was performed to analyze age related differences between 2 groups and one-way ANOVA followed by Bonferroni posttest was used to analyze age related differences when 3 or more groups were analyzed. p -values ≤ 0.05 were considered statistically significant.

Patient demographics
The patient characteristics are presented in Table 1

HPV-status in tonsillar tissue
The HPV-DNA status of the patients' tonsillar tissue is also shown in Table 1 [11][12][13][14]. Overall correlation between HPV-status and p16 INK4A -expresson of the TSCC-patients is summarized in Table 3 . p16 INK4A -expression was also analyzed in the non-neoplastic tonsils. Applying the mentioned algorithm classifying only tissue with strong ( > 75%) diffuse immunohistochemical staining as p16 INK4A -positive, resulted in 100% p16 INK4Anegative non-neoplastic cases. To make comparison to the literature on p16 INK4A -expression in non-neoplastic tonsillar tissue [32] possible, tis-  [30] . In case of HPV-DNA positivity the HPV-type is given. numbers in parenthesis are percentage. The patient population ( n = 215 patients) was comprised of patients with non-neoplastic chronic or recurrent tonsillitis (CRT; n = 107; 1 HPV-negative former smoker was excluded), tonsillar hyperplasia (H; n = 56; 1 HPV-positive former smoker was excluded; for 1 HPV-negative patient no information was available) and patients with tonsillar squamous cell carcinoma (TSCC; n = 52; for 1 HPV-negative and 1 HPV-positive patient no information was available). p-values were calculated by Fisher's Exact test.
sues with moderate (31-75%) positive cells were considered p16 INK4Apositive as well. This altered algorithm did not change the result obtained in the H-patients. However, in 20/107 CRT-patients (18.7%) moderate p16 INK4A -expression was detected, but did not correlate with HPV-expression in the tissues ( Table 3 ). Overall correlation between HPV-status and p16 INK4A -expresson of the CT and H-patients is also given in Table 3 .

HPV, p16 INK4A and smoking
The correlation between smoking habit of the patients and HPV-DNA-status of the patients' tonsillar tissue dependent on their disease is shown in Table 3 . In TSCC and CRT-patients there is a significant correlation between a negative smoking history and a positive HPV-DNA status which was not the case in patients with tonsillar hyperplasia. However, 8 (72.7%) of the 11 HPV-positive patients with tonsillar hyperplasia are never smokers. Similarly, but only in the TSCC-patients a significant correlation between a negative smoking history and a high ( > 75% of stained tumor cells) p16 INK4A -expression was seen.

HPV-infection in different biomaterials
Tonsillar tissue, sputum (total, supernatant, and cellular fraction) and swabs of the tonsillar tissue were analyzed for HPV-positivity in each patient. Noteworthy, when comparing HPV-status in tonsillar tissue, sputum or tonsillar swabs revealed HPV-infection of all affected biomaterials in a single patient were always infected with the same HPVtype.

HPV-infection of different biomaterials of the TSCC-patients
Correlating the HPV-status of tonsillar tissue with the HPV-status of the sputum and the tonsillar swabs of the TSCC-patients showed that  ( Table 4 , cases 9-12) had HPV-positive sputum and / or tonsillar swab samples. These 4 cases were reanalyzed by reextracting DNA and RNA of the tissues and RT-PCR was performed using HPV16 and HPV18 specific primers. Again, no HPV-DNA or -RNA could be detected in these tissues, despite positive signals for the positive controls. The discordance between tissue and sputum was 50%. In one patient with HPV-DNA positive but -RNA negative tissue all other biomaterials were HPV-DNA and -RNA positive ( Table 4 , case 8). A further patient is positive for HPV-DNA and -RNA in all biomaterials except for the tissue specimens ( Tab 4 , case 9). In 2/17 HPV-positive sputum samples HPV-DNA could be detected in the cell free supernatant, the corresponding cellular fractions, were HPV-DNA negative.

Discussion
The present study is unique in several aspects: Malignant and nonneoplastic lesions of palatal tonsils have been investigated in a prospective setting, with different patient biomaterials (tissue, swabs and sputum). All samples were analyzed not only for HPV DNA but also viral RNA, which was also performed in the swabs and sputum samples. In addition to the total sputum, the cellular fraction and the supernatant were tested separately for HPV. The findings obtained in this study are correspondingly diverse and are addressed in detail below. The high quality and validity of the results of the present study are based on the fact that if HPV was detected, the same HPV genotype has always been verified in different biomaterials of the same patients.
The immunohistochemically confirmed overexpression of the cellular protein p16 INK4A is considered a surrogate marker for active HPV infection in HNSCC, which is based on a negative feedback mechanism by viral oncogene intervention in the cell cycle [ 8 , 33 ]. At least since the inclusion of p16 INK4A -immunochemistry in the 8th edition of the TNM Classification (AJCC/UICC) for the determination of HPV status in oropharyngeal carcinoma [14] , p16 INK4A -analysis has become part of clinical routine. One aim of the study was to test the quality of p16 INK4A -expression in tonsillar tissue specimens as a surrogate marker for active HPV infection and to investigate to what extent other approaches such as HPV detection in tonsillar swabs or sputum might be better suited as surrogate markers for the true HPV status of tonsillar tissue. Along with other groups we have repeatedly demonstrated, that p16 INK4A -expression and HPV DNA/RNA detection show clear discrepancies [ 9 , 10 , 13 , 33 ]. Again, this was confirmed by the present study. Based on the p16 INK4A -analysis alone, 14/52 (26.2%) TSCC-patients would have been falsely classified as HPV positive or negative, with possible (adverse) effects on therapy planning and outcome. In addition, p16 INK4A -expression was correlated with patients' smoking habit. The correlation between strong p16 INK4A -expression and negative smoking history of the TSCC patients adds to the controversial data. Similar to the here presented data, Mazul and coworkers found a significant correlation between strong p16 INK4A -staining and a negative smoking history [34] , while no correlation between these parameters was seen by Pitak-Arnnop and coworkers [35] . Unfortunately, the present study's aim to establish sputum and tonsillar swabs as surrogate marker did not lead to a promising avenue. At least in our TSCC cohort, HPV sputum and/or swab results show similar high rates of discrepancies as seen for p16 INK4A -staining with possibly similar (adverse) consequences if sputum and/or tonsillar swabs were used to determine the HPV status of the tonsillar tissue itself.
HPV-DNA prevalence in malignant tonsillar samples examined in the present study were 40.4%. 81% of these, were active infections and were thus, as expected, in the range of data shown previously for our [ 8 , 10 , 13 , 33 ] and other comparable patient cohorts [ 5-7 , 12 ]. It is all the more remarkable, that of the 163 non-neoplastic tonsillar samples, not only 23 (14.2%) showed HPV-DNA infection (7 of them with high risk-HPV genotypes), but 5 of them [3.0% (3 HPV16, 2 HPV11)] carried active infections, based on RNA detected. Viral activity was proven by the presence of viral RNA, since p16 INK4A -immunohistochemistry in non-neoplastic tonsillar samples showed -if at all -moderate staining (solely in CRT) in 18.7% and this did not correlate with HPV status. Despite the very low rate of active HPV infection in the non-neoplastic specimens investigated, this result is nonetheless very interesting. The question arises what information these cases can provide within the context of the hitherto poorly understood transition of the natural history of HPV in the oral cavity and oropharynx to carcinogenesis. In particular, will these 3 HPV16 positive non-neoplastic cases progress to carcinoma and has tonsillectomy spared these patients from this? This notion is supported by studies on TSCC-patients and healthy volunteers, who carry 2-3% virus-specific antibodies and these antibodies can sometimes be measured decades before the appearance of a tumor [36,37] . Moreover, a nation-wide cohort study in Sweden comparing cancer incidence in a tonsillectomy cohort ( n = 225.718) with Sweden´s general population detected a reduced risk of tonsillar cancer but unrelated to other oropharyngeal or other head and neck cancers [34] which could be another argument for tonsillectomy potentially preventing TSCC. Tables 4 and 5 show the discordant HPV DNA/RNA results in the different biomaterials of the respective patients. Case 3 and 4 as well as cases 8 and 9 in Table 4 are particularly noteworthy. The detection of HPV16 DNA in the supernatant of the sputum only, but not in the cell fraction of case 3 and 4, shows that HPV DNA can not only be localized extracellularly, but is also technically detectable in the cell free supernatant. It can therefore be concluded that not every HPV DNA detection in the total sputum is equivalent to the detection of a cellular infection. Given that in these cases tissues and swabs are HPV DNA/RNA positive, an experimental error cannot fully be excluded despite the accuracy of the methods used. Case 8 and 9 of Table 4 are positive for HPV RNA in all biomaterials with the exception of the tissue samples themselves despite repeated DNA/RNA extraction and analysis. In these cases, the tissue result is unlikely to be false negative and these patients possibly have active HPV infection at a different site of the upper aerodigestive tract.
Based on our results sputum-or swab-based screening tests do not seem suitable to identify individuals at increased risk for HNSCC nor as a screening instrument during surveillance following HNSCC therapy. This is in line with data from Martin-Gomez and co-workers [25] and Gipson and co-workers [26] who also addressed this topic, but either used gargle instead of sputum and did not test for RNA [25] or performed a meta-analysis summarizing existing data [26] . Therefore, at present the situation remains that HPV-associated diseases of the head and neck only become evident and can be diagnosed when clinical symptoms and the tumor itself became apparent.
HPV-positive patients with non-neoplastic tonsils are significantly older than HPV-negative patients. This can most likely be attributed to observations from natural history studies, describing a continuous but with age increasing HPV detection rate in the oral cavity [ 38 , 39 ]. This is often explained by the increasing number of sexual partners with age [38] . We would like to refer to our recently published article on this topic, in which we discuss this correlation and its significance for the natural history of HPV and HPV-associated carcinogenesis in detail [40] . In the present study there is no age difference between HPV-positive and -negative TSCC patients. This phenomenon, which we have repeatedly seen in our analyses [ 8 , 10 , 13 , 33 ] contradicts a large part of the US-American literature [ 5 , 38 , 39 , 41 ]. The often significant age differences are explained by the fact that carcinogenesis initiated by the ingredients of tobacco smoke and/or alcohol takes longer, thus, patients being older at cancer diagnosis. However, there is increasing evidence that the observation that HPV-positive patients are significantly younger than HPV-negative patients [ 5 , 38 , 39 , 41 ] is not consistent in the literature, as different studies report no significant age-related differences between HPV-positive and HPV-negative patients, not only in European [ 10 , 42 , 43 ] but also in US-American study populations [44] .
The well-established correlation of smoking and HPV negative and non-smoking and HPV positive tissue samples [ 10 , 13 , 19 , 39 , 45 ] is confirmed by the present study, both in TSCC and CRT cases. Among the H-cases, 8 of 11 HPV-positive patients are also non-smokers. It can be assumed that the association between smoking behavior and HPV status is based on the interaction between HPV and two proteins, SLPI and Annexin A2, both of which are increasingly expressed in smokers [46] . The authors currently analyze these parameters in this study population.
In summary, neither p16 INK4A -immunohistochemistry integrated into the TNM classification nor any other analytical procedure performed in the present study can be recommended as a surrogate for the true HPV status of the tonsillar tissue. The most reliable detection method of active HPV infections, which are responsible for neoplastic lesions, is the detection of viral RNA. All investigated HPV-positive TSCC patients are infected with high-risk HPV genotypes and the infections are active with few exceptions, while the majority of patients with nonneoplastic tonsillar disease are infected with low-risk HPV genotypes while infections are predominantly inactive. However, three percent of non-neoplastic tonsillar tissues show active mostly high-risk HPV infection. Future studies should try to answer the question, how the latter infections mark a potential transformation of oral HPV natural history to carcinogenesis and whether tonsillectomy may protect against (HPVdriven) TSCC.

Declaration of Competing Interest
No potential conflict of interest relevant to this article was reported.

Data for reference
Data available on request due to privacy/ethical restrictions.