Accuracy of High-risk HPV DNA PCR, p16INK4a Immunohistochemistry or the Combination of Both to Diagnose HPV-driven Oropharyngeal Cancer

Accuracy of High-risk HPV DNA PCR, p16INK4a Immunohistochemistry or the Combination of Both to Diagnose HPV-driven Oropharyngeal Cancer | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Accuracy of High-risk HPV DNA PCR, p16INK4a Immunohistochemistry or the Combination of Both to Diagnose HPV-driven Oropharyngeal Cancer Cindy Simoens, Tarik Gheit, Ruediger Ridder, Ivana Gorbaslieva, and 20 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1008778/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 8 You are reading this latest preprint version Abstract BACKGROUND The incidence of high-risk human papillomavirus (hrHPV)-driven head and neck squamous cell carcinoma, in particular oropharyngeal cancers (OPC), is increasing in high-resource countries. Patients with HPV-induced cancer respond better to treatment and consequently have lower case-fatality rates than patients with HPV-unrelated OPC. These considerations highlight the importance of reliable and accurate markers to diagnose truly HPV-induced OPC. METHODS The accuracy of three possible test strategies, i.e. a) hrHPV DNA PCR (DNA), b) p16 INK4a immunohistochemistry (IHC) (p16), and c) the combination of both tests (considering joint DNA and p16 positivity as positivity criterion), was analysed in tissue samples from 99 Belgian OPC patients enrolled in the HPV-AHEAD study. Presence of HPV E6*I mRNA (mRNA) was considered as the reference, indicating HPV etiology. RESULTS Ninety-nine OPC patients were included, for which the positivity rates were 36.4%, 34.0% and 28.9% for DNA, p16 and mRNA, respectively. Ninety-five OPC patients had valid test results for all three tests (DNA, p16 and mRNA). Using mRNA status as the reference, DNA testing showed 100% (28/28) sensitivity, and 92.5% (62/67) specificity for the detection of HPV-driven cancer. p16 was 96.4% (27/28) sensitive and equally specific (92.5%; 62/67). The sensitivity and specificity of combined p16+DNA testing was 96.4% (27/28) and 97.0% (65/67), respectively. In this series, p16 alone and combined p16+DNA missed 1 in 28 HPV driven cancers, but p16 alone misclassified 5 in 67 non-HPV driven as positive, whereas combined testing would misclassify only 2 in 67. CONCLUSIONS Single hrHPV DNA PCR and p16 INK4a IHC are highly sensitive but less specific than using combined testing to diagnose HPV-driven OPC patients. Since HPV-driven OPC cancer can be treated less aggressively, combined testing can reduce treatment sequelae at the expense of a small number of patients that may receive less effective therapy. Infectious Diseases HPV DNA PCR p16INK4a IHC HPV mRNA oropharyngeal carcinoma HPV-AHEAD Background In high-resource countries, human papillomavirus (HPV)-driven head and neck squamous cell carcinoma represents an increasing health problem, particularly in cancers of the oropharyngeal region, including base of tongue and tonsils ( 1 – 4 ). An estimated 20-40% of oropharyngeal cancers (OPC) is believed to be caused by HPV infection, and the large majority of them (>80%) are due to HPV16 ( 2 , 5 ). Patients with HPV-induced cancers respond better to treatment and consequently have better survival than patients with HPV-unrelated OPC ( 6 – 11 ). Therefore, therapy may be de-escalated with reduced toxicity and less sequelae. However, treatment de-escalation may negatively impact patient survival if HPV was not the carcinogenic driver of the respective tumour. These considerations highlight the importance of reliable and accurate markers, or marker combinations, to diagnose truly HPV-induced OPC. The most widely applied detection methods thus far are based on PCR amplification of viral DNA to determine HPV-positivity. However, several independent studies have highlighted that PCR-based assays for the detection of HPV DNA are not sufficiently accurate to establish the viral causality ( 12 – 16 ). These PCR-based methods are highly sensitive and can detect even a few DNA copies per sample, which might yield false-positive results mainly reflecting transient infections ( 17 – 19 ). Additional markers, such as the presence of viral E6/E7 mRNA transcripts and p16 INK4a expression as surrogates for HPV-induced transformation, allow a more accurate classification of HPV-driven head and neck cancers (HNC) ( 14 , 20 – 24 ). The detection of HPV E6/E7 oncogene transcripts is currently regarded as gold standard in this context, as HPV-driven carcinomas critically depend on the carcinogenic action of the HPV E6 and E7 oncoproteins ( 22 , 25 ). However, the detection of viral transcripts is laborious and may not be routinely feasible in all laboratories. This is particularly true for mRNA transcript detection from formalin-fixed, paraffin-embedded (FFPE) tissue specimens, which are frequently used during routine diagnostic work up; however, RNA integrity may be affected by the fixation protocol. HPV E7 oncogenic signaling induces strong overexpression of the cellular protein p16 INK4a in HPV-transformed cells ( 26 ). The immunohistochemical detection of p16 INK4a overexpression is now used as a surrogate biomarker of HPV-transformed epithelium of the uterine cervix ( 27 – 29 ). Similarly, p16 INK4a immunohistochemistry (IHC) is frequently applied to determine HPV association in HNC ( 21 , 22 , 30 – 32 ). Apart from single p16 INK4a IHC, combined detection of p16 INK4a IHC and HPV DNA detection by PCR is frequently used. HPV-AHEAD (FP7 funded network) The HPV-AHEAD study (“Role of human papillomavirus infection and other co-factors in the aetiology of head and neck cancer in Europe and India”) group comprised partners from six European countries and from India (website HPV-AHEAD: https://hpv-ahead.iarc.fr/ ). The main goal of the study was to perform a comprehensive analysis on a large number of HNC cases to clarify pathogenic pathways in HNC carcinogenesis, and to identify clinically useful biomarkers. In a previous publication ( 33 ), the results of the histological and molecular assessment of 1039 archived HNC specimens from Belgian patients were described, primarily using the detection of HPV DNA, mRNA, and p16 INK4a IHC. In this study, we focus on the accuracy of these tests - individually and in combination - to diagnose hrHPV-driven oropharyngeal cancer. Methods All methods were carried out in accordance with relevant guidelines and regulations. Patient selection, clinical information and tissue specimen collection for the Belgian cohort The overall study included patients with oropharyngeal, oral cavity, laryngeal, hypopharyngeal, and unspecified head and neck squamous cell carcinoma. For a detailed description of the patient characteristics, we refer to the previous publication of the Belgian HPV-AHEAD study data (Simoens, 2021, Cancer Epidemiology). For the analyses in this study, the focus was on the oropharyngeal cancers (ICD-O-3 codes: C01 (base of tongue); C02.4 (lingual tonsil); C05.1 (soft palate); C05.2 (uvula); C09 (tonsil): C09.0, C09.1, C09.8 & C09.9; C10 (oropharynx): C10.0-10.4, C10.8 & C10.9). FFPE tumour blocks and clinical information were collected from OPC patients treated in two Belgian hospitals (GZA and UZA), diagnosed between 1980 and 2010. Ethical clearance was obtained from the Ethical Committees of ‘GZA hospitals’ (ref nb: BVDE/hp/2013/01.147), ‘UZA & UA (University of Antwerp)’ (ref nb: 11/47/362) and IARC, Lyon, France (ref nb: 11-30). Preparation of tissue sections (University of Antwerp, Antwerp, Belgium) FFPE-tissue blocks were all processed at the UA laboratory of cell biology and histology, following the optimized HPV-AHEAD sectioning protocol ( 33 , 34 ). Briefly, minimal ten sections (S) were prepared from each FFPE block. The first (S1) and the last (S10) 5µm sections were hematoxylin and eosin (H&E) stained for morphologic histology interpretation and used to check for the presence of tumour. S2 and S9 (5µm) were used for p16 INK4a IHC staining, while the 10µm sections S3-5 and S6-8 were used for the extraction of RNA and DNA, respectively. Histological review All sections were re-evaluated by the HPV-AHEAD pathology review panel. The review was blinded with respect to the original local diagnosis. Only FFPE blocks where S1 and S10 H&E sections reflected squamous tumour tissue were included in the analysis. HPV E6*I mRNA analysis (DKFZ, Heidelberg, Germany) All OPC cases were analysed for the presence of: (i) HPV16 E6*I mRNA, and (ii) ubiquitin C (ubC) mRNA as a cellular mRNA positive control (housekeeping gene used for RNA quality control). OPC cases positive for DNA of a non-HPV16 genotype were additionally analysed for E6*I mRNA of the respective genotype. Specimens that were HPV E6*I and/or ubC mRNA-positive (RNA+) were considered RNA valid. Reverse Transcription-PCR was carried out using the QuantiTect Virus Kit (Qiagen, Hilden, Germany), as described in full in former HPV-AHEAD publications ( 33 , 35 ). The type-specific E6*I mRNA assays identifying transcripts of fourteen high-risk and six possible/probable high-risk HPV genotypes ( 36 ) were applied. HPV DNA genotyping (IARC, Lyon, France) HPV DNA genotypes were detected by a E7 type-specific multiplex genotyping (E7-MPG) assay, which combines multiplex PCR and bead-based Luminex technology (Luminex Corporation, Austin, TX), as previously described ( 37 , 38 ). Type specific-MPG uses HPV type-specific primers targeting the E7 region of thirteen high-risk and six possible/probable high-risk HPV genotypes (HPV 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68a and b, 70, 73 and 82), as well as two low-risk HPV genotypes (HPV 6 and 11). Two primers for amplification of the beta-globin (β-globin) gene were also included to control for the DNA quality of each specimen. A detailed description of the DNA extraction and further HPV genotyping test characteristics can be found in the previous publication on the Belgian HPV-AHEAD study ( 33 ). Genotyping controls and DNA preparation were blindly analysed, and no sign of contamination of negative controls was detected during the laboratory work. p16 INK4a expression (Roche mtm laboratories, Mannheim, Germany) Expression of p16 INK4a was evaluated by IHC, with a dual-immunostaining protocol for the simultaneous immunostaining of both p16 INK4a and Ki-67 biomarkers (CINtec PLUS kit, Roche mtm laboratories AG, Mannheim, Germany), as previously described in the HPV-AHEAD publications ( 33 , 35 ). As in all HPV-AHEAD studies ( 35 , 39 ), a continuous, diffuse staining for p16 INK4a within the cancer area of the tissue sections was considered as positive, while a focal staining or no staining was considered negative. IHC slides were analysed without knowledge of any other clinical information (including HPV DNA and RNA status) by the scientists RR or DH and reviewed by one of the European members of the HPV-AHEAD pathology review panel (JPB, BLR, or FM). Discrepant cases were re-evaluated by a pathologist outside the review panel (AC), and final classification of the staining was based on majority consensus results. Corresponding to the former Belgian HPV-AHEAD publication ( 33 ), the p16 slides with technical issues were restained and re-evaluated, to minimize the number of missing results. Statistical analysis Presence of viral mRNA was considered as the reference indicating HPV etiology. The accuracy of three possible test strategies was analysed: a) hrHPV DNA PCR alone, b) p16 INK4a IHC alone, and c) the combination of p16 INK4a IHC and hrHPV DNA PCR; where positivity is defined by a co-positive result with both tests, a negative result by a co-negative result for both tests, and a discordant result by only one test being positive and the other being negative. Test strategy (c) involves three algorithms: ALGORITHM 1 (p16+DNA) consists of p16 INK4a IHC and hrHPV DNA PCR on all samples; ALGORITHM 2 (p16->DNA) involves p16 INK4a staining of all samples, followed by the HPV DNA test only on the p16 INK4a -positive samples; ALGORITHM 3 (DNA->p16) involves HPV DNA PCR on all samples, followed by p16 INK4a staining only on the HPV DNA-positive samples. Sensitivity was defined as the proportion of test positive samples among HPV RNA-positive patients. Specificity was defined as the proportion of negative test results among HPV RNA-negative patients. Relative sensitivity and specificity of each test compared to the other tests were also assessed. Binomial 95% confidence intervals (95% CIs) were computed for proportions. Given the matched testing of the same patient specimens, McNemar 95% CIs were computed for relative accuracy parameters. Continuous variables were summarized by their mean and 95% CIs. ANOVA was used to compare mean age by gender. Statistical analyses were performed using STATA 16 (StataCorp, College Station, TX). p-values were two sided and statistical significance was set at p equal or less than 0.05. Results Study population characteristics Ninety-nine of 116 OPC patients were included in the analysis, after exclusions based on the pathology review (for reasons of not being a squamous cell carcinoma or not reflecting invasive cancer, N=13) and beta-globin PCR negativity (N=4). 71.7% (71/99) of the patients were male, 26.3% (26/99) were female, and for the remaining two samples the gender of the patients was not specified. The mean age at diagnosis was 67.3 years (95%CI: 64.9-69.7) and was not different between males and females (p=0.47). HPV prevalence in OPC cases HPV prevalence was determined by HPV DNA PCR, p16 INK4a IHC, E6*I mRNA, and all combinations of tests in the HPV-AHEAD study. Out of 99 OPC cases, 36.4% (36/99) contained HPV DNA, 34.0% (33/97) showed p16 INK4a positivity, and 28.9% (28/97) were HPV RNA-positive (Table 1). Twenty-six of the 28 HPV RNA-positive samples were positive for HPV16 (92.9%), the remaining 2 samples contained HPV18 (7.1%). The proportion of HPV-positive OPCs varied between 28.4% (27/95) and 28.9% (28/97) for all combinations of tests that included RNA testing (DNA+RNA, p16+RNA, DNA+p16+RNA). For the combination of testing for HPV DNA and p16 INK4a IHC, the overall prevalence was slightly higher (30.9%, 30/97). Absolute and relative accuracy in the oropharynx and its subsites Both absolute and relative accuracy of hrHPV DNA detection by PCR (hrHPV DNA), p16 INK4a IHC (p16), and combined p16 and DNA testing algorithms (p16+DNA, p16->DNA and DNA->p16), to identify a transforming HPV infection were calculated. In the following tables, only the results are shown for the samples that had valid results on all three tests performed (N=95). All RNA-positive OPC cases were positive for hrHPV DNA (100%, 28/28), in contrast to one out of 28 HPV RNA-positive samples that did not have a positive p16 INK4a result (3.6%). Five out of 67 RNA-negative cases showed hrHPV DNA-positivity (7.5%), the same as observed for p16 INK4a IHC. This number would be further lowered to only 2 positive cases out of 67 RNA-negatives (3.0%), if hrHPV DNA and p16 INK4a testing (p16+DNA) were combined. The same holds for the other algorithms where all samples would initially be tested on p16 INK4a (p16->DNA) or hrHPV DNA (DNA->p16), followed by the other test if the initial test result turned out positive. Data are detailed in Table 2, for all OPC cases, and for the tonsils and the base of tongue in specific. The accuracy of hrHPV DNA PCR and p16 INK4a IHC to detect a transforming HPV infection in OPCs was calculated (Table 3 a). Highest sensitivity (100.0%, 28/28) was noted for hrHPV DNA testing, while specificity was equal for both hrHPV DNA and p16 INK4a (92.5%, 62/67). The best compromise was found in the combined testing of hrHPV DNA PCR and p16 INK4a staining, with 96.4% (27/28) sensitivity and 97.0% (65/67) specificity. When looking in more detail to the proposed algorithms, all provided equal accuracy, however, with a 60% reduction in the number of HPV DNA PCR or p16 INK4a IHC tests needed in the sequential testing algorithms. For the subgroup of the tonsils, p16 INK4a staining alone was as sensitive (94.7%, 18/19) and specific (91.3%, 21/23) as the combined testing, in contrast to the base of tongue group, where all tests were 100% sensitive and specific, except for the p16 INK4a specificity (85.7%, 12/14). hrHPV DNA PCR testing was slightly more sensitive compared to p16 INK4a IHC (ratio: 1.04, 95% CI: 0.97-1.11), but equally specific (ratio: 1.00, 95% CI not computable) (see Table 3 b and the Supplementary Table). The combination of hrHPV DNA and p16 INK4a was slightly more specific (ratio: 1.05, 95% CI: 0.97-1.21) compared to both single tests. Discussion In these Belgian OPC patients, PCR testing for HPV DNA showed the highest proportion of positive cases (36%). Due to its high sensitivity, HPV DNA PCR-based assays can detect low viral copy numbers, which may not trigger carcinogenesis. These assays cannot distinguish between transcriptionally-active and passenger HPV infections. Therefore, other assays (HPV E6*I mRNA, and p16 INK4a staining) were evaluated to assess whether combined testing could improve the specificity. Only 28% of the samples with a valid HPV E6*I mRNA result were positive for HPV DNA and p16 INK4a , which might be the fraction of oropharyngeal cancers caused by HPV. After all, the detection of HPV E6 and/or E7 mRNA is seen as the gold standard in this context, because HPV-driven carcinomas critically depend on the continuous expression of E6/E7 oncogenes of hrHPVs ( 22 , 25 , 40 ). However, the detection of viral transcripts is challenging, not only because the RNA extraction step is laborious and time consuming, but specific infrastructures and equipment are needed. Therefore, HPV RNA assays may not be routinely feasible in all laboratories or on all tissue samples. HPV-driven OPC represents an increasing health problem, and therefore, reliable and accurate diagnosis becomes essential. In our Belgian HPV-AHEAD study, HPV DNA PCR testing alone was 100% sensitive, but less specific (92.5%) compared to mRNA testing. The inherent strength of the PCR-based methodology lies in its capacity to detect very small amounts of HPV DNA. At the same time, strict laboratory procedures and controls are critical in reducing contamination-related false-positive findings ( 41 ). Immunohistochemistry for p16 ink4a is most widely used as a surrogate marker for hrHPV infection in FFPE tissues, also for oropharyngeal squamous cell carcinoma. p16 IHC testing alone was a bit less sensitive (96.4%), but co-presence of hrHPV DNA and p16 INK4a positivity was similarly sensitive (96.4%) and more specific (97.0%) compared to each test separately. In literature, several authors have advocated against the use of either p16 INK4a or hrHPV DNA alone as indicators of HPV-induced etiology in cancers, but recommended their combined use as a reliable and practical approach to differentiate HPV-induced from HPV-unrelated tumours ( 25 , 42 – 44 ). The meta-analysis performed by Prigge et al. ( 45 ) also showed a similarly high (pooled) sensitivity of the combined testing (93%) and either p16 (94%) or HPV DNA (98%) alone, and the (pooled) specificity (96%) was significantly higher than either testing method alone (83% and 84%, respectively). These accuracy data were confirmed by these Belgian results. Relative sensitivities and specificities were not significantly different from unity in our study, nor in the meta-analysis of Prigge et al. ( 45 ), where significance was only reached for the relative specificity of the combination of HPV DNA PCR and p16 INK4a tests versus p16 INK4a IHC (rel. spec.: 1.13 (95% CI: 1.04-1.23)), by pooling from multiple studies. Furthermore, better clinical outcomes have been reported for patients with HPV-induced compared to HPV-unrelated OPC ( 6 – 11 ). Given the ongoing discussions about the value of therapy de-escalation in patients with HPV-induced OPC, the reliable identification of viral etiology in the tumour and its impact on patient prognosis gain particular momentum. Improved prognostication by combined p16 INK4a and hrHPV DNA detection compared to single marker analysis has been demonstrated in a large meta-analysis on tumours in the head and neck region ( 46 ). In our study, 97% (92/95) of the patients would have been correctly diagnosed with the combined testing approach. However, one 58-year old male (current smoker and alcohol consumer) with a T1 (N0 M0) microinvasive tonsillar tumour might have been treated too aggressively by applying this combined test strategy, as his tumour would have been classified as a HPV-unrelated case, being p16 negative while actually both DNA and mRNA HPV16 positive. More than six years after diagnosis, the patient was alive without any evidence of disease after having had radical wide excision surgery. On the other hand, treatment de-escalation may negatively impact disease-specific patient survival rates if HPV in the tumour was not the cause of carcinogenesis. In our Belgian OPC patients, the number of false positive cancers would be reduced from five to two with the combined p16+DNA detection, who may otherwise receive a less aggressive treatment regimen. These two HPV-unrelated cancer patients were male and diagnosed with a tonsillar cancer. The first patient, with a Tx N2 M1 tonsil NOS tumour, was a heavy drinker and current smoker who only underwent surgery and had a recurrence within six months. He died two years after being diagnosed. The other patient, with a T3 N2 tonsil NOS tumour, received surgery followed by radiotherapy and concurrent chemotherapy for two months. At the last follow-up date, this patient was alive, but with evidence of residual disease. Of note, these two HPV-unrelated patients clearly had a much worse disease status at diagnosis and thereby reduced survival chances compared to the above described HPV-driven tonsillar cancer patient. Ninety-seven percent of the OPC patients in our study would have been correctly diagnosed as patients with a HPV-driven cancer by combined p16 INK4a and hrHPV DNA (p16+DNA) detection. Sequential testing algorithms (p16->DNA and DNA->p16) resulted in equally accurate results, however, with a 60% reduction in the number of tests needed to be performed. This will cause a substantial reduction in costs and laboratory time, while providing the same clinical value. Especially the algorithm of p16 INK4a on all samples followed by HPV DNA PCR on p16 INK4a -positive samples only would be a practical strategy. After all, p16 INK4a IHC can easily be combined with standard histology when a H&E-stained tissue section is prepared for examination by a pathologist. It is a routine diagnostic procedure, at a relatively low cost, available as a validated in-vitro diagnostic reagent and a fully automated protocol, which generates results within several hours after the procedure has been requested. HPV DNA PCR is also a standard laboratory procedure with high throughput and quick results, although against a higher cost. Preselection by p16 INK4a -staining therefore reduces the workload and associated costs, and the combination of HPV DNA and p16 INK4a testing leads to an important reduction of the number of false-positive observations versus the use of either of these assays alone. Conclusions In conclusion, combined testing for hrHPV DNA and p16 INK4a enhances specificity up to 97%, while maintaining high sensitivity (96%), compared to single testing. The diagnostic test combination represents an accurate, accessible and high-throughput testing strategy for the diagnosis of HPV-induced OPC in the clinical setting and for disease prognostication. Treatment adjustments can be encouraged based on this combined test result. Abbreviations CI Confidence Interval FFPE Formalin-Fixed, Paraffin-Embedded H&E Hematoxylin and Eosin HNC Head and Neck Cancers HPV Human Papillomavirus hrHPV high-risk Human Papillomavirus IHC Immunohistochemistry OPC Oropharyngeal Cancer p16 p16 INK4a PCR Polymerase Chain Reaction RNA+ mRNA-positive S Section ubC ubiquitin C Declarations Ethics approval and consent to participate Ethical clearance was obtained from the Ethical Committees of ‘GZA hospitals’ (ref nb: BVDE/hp/2013/01.147), ‘UZA & UA (University of Antwerp)’ (ref nb: 11/47/362) and IARC, Lyon, France (ref nb: 11-30). Informed consent of the individual patient was not necessary, as it concerns biobank-based research. The ethical principles, detailed by a European network specialized in biobanking (Hansson MG, Methods Mol.Biol 2011; 675:39-59), are respected. The need of informed consent was waived by the Ethical Committees of ‘GZA hospitals’ and ‘UZA & UA (University of Antwerp)’ for the Belgian patients. Consent for publication Not applicable Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Competing interests RR and SR are employees of Roche. All other authors declare that they have no competing interests. Funding The global HPV-AHEAD study was initially funded by the European Commission, with grant number: FP7-HEALTH-2011-282562. Sciensano, the employer of CS and MA, received funding for the analysis of the Belgian part of the HPV-AHEAD study, in part, by a research grant from the Investigator Initiated Studies Program of Merck Sharp & Dohme Corp. The opinions expressed in this paper are those of the authors and do not necessarily represent those of Merck Sharp & Dohme Corp. MA was also supported by the Horizon 2020 Framework Programme for Research and Innovation of the European Commission, through the RISCC Network (grant no. 847845); and the Belgian Foundation Against Cancer through the IHUVACC project. Authors' contributions CS, MA and JB designed the Belgian part of the HPV-AHEAD study; CS and MA performed the statistical analysis and interpreted the findings; CS also compiled the database, managed the Belgian part of the project and wrote the manuscript. IG collected all patient samples and the corresponding clinical information; PV, ML and OMV facilitated and supervised the specimen and clinical data collection. TG, DH, SR, AC, DA, and PB performed the respective DNA, RNA or p16 tests, and interpreted the data; they were supervised by MT, MP, RR, and MRP. RVK, NG, FM, BMLR, and JB were responsible for the histological review of all specimens. EL managed the overall HPV-AHEAD database, and validated the data entry. MT, RS, MP, MA, JB, SC, and RR were the PI’s of the HPV-AHEAD study and therefore responsible for the largest funding acquisition. TG, RR, IG, EL, FM, SC, JB, MT, and MA reviewed and/or edited the manuscript. All co-authors approved the final manuscript and its submission to this journal. Acknowledgements We are very grateful for the excellent work performed by Sofie Thys (University of Antwerp, Laboratory of cell biology and histology), for her meticulous work in preparing FFPE slide sections and logistical coordination and support of the study. Where authors are identified as personnel of the International Agency for Research on Cancer/World Health Organization, the authors alone are responsible for the views expressed in this article and they do not necessarily represent the decisions, policy, or views of the International Agency for Research on Cancer/World Health Organization. The HPV-AHEAD study group: Christel Herold-Mende (Dpt. of Neurosurgery and Dpt. of Otorhinolaryngology, Head and Neck Surgery, University of Heidelberg, Germany); Gerhard Dyckhoff (Dpt. of Otorhinolaryngology, Head and Neck Surgery, University of Heidelberg, Germany); George Mosialos (Aristotle University of Thessaloniki, Greece); Fausto Chiesa, Marta Tagliabue, Mohssen Ansarin (European Institute of Oncology, Milan, Italy); Heiner Boeing (German Institute of Human Nutrition, Berlin, Germany); Xavier Castellsagué†, Silvia de Sanjosé, Marisa Mena, Francesc Xavier Bosch, Laia Alemany (Catalan Institute of OncologyIDIBELL, L'Hospitalet de Llobregat, Spain); Pulikottil Okkuru Esmy (Christian Fellowship Community Health Centre, Ambillikai, India); Rudrapatna S. Jayshree, Kortikere S. Sabitha, Ashok M. Shenoy (Kidwai Memorial Institute of Oncology, Bangalore, India); Manavalan Vijayakumar (YEN ONCO Centre, Yenepoya University, Deralakatte, Mangalore 575018, Karnataka, India); Aruna S. Chiwate, Ranjit V. Thorat, Girish G. Hublikar, Shashikant S. Lakshetti, Bhagwan M. Nene (Nargis Dutt Memorial Cancer Hospital, Barshi 413401, India); Amal Ch Kataki, Ashok Kumar Das (Dr B. Borooah Cancer Institute, Guwahati, Assam, India); Subha Sankaran (Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram, India); Kunnambath Ramadas (Regional Cancer Centre, Thiruvananthapuram, India); Christine Carreira, Sandrine McKay-Chopin (International Agency for Research on Cancer (IARC), Lyon, France), Thara Somanathan (Regional Cancer Centre, Thiruvananthapuram, 695011, India), Alfredo Zito (Istituto Tumori "Giovanni Paolo II", Bari). †Dr Castellsagué passed away on June 12th, 2016. References 1. Gillison ML, Castellsague X, Chaturvedi A, Goodman MT, Snijders P, Tommasino M, et al. Comparative epidemiology of HPV infection and associated cancers of the head and neck and cervix. Int J Cancer. 2014;134(3):497-507. 2. Arbyn M, de Sanjose S, Saraiya M, Sideri M, Palefsky JM, Lacey C, et al. 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Detection and significance of human papillomavirus, CDKN2A(p16) and CDKN1A(p21) expression in squamous cell carcinoma of the larynx. Mod Pathol. 2013;26(2):223-31. 16. Ndiaye C, Mena M, Alemany L, Arbyn M, Castellsague X, Laporte L, et al. HPV DNA, E6/E7 mRNA, and p16INK4a detection in head and neck cancers: a systematic review and meta-analysis. Lancet Oncol 2014;15(12):1319-31. 17. Gillison ML, Koch WM, Capone RB, Spafford M, Westra WH, Wu L, et al. Evidence for a causal association between human papillomavirus and a subset of head and neck cancers. J Natl Cancer Inst 2000;92(9):709-20. 18. Kreimer AR, Clifford GM, Boyle P, Franceschi S. Human papillomavirus types in head and neck squamous cell carcinomas worldwide: a systematic review. Cancer Epidemiol Biomarkers Prev. 2005;14(2):467-75. 19. Ha PK, Pai SI, Westra WH, Gillison ML, Tong BC, Sidransky D, et al. Real-time quantitative PCR demonstrates low prevalence of human papillomavirus type 16 in premalignant and malignant lesions of the oral cavity. Clin Cancer Res. 2002;8(5):1203-9. 20. Anantharaman D, Gheit T, Waterboer T, Abedi-Ardekani B, Carreira C, McKay-Chopin S, et al. Human papillomavirus infections and upper aero-digestive tract cancers: the ARCAGE study. J Natl Cancer Inst 2013;105(8):536-45. 21. Jordan RC, Lingen MW, Perez-Ordonez B, He X, Pickard R, Koluder M, et al. Validation of Methods for Oropharyngeal Cancer HPV Status Determination in US Cooperative Group Trials. Am J Surg Pathol 2012;36(7):945-54. 22. Smeets SJ, Hesselink AT, Speel EJ, Haesevoets A, Snijders PJ, Pawlita M, et al. A novel algorithm for reliable detection of human papillomavirus in paraffin embedded head and neck cancer specimen. Int J Cancer. 2007;121(11):2465-72. 23. Rietbergen MM, Snijders PJ, Beekzada D, Braakhuis BJ, Brink A, Heideman DA, et al. 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Bergeron C, Ronco G, Reuschenbach M, Wentzensen N, Arbyn M, Stoler M, et al. The clinical impact of using p16(INK4a) immunochemistry in cervical histopathology and cytology: an update of recent developments. Int J Cancer. 2015;136(12):2741-51. 29. Roelens J, Reuschenbach M, von Knebel-Doeberitz M, Wentzensen N, Bergeron C, Arbyn M. p16INK4a immunocytochemistry versus HPV testing for triage of women with minor cytological abnormalities: A systematic review and meta-analysis. Cancer. 2012;120(5):294-307. 30. Klussmann JP, Gultekin E, Weissenborn SJ, Wieland U, Dries V, Dienes HP, et al. Expression of p16 protein identifies a distinct entity of tonsillar carcinomas associated with human papillomavirus. Am J Pathol 2003;162(3):747-53. 31. Prigge ES, Toth C, Dyckhoff G, Wagner S, Müller F, Wittekindt C, et al. p16(INK4a) /Ki-67 co-expression specifically identifies transformed cells in the head and neck region. Int J Cancer. 2015;136(7):1589-99. 32. 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Halec G, Schmitt M, Dondog B, Sharkhuu E, Wentzensen N, Gheit T, et al. Biological activity of probable/possible high-risk human papillomavirus types in cervical cancer. Int J Cancer. 2013;132(1):63-71. 37. Gheit T, Landi S, Gemignani F, Snijders PJ, Vaccarella S, Francheschi S, et al. Development of a Sensitive and Specific Assay Combining Multiplex PCR and DNA Microarray Primer Extension To Detect High-Risk Mucosal Human Papillomavirus Types. J Clin Microbiol 2006;44(6):2025-31. 38. Schmitt M, Bolormaa D, Waterboer T, Pawlita M, Tommasino M, Gheit T. Abundance of Multiple High-Risk Human Papillomavirus (HPV) Infections Found in Cervical Cells Analyzed by Use of an Ultrasensitive HPV Genotyping Assay. J Clin Microbiol 2010;48(1):143-9. 39. Tagliabue M, Mena M, Maffini F, Gheit T, Quiros Blasco B, Holzinger D, et al. Role of Human Papillomavirus Infection in Head and Neck Cancer in Italy: The HPV-AHEAD Study. Cancers (Basel). 2020;12(12). 40. Johansson H, Bjelkenkrantz K, Darlin L, Dilllner J, Forslund O. Presence of High-Risk HPV mRNA in Relation to Future High-Grade Lesions among High-Risk HPV DNA Positive Women with Minor Cytological Abnormalities. PloS one. 2015;10(4):e0124460-/. 41. Victor T, Jordaan A, du Toit R, Van Helden PD. Laboratory experience and guidelines for avoiding false positive polymerase chain reaction results. Eur J Clin Chem Clin Biochem. 1993;31(8):531-5. 42. de Sanjose S, Alemany L, Ordi J, Tous S, Alejo M, Bigby SM, et al. Worldwide human papillomavirus genotype attribution in over 2000 cases of intraepithelial and invasive lesions of the vulva. Eur J Cancer. 2013;49(16):3450-61. 43. Orosco RK, Califano JA. HPV status, like politics, is local-evaluating p16 staining and a new staging system in a Dutch cohort of oropharynx cancer. Ann Oncol. 2018;29(5):1089-90. 44. Wagner S, Prigge ES, Wuerdemann N, Reder H, Bushnak A, Sharma SJ, et al. Evaluation of p16(INK4a) expression as a single marker to select patients with HPV-driven oropharyngeal cancers for treatment de-escalation. Br J Cancer. 2020;123(7):1114-22. 45. Prigge ES, Arbyn M, von Knebel Doeberitz M, Reuschenbach M. Diagnostic accuracy of p16(INK4a) immunohistochemistry in oropharyngeal squamous cell carcinomas: A systematic review and meta-analysis. Int J Cancer. 2017;140(5):1186-98. 46. Albers AE, Qian X, Kaufmann AM, Coordes A. Meta analysis: HPV and p16 pattern determines survival in patients with HNSCC and identifies potential new biologic subtype. Sci Rep. 2017;7(1):16715. Tables Table 1: Prevalence of HPV determined by HPV DNA PCR (DNA), p16 INK4a IHC (p16), E6*I mRNA (RNA), and all combinations of tests, in oropharyngeal cancer Oropharyngeal cancer Marker positivity Positive Proportion (95% CI) DNA (N=99) 36° 36.4% (27.4-46.4) p16 *(N=97) 33 34.0% (25.2-44.1) RNA £ (N=97) 28 28.9% (20.6-38.8) p16+DNA (N=97) 30 30.9% (22.5-40.9) DNA+RNA (N=97) 28 28.9% (20.6-38.8) RNA+p16 (N=95) 27 28.4% (20.2-38.4) p16+DNA+RNA (N=95) 27 28.4% (20.2-38.4) ° 1 lrHPV (HPV6) and 35 hrHPV * 2 equivocal results after final re-review £ 2 samples gave an invalid RNA test result Table 2: hrHPV DNA, p16 INK4a (p16) and combined hrHPV DNA/p16 INK4a test results by hrHPV RNA status in cancers of the oropharynx, tonsils and base of the tongue hrHPV RNA Pos Neg N N Total hrHPV DNA Oropharynx Pos 28 5 33 Neg 0 62 62 Total 28 67 95 Tonsil Pos 19 3 22 Neg 0 20 20 Total 19 23 42 Base of tongue Pos 7 0 7 Neg 0 14 14 Total 7 14 21 p16 Oropharynx Pos 27 5 32 Neg 1 62 63 Total 28 67 95 Tonsil Pos 18 2 20 Neg 1 21 22 Total 19 23 42 Base of tongue Pos 7 2 9 Neg 0 12 12 Total 7 14 21 p16+DNA Oropharynx Pos 27 2 29 Neg 1 65 66 Total 28 67 95 Tonsil Pos 18 2 20 Neg 1 21 22 Total 19 23 42 Base of tongue Pos 7 0 7 Neg 0 14 14 Total 7 14 21 p16->DNA Oropharynx Pos 27 2 29 Neg 1 65 66 Total 28 67 95 Tonsil Pos 18 2 20 Neg 1 21 22 Total 19 23 42 Base of tongue Pos 7 0 7 Neg 0 14 14 Total 7 14 21 DNA->p16 Oropharynx Pos 27 2 29 Neg 1 65 66 Total 28 67 95 Tonsil Pos 18 2 20 Neg 1 21 22 Total 19 23 42 Base of tongue Pos 7 0 7 Neg 0 14 14 Total 7 14 21 Table 3a: Absolute sensitivity and specificity of hrHPV DNA PCR (hrHPV DNA), p16 INK4a IHC (p16) and the combined testing (p16+DNA) to identify a HPV-driven oropharyngeal cancer Absolute Sensitivity Specificity hrHPV DNA n/N % (95% CI) 28/28 100% (87.7-100) 62/67 92.5% (83.4-97.5) p16 n/N % (95% CI) 27/28 96.4% (81.7-99.9) 62/67 92.5% (83.4-97.5) p16+DNA n/N % (95% CI) 27/28 96.4% (81.7-99.9) 65/67 97.0% (89.9-99.6) Table 3b: Relative sensitivity and specificity of hrHPV DNA PCR (DNA) vs p16 INK4a IHC (p16), and of DNA and p16 vs combined testing (p16+DNA), to identify a HPV-driven oropharyngeal cancer Relative Sensitivity Specificity hrHPV DNA vs p16 1.04 1.00 (95% CI) (0.97-1.11) (1.00-1.00) p16+DNA vs p16 1.00 1.05 (95% CI) (1.00-1.00) (0.87-1.26) p16+DNA vs hrHPV DNA 0.96 1.05 (95% CI) (0.90-1.04) (0.87-1.26) Additional Declarations Competing interest reported. RR and SR are employees of Roche. All other authors declare that they have no competing interests. Supplementary Files suppltableBMCInfDis.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Major revision 14 Dec, 2021 Reviews received at journal 25 Nov, 2021 Reviewers agreed at journal 22 Nov, 2021 Reviewers invited by journal 18 Nov, 2021 Editor assigned by journal 18 Nov, 2021 Editor invited by journal 18 Nov, 2021 Submission checks completed at journal 18 Nov, 2021 First submitted to journal 22 Oct, 2021 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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RR and SR are employees of Roche. All other authors declare that they have no competing interests.","formattedTitle":"\u003cp\u003eAccuracy of High-risk HPV DNA PCR, p16INK4a Immunohistochemistry or the Combination of Both to Diagnose HPV-driven Oropharyngeal Cancer\u003c/p\u003e","fulltext":[{"header":"Background","content":"\u003cp\u003eIn high-resource countries, human papillomavirus (HPV)-driven head and neck squamous cell carcinoma represents an increasing health problem, particularly in cancers of the oropharyngeal region, including base of tongue and tonsils (\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). An estimated 20-40% of oropharyngeal cancers (OPC) is believed to be caused by HPV infection, and the large majority of them (\u0026gt;80%) are due to HPV16 (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). Patients with HPV-induced cancers respond better to treatment and consequently have better survival than patients with HPV-unrelated OPC (\u003cspan additionalcitationids=\"CR7 CR8 CR9 CR10\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). Therefore, therapy may be de-escalated with reduced toxicity and less sequelae. However, treatment de-escalation may negatively impact patient survival if HPV was not the carcinogenic driver of the respective tumour. These considerations highlight the importance of reliable and accurate markers, or marker combinations, to diagnose truly HPV-induced OPC.\u003c/p\u003e \u003cp\u003eThe most widely applied detection methods thus far are based on PCR amplification of viral DNA to determine HPV-positivity. However, several independent studies have highlighted that PCR-based assays for the detection of HPV DNA are not sufficiently accurate to establish the viral causality (\u003cspan additionalcitationids=\"CR13 CR14 CR15\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). These PCR-based methods are highly sensitive and can detect even a few DNA copies per sample, which might yield false-positive results mainly reflecting transient infections (\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). Additional markers, such as the presence of viral E6/E7 mRNA transcripts and p16\u003csup\u003eINK4a\u003c/sup\u003e expression as surrogates for HPV-induced transformation, allow a more accurate classification of HPV-driven head and neck cancers (HNC) (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan additionalcitationids=\"CR21 CR22 CR23\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe detection of HPV E6/E7 oncogene transcripts is currently regarded as gold standard in this context, as HPV-driven carcinomas critically depend on the carcinogenic action of the HPV E6 and E7 oncoproteins (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). However, the detection of viral transcripts is laborious and may not be routinely feasible in all laboratories. This is particularly true for mRNA transcript detection from formalin-fixed, paraffin-embedded (FFPE) tissue specimens, which are frequently used during routine diagnostic work up; however, RNA integrity may be affected by the fixation protocol.\u003c/p\u003e \u003cp\u003eHPV E7 oncogenic signaling induces strong overexpression of the cellular protein p16\u003csup\u003eINK4a\u003c/sup\u003e in HPV-transformed cells (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). The immunohistochemical detection of p16\u003csup\u003eINK4a\u003c/sup\u003e overexpression is now used as a surrogate biomarker of HPV-transformed epithelium of the uterine cervix (\u003cspan additionalcitationids=\"CR28\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). Similarly, p16\u003csup\u003eINK4a\u003c/sup\u003e immunohistochemistry (IHC) is frequently applied to determine HPV association in HNC (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan additionalcitationids=\"CR31\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). Apart from single p16\u003csup\u003eINK4a\u003c/sup\u003e IHC, combined detection of p16\u003csup\u003eINK4a\u003c/sup\u003e IHC and HPV DNA detection by PCR is frequently used.\u003c/p\u003e \u003cdiv id=\"Sec2\" class=\"Section2\"\u003e \u003ch2\u003eHPV-AHEAD (FP7 funded network)\u003c/h2\u003e \u003cp\u003eThe HPV-AHEAD study (\u0026ldquo;Role of human papillomavirus infection and other co-factors in the aetiology of head and neck cancer in Europe and India\u0026rdquo;) group comprised partners from six European countries and from India (website HPV-AHEAD: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://hpv-ahead.iarc.fr/\u003c/span\u003e\u003c/span\u003e). The main goal of the study was to perform a comprehensive analysis on a large number of HNC cases to clarify pathogenic pathways in HNC carcinogenesis, and to identify clinically useful biomarkers.\u003c/p\u003e \u003cp\u003eIn a previous publication (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e), the results of the histological and molecular assessment of 1039 archived HNC specimens from Belgian patients were described, primarily using the detection of HPV DNA, mRNA, and p16\u003csup\u003eINK4a\u003c/sup\u003e IHC. In this study, we focus on the accuracy of these tests - individually and in combination - to diagnose hrHPV-driven oropharyngeal cancer.\u003c/p\u003e \u003c/div\u003e"},{"header":"Methods","content":"\u003cp\u003e All methods were carried out in accordance with relevant guidelines and regulations.\u003c/p\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003ePatient selection, clinical information and tissue specimen collection for the Belgian cohort\u003c/h2\u003e \u003cp\u003eThe overall study included patients with oropharyngeal, oral cavity, laryngeal, hypopharyngeal, and unspecified head and neck squamous cell carcinoma. For a detailed description of the patient characteristics, we refer to the previous publication of the Belgian HPV-AHEAD study data (Simoens, 2021, Cancer Epidemiology). For the analyses in this study, the focus was on the oropharyngeal cancers (ICD-O-3 codes: C01 (base of tongue); C02.4 (lingual tonsil); C05.1 (soft palate); C05.2 (uvula); C09 (tonsil): C09.0, C09.1, C09.8 \u0026amp; C09.9; C10 (oropharynx): C10.0-10.4, C10.8 \u0026amp; C10.9).\u003c/p\u003e \u003cp\u003eFFPE tumour blocks and clinical information were collected from OPC patients treated in two Belgian hospitals (GZA and UZA), diagnosed between 1980 and 2010.\u003c/p\u003e \u003cp\u003eEthical clearance was obtained from the Ethical Committees of \u0026lsquo;GZA hospitals\u0026rsquo; (ref nb: BVDE/hp/2013/01.147), \u0026lsquo;UZA \u0026amp; UA (University of Antwerp)\u0026rsquo; (ref nb: 11/47/362) and IARC, Lyon, France (ref nb: 11-30).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003ePreparation of tissue sections (University of Antwerp, Antwerp, Belgium)\u003c/h2\u003e \u003cp\u003eFFPE-tissue blocks were all processed at the UA laboratory of cell biology and histology, following the optimized HPV-AHEAD sectioning protocol (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e). Briefly, minimal ten sections (S) were prepared from each FFPE block. The first (S1) and the last (S10) 5\u0026micro;m sections were hematoxylin and eosin (H\u0026amp;E) stained for morphologic histology interpretation and used to check for the presence of tumour. S2 and S9 (5\u0026micro;m) were used for p16\u003csup\u003eINK4a\u003c/sup\u003e IHC staining, while the 10\u0026micro;m sections S3-5 and S6-8 were used for the extraction of RNA and DNA, respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eHistological review\u003c/h2\u003e \u003cp\u003eAll sections were re-evaluated by the HPV-AHEAD pathology review panel. The review was blinded with respect to the original local diagnosis. Only FFPE blocks where S1 and S10 H\u0026amp;E sections reflected squamous tumour tissue were included in the analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eHPV E6*I mRNA analysis (DKFZ, Heidelberg, Germany)\u003c/h2\u003e \u003cp\u003eAll OPC cases were analysed for the presence of: (i) HPV16 E6*I mRNA, and (ii) ubiquitin C (ubC) mRNA as a cellular mRNA positive control (housekeeping gene used for RNA quality control). OPC cases positive for DNA of a non-HPV16 genotype were additionally analysed for E6*I mRNA of the respective genotype. Specimens that were HPV E6*I and/or ubC mRNA-positive (RNA+) were considered RNA valid. Reverse Transcription-PCR was carried out using the QuantiTect Virus Kit (Qiagen, Hilden, Germany), as described in full in former HPV-AHEAD publications (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e). The type-specific E6*I mRNA assays identifying transcripts of fourteen high-risk and six possible/probable high-risk HPV genotypes (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e) were applied.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eHPV DNA genotyping (IARC, Lyon, France)\u003c/h2\u003e \u003cp\u003eHPV DNA genotypes were detected by a E7 type-specific multiplex genotyping (E7-MPG) assay, which combines multiplex PCR and bead-based Luminex technology (Luminex Corporation, Austin, TX), as previously described (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). Type specific-MPG uses HPV type-specific primers targeting the E7 region of thirteen high-risk and six possible/probable high-risk HPV genotypes (HPV 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68a and b, 70, 73 and 82), as well as two low-risk HPV genotypes (HPV 6 and 11). Two primers for amplification of the beta-globin (β-globin) gene were also included to control for the DNA quality of each specimen. A detailed description of the DNA extraction and further HPV genotyping test characteristics can be found in the previous publication on the Belgian HPV-AHEAD study (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). Genotyping controls and DNA preparation were blindly analysed, and no sign of contamination of negative controls was detected during the laboratory work.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003ep16\u003csup\u003eINK4a\u003c/sup\u003e expression (Roche mtm laboratories, Mannheim, Germany)\u003c/h2\u003e \u003cp\u003eExpression of p16\u003csup\u003eINK4a\u003c/sup\u003e was evaluated by IHC, with a dual-immunostaining protocol for the simultaneous immunostaining of both p16\u003csup\u003eINK4a\u003c/sup\u003e and Ki-67 biomarkers (CINtec PLUS kit, Roche mtm laboratories AG, Mannheim, Germany), as previously described in the HPV-AHEAD publications (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e). As in all HPV-AHEAD studies (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e), a continuous, diffuse staining for p16\u003csup\u003eINK4a\u003c/sup\u003e within the cancer area of the tissue sections was considered as positive, while a focal staining or no staining was considered negative. IHC slides were analysed without knowledge of any other clinical information (including HPV DNA and RNA status) by the scientists RR or DH and reviewed by one of the European members of the HPV-AHEAD pathology review panel (JPB, BLR, or FM). Discrepant cases were re-evaluated by a pathologist outside the review panel (AC), and final classification of the staining was based on majority consensus results.\u003c/p\u003e \u003cp\u003eCorresponding to the former Belgian HPV-AHEAD publication (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e), the p16 slides with technical issues were restained and re-evaluated, to minimize the number of missing results.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003ePresence of viral mRNA was considered as the reference indicating HPV etiology. The accuracy of three possible test strategies was analysed: a) hrHPV DNA PCR alone, b) p16\u003csup\u003eINK4a\u003c/sup\u003e IHC alone, and c) the combination of p16\u003csup\u003eINK4a\u003c/sup\u003e IHC and hrHPV DNA PCR; where positivity is defined by a co-positive result with both tests, a negative result by a co-negative result for both tests, and a discordant result by only one test being positive and the other being negative. Test strategy (c) involves three algorithms: ALGORITHM 1 (p16+DNA) consists of p16\u003csup\u003eINK4a\u003c/sup\u003e IHC and hrHPV DNA PCR on all samples; ALGORITHM 2 (p16-\u0026gt;DNA) involves p16\u003csup\u003eINK4a\u003c/sup\u003e staining of all samples, followed by the HPV DNA test only on the p16\u003csup\u003eINK4a\u003c/sup\u003e-positive samples; ALGORITHM 3 (DNA-\u0026gt;p16) involves HPV DNA PCR on all samples, followed by p16\u003csup\u003eINK4a\u003c/sup\u003e staining only on the HPV DNA-positive samples.\u003c/p\u003e \u003cp\u003eSensitivity was defined as the proportion of test positive samples among HPV RNA-positive patients. Specificity was defined as the proportion of negative test results among HPV RNA-negative patients. Relative sensitivity and specificity of each test compared to the other tests were also assessed. Binomial 95% confidence intervals (95% CIs) were computed for proportions. Given the matched testing of the same patient specimens, McNemar 95% CIs were computed for relative accuracy parameters. Continuous variables were summarized by their mean and 95% CIs. ANOVA was used to compare mean age by gender.\u003c/p\u003e \u003cp\u003eStatistical analyses were performed using STATA 16 (StataCorp, College Station, TX). p-values were two sided and statistical significance was set at p equal or less than 0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eStudy population characteristics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNinety-nine of 116 OPC patients were included in the analysis, after exclusions based on the pathology review (for reasons of not being a squamous cell carcinoma or not reflecting invasive cancer, N=13) and beta-globin PCR negativity (N=4). 71.7% (71/99) of the patients were male, 26.3% (26/99) were female, and for the remaining two samples the gender of the patients was not specified. The mean age at diagnosis was 67.3 years (95%CI: 64.9-69.7) and was not different between males and females (p=0.47).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHPV prevalence in OPC cases\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHPV prevalence was determined by HPV DNA PCR, p16\u003csup\u003eINK4a\u003c/sup\u003e IHC, E6*I mRNA, and all combinations of tests in the HPV-AHEAD study. Out of 99 OPC cases, 36.4% (36/99) contained HPV DNA, 34.0% (33/97) showed p16\u003csup\u003eINK4a\u003c/sup\u003e positivity, and 28.9% (28/97) were HPV RNA-positive (Table 1). Twenty-six of the 28 HPV RNA-positive samples were positive for HPV16 (92.9%), the remaining 2 samples contained HPV18 (7.1%).\u003c/p\u003e\n\u003cp\u003eThe proportion of HPV-positive OPCs varied between 28.4% (27/95) and 28.9% (28/97) for all combinations of tests that included RNA testing (DNA+RNA, p16+RNA, DNA+p16+RNA). For the combination of testing for HPV DNA and p16\u003csup\u003eINK4a\u003c/sup\u003e IHC, the overall prevalence was slightly higher (30.9%, 30/97).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAbsolute and relative accuracy in the oropharynx and its subsites\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBoth absolute and relative accuracy of hrHPV DNA detection by PCR (hrHPV DNA), p16\u003csup\u003eINK4a\u003c/sup\u003e IHC (p16), and combined p16 and DNA testing algorithms (p16+DNA, p16-\u0026gt;DNA and DNA-\u0026gt;p16), to identify a transforming HPV infection were calculated. In the following tables, only the results are shown for the samples that had valid results on all three tests performed (N=95).\u003c/p\u003e\n\u003cp\u003eAll RNA-positive OPC cases were positive for hrHPV DNA (100%, 28/28), in contrast to one out of 28 HPV RNA-positive samples that did not have a positive p16\u003csup\u003eINK4a\u003c/sup\u003e result (3.6%). Five out of 67 RNA-negative cases showed hrHPV DNA-positivity (7.5%), the same as observed for p16\u003csup\u003eINK4a\u003c/sup\u003e IHC. This number would be further lowered to only 2 positive cases out of 67 RNA-negatives (3.0%), if hrHPV DNA and p16\u003csup\u003eINK4a\u003c/sup\u003e testing (p16+DNA) were combined. The same holds for the other algorithms where all samples would initially be tested on p16\u003csup\u003eINK4a\u003c/sup\u003e (p16-\u0026gt;DNA) or hrHPV DNA (DNA-\u0026gt;p16), followed by the other test if the initial test result turned out positive. Data are detailed in Table 2, for all OPC cases, and for the tonsils and the base of tongue in specific.\u003c/p\u003e\n\u003cp\u003eThe accuracy of hrHPV DNA PCR and p16\u003csup\u003eINK4a\u003c/sup\u003e IHC to detect a transforming HPV infection in OPCs was calculated (Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ea). Highest sensitivity (100.0%, 28/28) was noted for hrHPV DNA testing, while specificity was equal for both hrHPV DNA and p16\u003csup\u003eINK4a\u003c/sup\u003e (92.5%, 62/67). The best compromise was found in the combined testing of hrHPV DNA PCR and p16\u003csup\u003eINK4a\u003c/sup\u003e staining, with 96.4% (27/28) sensitivity and 97.0% (65/67) specificity. When looking in more detail to the proposed algorithms, all provided equal accuracy, however, with a 60% reduction in the number of HPV DNA PCR or p16\u003csup\u003eINK4a\u003c/sup\u003e IHC tests needed in the sequential testing algorithms.\u003c/p\u003e\n\n\u003cp\u003eFor the subgroup of the tonsils, p16\u003csup\u003eINK4a\u003c/sup\u003e staining alone was as sensitive (94.7%, 18/19) and specific (91.3%, 21/23) as the combined testing, in contrast to the base of tongue group, where all tests were 100% sensitive and specific, except for the p16\u003csup\u003eINK4a\u003c/sup\u003e specificity (85.7%, 12/14).\u003c/p\u003e\n\u003cp\u003ehrHPV DNA PCR testing was slightly more sensitive compared to p16\u003csup\u003eINK4a\u003c/sup\u003e IHC (ratio: 1.04, 95% CI: 0.97-1.11), but equally specific (ratio: 1.00, 95% CI not computable) (see Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eb and the Supplementary Table). The combination of hrHPV DNA and p16\u003csup\u003eINK4a\u003c/sup\u003e was slightly more specific (ratio: 1.05, 95% CI: 0.97-1.21) compared to both single tests.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn these Belgian OPC patients, PCR testing for HPV DNA showed the highest proportion of positive cases (36%). Due to its high sensitivity, HPV DNA PCR-based assays can detect low viral copy numbers, which may not trigger carcinogenesis. These assays cannot distinguish between transcriptionally-active and passenger HPV infections. Therefore, other assays (HPV E6*I mRNA, and p16\u003csup\u003eINK4a\u003c/sup\u003e staining) were evaluated to assess whether combined testing could improve the specificity. Only 28% of the samples with a valid HPV E6*I mRNA result were positive for HPV DNA and p16\u003csup\u003eINK4a\u003c/sup\u003e, which might be the fraction of oropharyngeal cancers caused by HPV. After all, the detection of HPV E6 and/or E7 mRNA is seen as the gold standard in this context, because HPV-driven carcinomas critically depend on the continuous expression of E6/E7 oncogenes of hrHPVs (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e). However, the detection of viral transcripts is challenging, not only because the RNA extraction step is laborious and time consuming, but specific infrastructures and equipment are needed. Therefore, HPV RNA assays may not be routinely feasible in all laboratories or on all tissue samples.\u003c/p\u003e \u003cp\u003eHPV-driven OPC represents an increasing health problem, and therefore, reliable and accurate diagnosis becomes essential. In our Belgian HPV-AHEAD study, HPV DNA PCR testing alone was 100% sensitive, but less specific (92.5%) compared to mRNA testing. The inherent strength of the PCR-based methodology lies in its capacity to detect very small amounts of HPV DNA. At the same time, strict laboratory procedures and controls are critical in reducing contamination-related false-positive findings (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e). Immunohistochemistry for p16\u003csup\u003eink4a\u003c/sup\u003e is most widely used as a surrogate marker for hrHPV infection in FFPE tissues, also for oropharyngeal squamous cell carcinoma. p16 IHC testing alone was a bit less sensitive (96.4%), but co-presence of hrHPV DNA and p16\u003csup\u003eINK4a\u003c/sup\u003e positivity was similarly sensitive (96.4%) and more specific (97.0%) compared to each test separately. In literature, several authors have advocated against the use of either p16\u003csup\u003eINK4a\u003c/sup\u003e or hrHPV DNA alone as indicators of HPV-induced etiology in cancers, but recommended their combined use as a reliable and practical approach to differentiate HPV-induced from HPV-unrelated tumours (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan additionalcitationids=\"CR43\" citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e). The meta-analysis performed by Prigge et al. (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e) also showed a similarly high (pooled) sensitivity of the combined testing (93%) and either p16 (94%) or HPV DNA (98%) alone, and the (pooled) specificity (96%) was significantly higher than either testing method alone (83% and 84%, respectively). These accuracy data were confirmed by these Belgian results.\u003c/p\u003e \u003cp\u003eRelative sensitivities and specificities were not significantly different from unity in our study, nor in the meta-analysis of Prigge et al. (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e), where significance was only reached for the relative specificity of the combination of HPV DNA PCR and p16\u003csup\u003e\u003cem\u003eINK4a\u003c/em\u003e\u003c/sup\u003e tests versus p16\u003csup\u003e\u003cem\u003eINK4a\u003c/em\u003e\u003c/sup\u003e IHC (rel. spec.: 1.13 (95% CI: 1.04-1.23)), by pooling from multiple studies.\u003c/p\u003e \u003cp\u003eFurthermore, better clinical outcomes have been reported for patients with HPV-induced compared to HPV-unrelated OPC (\u003cspan additionalcitationids=\"CR7 CR8 CR9 CR10\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). Given the ongoing discussions about the value of therapy de-escalation in patients with HPV-induced OPC, the reliable identification of viral etiology in the tumour and its impact on patient prognosis gain particular momentum. Improved prognostication by combined p16\u003csup\u003eINK4a\u003c/sup\u003e and hrHPV DNA detection compared to single marker analysis has been demonstrated in a large meta-analysis on tumours in the head and neck region (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e). In our study, 97% (92/95) of the patients would have been correctly diagnosed with the combined testing approach. However, one 58-year old male (current smoker and alcohol consumer) with a T1 (N0 M0) microinvasive tonsillar tumour might have been treated too aggressively by applying this combined test strategy, as his tumour would have been classified as a HPV-unrelated case, being p16 negative while actually both DNA and mRNA HPV16 positive. More than six years after diagnosis, the patient was alive without any evidence of disease after having had radical wide excision surgery.\u003c/p\u003e \u003cp\u003eOn the other hand, treatment de-escalation may negatively impact disease-specific patient survival rates if HPV in the tumour was not the cause of carcinogenesis. In our Belgian OPC patients, the number of false positive cancers would be reduced from five to two with the combined p16+DNA detection, who may otherwise receive a less aggressive treatment regimen. These two HPV-unrelated cancer patients were male and diagnosed with a tonsillar cancer. The first patient, with a Tx N2 M1 tonsil NOS tumour, was a heavy drinker and current smoker who only underwent surgery and had a recurrence within six months. He died two years after being diagnosed. The other patient, with a T3 N2 tonsil NOS tumour, received surgery followed by radiotherapy and concurrent chemotherapy for two months. At the last follow-up date, this patient was alive, but with evidence of residual disease. Of note, these two HPV-unrelated patients clearly had a much worse disease status at diagnosis and thereby reduced survival chances compared to the above described HPV-driven tonsillar cancer patient.\u003c/p\u003e \u003cp\u003eNinety-seven percent of the OPC patients in our study would have been correctly diagnosed as patients with a HPV-driven cancer by combined p16\u003csup\u003eINK4a\u003c/sup\u003e and hrHPV DNA (p16+DNA) detection. Sequential testing algorithms (p16-\u0026gt;DNA and DNA-\u0026gt;p16) resulted in equally accurate results, however, with a 60% reduction in the number of tests needed to be performed. This will cause a substantial reduction in costs and laboratory time, while providing the same clinical value. Especially the algorithm of p16\u003csup\u003eINK4a\u003c/sup\u003e on all samples followed by HPV DNA PCR on p16\u003csup\u003eINK4a\u003c/sup\u003e-positive samples only would be a practical strategy. After all, p16\u003csup\u003eINK4a\u003c/sup\u003e IHC can easily be combined with standard histology when a H\u0026amp;E-stained tissue section is prepared for examination by a pathologist. It is a routine diagnostic procedure, at a relatively low cost, available as a validated \u003cem\u003ein-vitro\u003c/em\u003e diagnostic reagent and a fully automated protocol, which generates results within several hours after the procedure has been requested. HPV DNA PCR is also a standard laboratory procedure with high throughput and quick results, although against a higher cost. Preselection by p16\u003csup\u003eINK4a\u003c/sup\u003e-staining therefore reduces the workload and associated costs, and the combination of HPV DNA and p16\u003csup\u003eINK4a\u003c/sup\u003e testing leads to an important reduction of the number of false-positive observations versus the use of either of these assays alone.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn conclusion, combined testing for hrHPV DNA and p16\u003csup\u003eINK4a\u003c/sup\u003e enhances specificity up to 97%, while maintaining high sensitivity (96%), compared to single testing. The diagnostic test combination represents an accurate, accessible and high-throughput testing strategy for the diagnosis of HPV-induced OPC in the clinical setting and for disease prognostication. Treatment adjustments can be encouraged based on this combined test result.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eCI\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Confidence Interval\u003c/p\u003e\n\u003cp\u003eFFPE\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Formalin-Fixed, Paraffin-Embedded\u003c/p\u003e\n\u003cp\u003eH\u0026amp;E\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Hematoxylin and Eosin\u003c/p\u003e\n\u003cp\u003eHNC\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Head and Neck Cancers\u003c/p\u003e\n\u003cp\u003eHPV\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Human Papillomavirus\u003c/p\u003e\n\u003cp\u003ehrHPV\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;high-risk Human Papillomavirus\u003c/p\u003e\n\u003cp\u003eIHC\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Immunohistochemistry\u003c/p\u003e\n\u003cp\u003eOPC\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Oropharyngeal Cancer\u003c/p\u003e\n\u003cp\u003ep16\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;p16\u003csup\u003eINK4a\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003ePCR\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Polymerase Chain Reaction\u003c/p\u003e\n\u003cp\u003eRNA+\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;mRNA-positive\u003c/p\u003e\n\u003cp\u003eS\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Section\u003c/p\u003e\n\u003cp\u003eubC \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;ubiquitin C\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEthical clearance was obtained from the Ethical Committees of \u0026lsquo;GZA hospitals\u0026rsquo; (ref nb: BVDE/hp/2013/01.147), \u0026lsquo;UZA \u0026amp; UA (University of Antwerp)\u0026rsquo; (ref nb: 11/47/362) and IARC, Lyon, France (ref nb: 11-30).\u003c/p\u003e\n\u003cp\u003eInformed consent of the individual patient was not necessary, as it concerns biobank-based research. The ethical principles, detailed by a European network specialized in biobanking (Hansson MG, Methods Mol.Biol 2011; 675:39-59), are respected. The need of informed consent was waived by the Ethical Committees of \u0026lsquo;GZA hospitals\u0026rsquo; and \u0026lsquo;UZA \u0026amp; UA (University of Antwerp)\u0026rsquo; for the Belgian patients.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRR and SR are employees of Roche. All other authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe global HPV-AHEAD study was initially funded by the European Commission, with grant number: FP7-HEALTH-2011-282562.\u003c/p\u003e\n\u003cp\u003eSciensano, the employer of CS and MA, received funding for the analysis of the Belgian part of the HPV-AHEAD study, in part, by a research grant from the Investigator Initiated Studies Program of Merck Sharp \u0026amp; Dohme Corp. The opinions expressed in this paper are those of the authors and do not necessarily represent those of Merck Sharp \u0026amp; Dohme Corp.\u003c/p\u003e\n\u003cp\u003eMA was also supported by the Horizon 2020 Framework Programme for Research and Innovation of the European Commission, through the RISCC Network (grant no. 847845); and the Belgian Foundation Against Cancer through the IHUVACC project.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCS, MA and JB designed the Belgian part of the HPV-AHEAD study; CS and MA performed the statistical analysis and interpreted the findings; CS also compiled the database, managed the Belgian part of the project and wrote the manuscript. IG collected all patient samples and the corresponding clinical information; PV, ML and OMV facilitated and supervised the specimen and clinical data collection. TG, DH, SR, AC, DA, and PB performed the respective DNA, RNA or p16 tests, and interpreted the data; they were supervised by MT, MP, RR, and MRP. RVK, NG, FM, BMLR, and JB were responsible for the histological review of all specimens. EL managed the overall HPV-AHEAD database, and validated the data entry. MT, RS, MP, MA, JB, SC, and RR were the PI\u0026rsquo;s of the HPV-AHEAD study and therefore responsible for the largest funding acquisition. TG, RR, IG, EL, FM, SC, JB, MT, and MA reviewed and/or edited the manuscript. All co-authors approved the final manuscript and its submission to this journal.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe are very grateful for the excellent work performed by Sofie Thys (University of Antwerp, Laboratory of cell biology and histology), for her meticulous work in preparing FFPE slide sections and logistical coordination and support of the study.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWhere authors are identified as personnel of the International Agency for Research on Cancer/World Health Organization, the authors alone are responsible for the views expressed in this article and they do not necessarily represent the decisions, policy, or views of the International Agency for Research on Cancer/World Health Organization.\u003c/p\u003e\n\u003cp\u003eThe HPV-AHEAD study group:\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eChristel Herold-Mende (Dpt. of Neurosurgery and Dpt. of Otorhinolaryngology, Head and Neck Surgery, University of Heidelberg, Germany); Gerhard Dyckhoff (Dpt. of Otorhinolaryngology, Head and Neck Surgery, University of Heidelberg, Germany); George Mosialos (Aristotle University of Thessaloniki, Greece); Fausto Chiesa, Marta Tagliabue, Mohssen Ansarin (European Institute of Oncology, Milan, Italy); Heiner Boeing (German Institute of Human Nutrition, Berlin, Germany); \u0026nbsp;Xavier Castellsagu\u0026eacute;\u0026dagger;, Silvia de Sanjos\u0026eacute;, Marisa Mena, Francesc Xavier Bosch, Laia Alemany (Catalan Institute of OncologyIDIBELL, L\u0026apos;Hospitalet de Llobregat, Spain); Pulikottil Okkuru Esmy (Christian Fellowship Community Health Centre, Ambillikai, India); Rudrapatna S. Jayshree, Kortikere S. Sabitha, Ashok M. Shenoy (Kidwai Memorial Institute of Oncology, Bangalore, India); Manavalan Vijayakumar (YEN ONCO Centre, Yenepoya University, Deralakatte, Mangalore 575018, Karnataka, India); Aruna S. Chiwate, Ranjit V. Thorat, Girish G. Hublikar, Shashikant S. Lakshetti, Bhagwan M. Nene (Nargis Dutt Memorial Cancer Hospital, Barshi 413401, India); Amal Ch Kataki, Ashok Kumar Das (Dr B. Borooah Cancer Institute, Guwahati, Assam, India); Subha Sankaran (Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram, India); Kunnambath Ramadas (Regional Cancer Centre, Thiruvananthapuram, India); Christine Carreira, Sandrine McKay-Chopin (International Agency for Research on Cancer (IARC), Lyon, France), Thara Somanathan (Regional Cancer Centre, Thiruvananthapuram, 695011, India), Alfredo Zito (Istituto Tumori \u0026quot;Giovanni Paolo II\u0026quot;, Bari).\u003c/p\u003e\n\u003cp\u003e\u0026dagger;Dr Castellsagu\u0026eacute; passed away on June 12th, 2016.\u003c/p\u003e"},{"header":"References","content":"\u003cp\u003e1.\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Gillison ML, Castellsague X, Chaturvedi A, Goodman MT, Snijders P, Tommasino M, et al. Comparative epidemiology of HPV infection and associated cancers of the head and neck and cervix. 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Cancer. 2012;120(5):294-307.\u003c/p\u003e\n\u003cp\u003e30.\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Klussmann JP, Gultekin E, Weissenborn SJ, Wieland U, Dries V, Dienes HP, et al.\u0026nbsp;Expression of p16 protein identifies a distinct entity of tonsillar carcinomas associated with human papillomavirus. Am J Pathol 2003;162(3):747-53.\u003c/p\u003e\n\u003cp\u003e31.\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Prigge ES, Toth C, Dyckhoff G, Wagner S, M\u0026uuml;ller F, Wittekindt C, et al. p16(INK4a) /Ki-67 co-expression specifically identifies transformed cells in the head and neck region. Int J Cancer. 2015;136(7):1589-99.\u003c/p\u003e\n\u003cp\u003e32.\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Castellsague X, Alemany L, Quer M, Halec G, Quiros B, Tous S, et al. HPV Involvement in Head and Neck Cancers: Comprehensive Assessment of Biomarkers in 3680 Patients. J Natl Cancer Inst 2016;108(6).\u003c/p\u003e\n\u003cp\u003e33.\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Simoens C, Gorbaslieva I, Gheit T, Holzinger D, Lucas E, Ridder R, et al. HPV DNA genotyping, HPV E6*I mRNA detection, and p16(INK4a)/Ki-67 staining in Belgian head and neck cancer patient specimens, collected within the HPV-AHEAD study. Cancer Epidemiol. 2021;72:101925.\u003c/p\u003e\n\u003cp\u003e34.\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Mena M, Lloveras B, Tous S, Bogers J, Maffini F, Gangane N, et al. Development and validation of a protocol for optimizing the use of paraffin blocks in molecular epidemiological studies: The example from the HPV-AHEAD study. PloS one. 2017;12(10):e0184520.\u003c/p\u003e\n\u003cp\u003e35.\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Gheit T, Anantharaman D, Holzinger D, Alemany L, Tous S, Lucas E, et al. Role of mucosal high-risk human papillomavirus types in head and neck cancers in central India. Int J Cancer. 2017;141(1):143-51.\u003c/p\u003e\n\u003cp\u003e36.\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Halec G, Schmitt M, Dondog B, Sharkhuu E, Wentzensen N, Gheit T, et al. Biological activity of probable/possible high-risk human papillomavirus types in cervical cancer. Int J Cancer. 2013;132(1):63-71.\u003c/p\u003e\n\u003cp\u003e37.\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Gheit T, Landi S, Gemignani F, Snijders PJ, Vaccarella S, Francheschi S, et al. Development of a Sensitive and Specific Assay Combining Multiplex PCR and DNA Microarray Primer Extension To Detect High-Risk Mucosal Human Papillomavirus Types. J Clin Microbiol 2006;44(6):2025-31.\u003c/p\u003e\n\u003cp\u003e38.\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Schmitt M, Bolormaa D, Waterboer T, Pawlita M, Tommasino M, Gheit T. Abundance of Multiple High-Risk Human Papillomavirus (HPV) Infections Found in Cervical Cells Analyzed by Use of an Ultrasensitive HPV Genotyping Assay. J Clin Microbiol 2010;48(1):143-9.\u003c/p\u003e\n\u003cp\u003e39.\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Tagliabue M, Mena M, Maffini F, Gheit T, Quiros Blasco B, Holzinger D, et al.\u0026nbsp;Role of Human Papillomavirus Infection in Head and Neck Cancer in Italy: The HPV-AHEAD Study. Cancers (Basel). 2020;12(12).\u003c/p\u003e\n\u003cp\u003e40.\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Johansson H, Bjelkenkrantz K, Darlin L, Dilllner J, Forslund O. Presence of High-Risk HPV mRNA in Relation to Future High-Grade Lesions among High-Risk HPV DNA Positive Women with Minor Cytological Abnormalities. PloS one. 2015;10(4):e0124460-/.\u003c/p\u003e\n\u003cp\u003e41.\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Victor T, Jordaan A, du Toit R, Van Helden PD.\u0026nbsp;Laboratory experience and guidelines for avoiding false positive polymerase chain reaction results. Eur J Clin Chem Clin Biochem. 1993;31(8):531-5.\u003c/p\u003e\n\u003cp\u003e42.\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;de Sanjose S, Alemany L, Ordi J, Tous S, Alejo M, Bigby SM, et al. Worldwide human papillomavirus genotype attribution in over 2000 cases of intraepithelial and invasive lesions of the vulva. Eur J Cancer. 2013;49(16):3450-61.\u003c/p\u003e\n\u003cp\u003e43.\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Orosco RK, Califano JA. HPV status, like politics, is local-evaluating p16 staining and a new staging system in a Dutch cohort of oropharynx cancer. Ann Oncol. 2018;29(5):1089-90.\u003c/p\u003e\n\u003cp\u003e44.\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Wagner S, Prigge ES, Wuerdemann N, Reder H, Bushnak A, Sharma SJ, et al. Evaluation of p16(INK4a) expression as a single marker to select patients with HPV-driven oropharyngeal cancers for treatment de-escalation. Br J Cancer. 2020;123(7):1114-22.\u003c/p\u003e\n\u003cp\u003e45.\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Prigge ES, Arbyn M, von Knebel Doeberitz M, Reuschenbach M. Diagnostic accuracy of p16(INK4a) immunohistochemistry in oropharyngeal squamous cell carcinomas: A systematic review and meta-analysis. Int J Cancer. 2017;140(5):1186-98.\u003c/p\u003e\n\u003cp\u003e46. \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Albers AE, Qian X, Kaufmann AM, Coordes A. Meta analysis: HPV and p16 pattern determines survival in patients with HNSCC and identifies potential new biologic subtype. Sci Rep. 2017;7(1):16715.\u003c/p\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable 1: \u0026nbsp; Prevalence of HPV determined by HPV DNA PCR (DNA), p16\u003csup\u003eINK4a\u003c/sup\u003e IHC (p16),\u003c/strong\u003e \u003cstrong\u003eE6*I mRNA\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e(RNA), and all combinations of tests, in oropharyngeal cancer\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"0\" cellpadding=\"0\" cellspacing=\"0\" width=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"27.75%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"14.25%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"bottom\" width=\"58%\"\u003e\n \u003cp\u003eOropharyngeal cancer\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" valign=\"bottom\" width=\"42%\"\u003e\n \u003cp\u003eMarker positivity\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"16.75%\"\u003e\n \u003cp\u003ePositive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"20.5%\"\u003e\n \u003cp\u003eProportion\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"20.75%\"\u003e\n \u003cp\u003e(95% CI)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"27.75%\"\u003e\n \u003cp\u003eDNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"14.25%\"\u003e\n \u003cp\u003e(N=99)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"16.75%\"\u003e\n \u003cp\u003e36\u0026deg;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"20.5%\"\u003e\n \u003cp\u003e36.4%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"20.75%\"\u003e\n \u003cp\u003e(27.4-46.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"27.75%\"\u003e\n \u003cp\u003ep16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"14.25%\"\u003e\n \u003cp\u003e*(N=97)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"16.75%\"\u003e\n \u003cp\u003e33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"20.5%\"\u003e\n \u003cp\u003e34.0%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"20.75%\"\u003e\n \u003cp\u003e(25.2-44.1)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"27.75%\"\u003e\n \u003cp\u003eRNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"14.25%\"\u003e\n \u003cp\u003e\u003csup\u003e\u0026pound;\u003c/sup\u003e(N=97)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"16.75%\"\u003e\n \u003cp\u003e28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"20.5%\"\u003e\n \u003cp\u003e28.9%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"20.75%\"\u003e\n \u003cp\u003e(20.6-38.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"27.75%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003ep16+DNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"14.25%\"\u003e\n \u003cp\u003e(N=97)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"16.75%\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"20.5%\"\u003e\n \u003cp\u003e30.9%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"20.75%\"\u003e\n \u003cp\u003e(22.5-40.9)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"27.75%\"\u003e\n \u003cp\u003eDNA+RNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"14.25%\"\u003e\n \u003cp\u003e(N=97)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"16.75%\"\u003e\n \u003cp\u003e28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"20.5%\"\u003e\n \u003cp\u003e28.9%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"20.75%\"\u003e\n \u003cp\u003e(20.6-38.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"27.75%\"\u003e\n \u003cp\u003eRNA+p16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"14.25%\"\u003e\n \u003cp\u003e(N=95)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"16.75%\"\u003e\n \u003cp\u003e27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"20.5%\"\u003e\n \u003cp\u003e28.4%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"20.75%\"\u003e\n \u003cp\u003e(20.2-38.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"27.75%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"14.25%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"16.75%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"20.5%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"20.75%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"27.75%\"\u003e\n \u003cp\u003ep16+DNA+RNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"14.25%\"\u003e\n \u003cp\u003e(N=95)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"16.75%\"\u003e\n \u003cp\u003e27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"20.5%\"\u003e\n \u003cp\u003e28.4%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"20.75%\"\u003e\n \u003cp\u003e(20.2-38.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026deg; \u0026nbsp;1 lrHPV (HPV6) and 35 hrHPV\u003c/p\u003e\n\u003cp\u003e* 2 equivocal results after final re-review\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e\u0026pound;\u003c/sup\u003e\u0026nbsp; 2 samples gave an invalid RNA test result\u003cstrong\u003e\u003cbr\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2: hrHPV DNA, p16\u003csup\u003eINK4a\u003c/sup\u003e (p16) and combined hrHPV DNA/p16\u003csup\u003eINK4a\u003c/sup\u003e test results by\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ehrHPV RNA status in cancers of the oropharynx, tonsils and base of the tongue\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"0\" cellpadding=\"0\" cellspacing=\"0\" width=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"22.33009708737864%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"bottom\" width=\"31.067961165048544%\"\u003e\n \u003cp\u003e\u003cstrong\u003ehrHPV RNA\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"22.33009708737864%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.29126213592233%\"\u003e\n \u003cp\u003ePos\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.776699029126213%\"\u003e\n \u003cp\u003eNeg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"22.33009708737864%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.29126213592233%\"\u003e\n \u003cp\u003e\u003cstrong\u003eN\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.776699029126213%\"\u003e\n \u003cp\u003e\u003cstrong\u003eN\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003eTotal\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"22.33009708737864%\"\u003e\n \u003cp\u003e\u003cstrong\u003ehrHPV DNA\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"bottom\" width=\"31.067961165048544%\"\u003e\n \u003cp\u003eOropharynx\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.29126213592233%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.776699029126213%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"22.33009708737864%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003ePos\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.29126213592233%\"\u003e\n \u003cp\u003e\u003cstrong\u003e28\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.776699029126213%\"\u003e\n \u003cp\u003e\u003cstrong\u003e5\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e33\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"22.33009708737864%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003eNeg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.29126213592233%\"\u003e\n \u003cp\u003e\u003cstrong\u003e0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.776699029126213%\"\u003e\n \u003cp\u003e\u003cstrong\u003e62\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e62\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"22.33009708737864%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003eTotal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.29126213592233%\"\u003e\n \u003cp\u003e\u003cstrong\u003e28\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.776699029126213%\"\u003e\n \u003cp\u003e\u003cstrong\u003e67\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e95\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"22.33009708737864%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003eTonsil\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.29126213592233%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.776699029126213%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"22.33009708737864%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003ePos\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.29126213592233%\"\u003e\n \u003cp\u003e\u003cstrong\u003e19\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.776699029126213%\"\u003e\n \u003cp\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e22\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"22.33009708737864%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003eNeg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.29126213592233%\"\u003e\n \u003cp\u003e\u003cstrong\u003e0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.776699029126213%\"\u003e\n \u003cp\u003e\u003cstrong\u003e20\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"22.33009708737864%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003eTotal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.29126213592233%\"\u003e\n \u003cp\u003e\u003cstrong\u003e19\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.776699029126213%\"\u003e\n \u003cp\u003e\u003cstrong\u003e23\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e42\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"22.33009708737864%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"bottom\" width=\"31.067961165048544%\"\u003e\n \u003cp\u003eBase of tongue\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.29126213592233%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.776699029126213%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"22.33009708737864%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003ePos\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.29126213592233%\"\u003e\n \u003cp\u003e\u003cstrong\u003e7\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.776699029126213%\"\u003e\n \u003cp\u003e\u003cstrong\u003e0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"22.33009708737864%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003eNeg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.29126213592233%\"\u003e\n \u003cp\u003e\u003cstrong\u003e0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.776699029126213%\"\u003e\n \u003cp\u003e\u003cstrong\u003e14\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"22.33009708737864%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003eTotal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.29126213592233%\"\u003e\n \u003cp\u003e\u003cstrong\u003e7\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.776699029126213%\"\u003e\n \u003cp\u003e\u003cstrong\u003e14\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e21\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"22.33009708737864%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.29126213592233%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.776699029126213%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"22.33009708737864%\"\u003e\n \u003cp\u003e\u003cstrong\u003ep16\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"bottom\" width=\"31.067961165048544%\"\u003e\n \u003cp\u003eOropharynx\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.29126213592233%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.776699029126213%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"22.33009708737864%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003ePos\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.29126213592233%\"\u003e\n \u003cp\u003e\u003cstrong\u003e27\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.776699029126213%\"\u003e\n \u003cp\u003e\u003cstrong\u003e5\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e32\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"22.33009708737864%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003eNeg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.29126213592233%\"\u003e\n \u003cp\u003e\u003cstrong\u003e1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.776699029126213%\"\u003e\n \u003cp\u003e\u003cstrong\u003e62\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e63\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"22.33009708737864%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003eTotal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.29126213592233%\"\u003e\n \u003cp\u003e\u003cstrong\u003e28\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.776699029126213%\"\u003e\n \u003cp\u003e\u003cstrong\u003e67\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e95\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"22.33009708737864%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003eTonsil\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.29126213592233%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.776699029126213%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"22.33009708737864%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003ePos\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.29126213592233%\"\u003e\n \u003cp\u003e\u003cstrong\u003e18\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.776699029126213%\"\u003e\n \u003cp\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"22.33009708737864%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003eNeg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.29126213592233%\"\u003e\n \u003cp\u003e\u003cstrong\u003e1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.776699029126213%\"\u003e\n \u003cp\u003e\u003cstrong\u003e21\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e22\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"22.33009708737864%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003eTotal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.29126213592233%\"\u003e\n \u003cp\u003e\u003cstrong\u003e19\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.776699029126213%\"\u003e\n \u003cp\u003e\u003cstrong\u003e23\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e42\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"22.33009708737864%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"bottom\" width=\"31.067961165048544%\"\u003e\n \u003cp\u003eBase of tongue\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.29126213592233%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.776699029126213%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"22.33009708737864%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003ePos\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.29126213592233%\"\u003e\n \u003cp\u003e\u003cstrong\u003e7\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.776699029126213%\"\u003e\n \u003cp\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"22.33009708737864%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003eNeg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.29126213592233%\"\u003e\n \u003cp\u003e\u003cstrong\u003e0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.776699029126213%\"\u003e\n \u003cp\u003e\u003cstrong\u003e12\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"22.33009708737864%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003eTotal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.29126213592233%\"\u003e\n \u003cp\u003e\u003cstrong\u003e7\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.776699029126213%\"\u003e\n \u003cp\u003e\u003cstrong\u003e14\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e21\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"0\" cellpadding=\"0\" cellspacing=\"0\" width=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"22.33009708737864%\"\u003e\n \u003cp\u003e\u003cstrong\u003ep16+DNA\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"bottom\" width=\"31.067961165048544%\"\u003e\n \u003cp\u003eOropharynx\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"22.33009708737864%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"18.932038834951456%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"12.135922330097088%\"\u003e\n \u003cp\u003ePos\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e\u003cstrong\u003e27\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e29\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"22.33009708737864%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"18.932038834951456%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"12.135922330097088%\"\u003e\n \u003cp\u003eNeg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e\u003cstrong\u003e1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e\u003cstrong\u003e65\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e66\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"22.33009708737864%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"18.932038834951456%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"12.135922330097088%\"\u003e\n \u003cp\u003eTotal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e\u003cstrong\u003e28\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e\u003cstrong\u003e67\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e95\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"22.33009708737864%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"18.932038834951456%\"\u003e\n \u003cp\u003eTonsil\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"12.135922330097088%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"22.33009708737864%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"18.932038834951456%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"12.135922330097088%\"\u003e\n \u003cp\u003ePos\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e\u003cstrong\u003e18\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"22.33009708737864%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"18.932038834951456%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"12.135922330097088%\"\u003e\n \u003cp\u003eNeg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e\u003cstrong\u003e1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e\u003cstrong\u003e21\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e22\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"22.33009708737864%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"18.932038834951456%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"12.135922330097088%\"\u003e\n \u003cp\u003eTotal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e\u003cstrong\u003e19\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e\u003cstrong\u003e23\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e42\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"22.33009708737864%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"bottom\" width=\"31.067961165048544%\"\u003e\n \u003cp\u003eBase of tongue\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"22.33009708737864%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"18.932038834951456%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"12.135922330097088%\"\u003e\n \u003cp\u003ePos\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e\u003cstrong\u003e7\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e\u003cstrong\u003e0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"22.33009708737864%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"18.932038834951456%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"12.135922330097088%\"\u003e\n \u003cp\u003eNeg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e\u003cstrong\u003e0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e\u003cstrong\u003e14\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"22.33009708737864%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"18.932038834951456%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"12.135922330097088%\"\u003e\n \u003cp\u003eTotal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e\u003cstrong\u003e7\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e\u003cstrong\u003e14\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e21\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"22.33009708737864%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"18.932038834951456%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"12.135922330097088%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"22.33009708737864%\"\u003e\n \u003cp\u003e\u003cstrong\u003ep16-\u0026gt;DNA\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"bottom\" width=\"31.067961165048544%\"\u003e\n \u003cp\u003eOropharynx\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"22.33009708737864%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"18.932038834951456%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"12.135922330097088%\"\u003e\n \u003cp\u003ePos\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e\u003cstrong\u003e27\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e29\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"22.33009708737864%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"18.932038834951456%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"12.135922330097088%\"\u003e\n \u003cp\u003eNeg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e\u003cstrong\u003e1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e\u003cstrong\u003e65\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e66\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"22.33009708737864%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"18.932038834951456%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"12.135922330097088%\"\u003e\n \u003cp\u003eTotal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e\u003cstrong\u003e28\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e\u003cstrong\u003e67\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e95\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"22.33009708737864%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"18.932038834951456%\"\u003e\n \u003cp\u003eTonsil\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"12.135922330097088%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"22.33009708737864%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"18.932038834951456%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"12.135922330097088%\"\u003e\n \u003cp\u003ePos\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e\u003cstrong\u003e18\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"22.33009708737864%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"18.932038834951456%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"12.135922330097088%\"\u003e\n \u003cp\u003eNeg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e\u003cstrong\u003e1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e\u003cstrong\u003e21\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e22\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"22.33009708737864%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"18.932038834951456%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"12.135922330097088%\"\u003e\n \u003cp\u003eTotal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e\u003cstrong\u003e19\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e\u003cstrong\u003e23\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e42\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"22.33009708737864%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"bottom\" width=\"31.067961165048544%\"\u003e\n \u003cp\u003eBase of tongue\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"22.33009708737864%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"18.932038834951456%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"12.135922330097088%\"\u003e\n \u003cp\u003ePos\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e\u003cstrong\u003e7\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e\u003cstrong\u003e0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"22.33009708737864%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"18.932038834951456%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"12.135922330097088%\"\u003e\n \u003cp\u003eNeg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e\u003cstrong\u003e0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e\u003cstrong\u003e14\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"22.33009708737864%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"18.932038834951456%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"12.135922330097088%\"\u003e\n \u003cp\u003eTotal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e\u003cstrong\u003e7\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e\u003cstrong\u003e14\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e21\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"22.33009708737864%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"18.932038834951456%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"12.135922330097088%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"22.33009708737864%\"\u003e\n \u003cp\u003e\u003cstrong\u003eDNA-\u0026gt;p16\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"bottom\" width=\"31.067961165048544%\"\u003e\n \u003cp\u003eOropharynx\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"22.33009708737864%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"18.932038834951456%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"12.135922330097088%\"\u003e\n \u003cp\u003ePos\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e\u003cstrong\u003e27\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e29\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"22.33009708737864%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"18.932038834951456%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"12.135922330097088%\"\u003e\n \u003cp\u003eNeg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e\u003cstrong\u003e1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e\u003cstrong\u003e65\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e66\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"22.33009708737864%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"18.932038834951456%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"12.135922330097088%\"\u003e\n \u003cp\u003eTotal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e\u003cstrong\u003e28\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e\u003cstrong\u003e67\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e95\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"22.33009708737864%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"18.932038834951456%\"\u003e\n \u003cp\u003eTonsil\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"12.135922330097088%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"22.33009708737864%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"18.932038834951456%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"12.135922330097088%\"\u003e\n \u003cp\u003ePos\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e\u003cstrong\u003e18\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"22.33009708737864%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"18.932038834951456%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"12.135922330097088%\"\u003e\n \u003cp\u003eNeg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e\u003cstrong\u003e1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e\u003cstrong\u003e21\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e22\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"22.33009708737864%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"18.932038834951456%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"12.135922330097088%\"\u003e\n \u003cp\u003eTotal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e\u003cstrong\u003e19\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e\u003cstrong\u003e23\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e42\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"22.33009708737864%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"bottom\" width=\"31.067961165048544%\"\u003e\n \u003cp\u003eBase of tongue\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"22.33009708737864%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"18.932038834951456%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"12.135922330097088%\"\u003e\n \u003cp\u003ePos\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e\u003cstrong\u003e7\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e\u003cstrong\u003e0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"22.33009708737864%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"18.932038834951456%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"12.135922330097088%\"\u003e\n \u003cp\u003eNeg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e\u003cstrong\u003e0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e\u003cstrong\u003e14\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"22.33009708737864%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"18.932038834951456%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"12.135922330097088%\"\u003e\n \u003cp\u003eTotal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e\u003cstrong\u003e7\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e\u003cstrong\u003e14\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"15.533980582524272%\"\u003e\n \u003cp\u003e21\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3a: Absolute sensitivity and specificity of hrHPV DNA PCR (hrHPV DNA), p16\u003csup\u003eINK4a\u003c/sup\u003e IHC (p16)\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eand the combined testing (p16+DNA) to identify a HPV-driven oropharyngeal cancer\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"0\" cellpadding=\"0\" cellspacing=\"0\" width=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"41.90231362467866%\"\u003e\n \u003cp\u003e\u003cstrong\u003eAbsolute\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"29.04884318766067%\"\u003e\n \u003cp\u003e\u003cstrong\u003eSensitivity\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"29.04884318766067%\"\u003e\n \u003cp\u003e\u003cstrong\u003eSpecificity\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"41.90231362467866%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"29.04884318766067%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"29.04884318766067%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"41.90231362467866%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"29.04884318766067%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"29.04884318766067%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"41.90231362467866%\"\u003e\n \u003cp\u003e\u003cstrong\u003ehrHPV DNA\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"29.04884318766067%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"29.04884318766067%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"41.90231362467866%\"\u003e\n \u003cp\u003en/N\u003c/p\u003e\n \u003cp\u003e%\u003c/p\u003e\n \u003cp\u003e(95% CI)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"29.04884318766067%\"\u003e\n \u003cp\u003e28/28\u003c/p\u003e\n \u003cp\u003e100%\u003c/p\u003e\n \u003cp\u003e(87.7-100)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"29.04884318766067%\"\u003e\n \u003cp\u003e62/67\u003c/p\u003e\n \u003cp\u003e92.5%\u003c/p\u003e\n \u003cp\u003e(83.4-97.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"41.90231362467866%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"29.04884318766067%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"29.04884318766067%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"41.90231362467866%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"29.04884318766067%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"29.04884318766067%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"41.90231362467866%\"\u003e\n \u003cp\u003e\u003cstrong\u003ep16\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"29.04884318766067%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"29.04884318766067%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"41.90231362467866%\"\u003e\n \u003cp\u003en/N\u003c/p\u003e\n \u003cp\u003e%\u003c/p\u003e\n \u003cp\u003e(95% CI)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"29.04884318766067%\"\u003e\n \u003cp\u003e27/28\u003c/p\u003e\n \u003cp\u003e96.4%\u003c/p\u003e\n \u003cp\u003e(81.7-99.9)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"29.04884318766067%\"\u003e\n \u003cp\u003e62/67\u003c/p\u003e\n \u003cp\u003e92.5%\u003c/p\u003e\n \u003cp\u003e(83.4-97.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"41.90231362467866%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"29.04884318766067%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"29.04884318766067%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"41.90231362467866%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"29.04884318766067%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"29.04884318766067%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"41.90231362467866%\"\u003e\n \u003cp\u003e\u003cstrong\u003ep16+DNA\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"29.04884318766067%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"29.04884318766067%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"41.90231362467866%\"\u003e\n \u003cp\u003en/N\u003c/p\u003e\n \u003cp\u003e%\u003c/p\u003e\n \u003cp\u003e(95% CI)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"29.04884318766067%\"\u003e\n \u003cp\u003e27/28\u003c/p\u003e\n \u003cp\u003e96.4%\u003c/p\u003e\n \u003cp\u003e(81.7-99.9)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"29.04884318766067%\"\u003e\n \u003cp\u003e65/67\u003c/p\u003e\n \u003cp\u003e97.0%\u003c/p\u003e\n \u003cp\u003e(89.9-99.6)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"41.90231362467866%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"29.04884318766067%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"29.04884318766067%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3b: Relative sensitivity and specificity of hrHPV DNA PCR (DNA) vs p16\u003csup\u003eINK4a\u003c/sup\u003e IHC (p16), and\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eof DNA and p16 vs combined testing (p16+DNA), to identify a HPV-driven\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eoropharyngeal cancer\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"0\" cellpadding=\"0\" cellspacing=\"0\" width=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"41.90231362467866%\"\u003e\n \u003cp\u003e\u003cstrong\u003eRelative\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"29.04884318766067%\"\u003e\n \u003cp\u003e\u003cstrong\u003eSensitivity\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"29.04884318766067%\"\u003e\n \u003cp\u003e\u003cstrong\u003eSpecificity\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"41.90231362467866%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"29.04884318766067%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"29.04884318766067%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"41.90231362467866%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"29.04884318766067%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"29.04884318766067%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"41.90231362467866%\"\u003e\n \u003cp\u003e\u003cstrong\u003ehrHPV DNA vs p16\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"29.04884318766067%\"\u003e\n \u003cp\u003e1.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"29.04884318766067%\"\u003e\n \u003cp\u003e1.00\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"41.90231362467866%\"\u003e\n \u003cp\u003e(95% CI)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"29.04884318766067%\"\u003e\n \u003cp\u003e(0.97-1.11)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"29.04884318766067%\"\u003e\n \u003cp\u003e(1.00-1.00)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"41.90231362467866%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"29.04884318766067%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"29.04884318766067%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"41.90231362467866%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"29.04884318766067%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"29.04884318766067%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"41.90231362467866%\"\u003e\n \u003cp\u003e\u003cstrong\u003ep16+DNA vs p16\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"29.04884318766067%\"\u003e\n \u003cp\u003e1.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"29.04884318766067%\"\u003e\n \u003cp\u003e1.05\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"41.90231362467866%\"\u003e\n \u003cp\u003e(95% CI)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"29.04884318766067%\"\u003e\n \u003cp\u003e(1.00-1.00)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"29.04884318766067%\"\u003e\n \u003cp\u003e(0.87-1.26)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"41.90231362467866%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"29.04884318766067%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"29.04884318766067%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"41.90231362467866%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"29.04884318766067%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"29.04884318766067%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"41.90231362467866%\"\u003e\n \u003cp\u003e\u003cstrong\u003ep16+DNA vs hrHPV DNA\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"29.04884318766067%\"\u003e\n \u003cp\u003e0.96\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"29.04884318766067%\"\u003e\n \u003cp\u003e1.05\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"41.90231362467866%\"\u003e\n \u003cp\u003e(95% CI)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"29.04884318766067%\"\u003e\n \u003cp\u003e(0.90-1.04)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"29.04884318766067%\"\u003e\n \u003cp\u003e(0.87-1.26)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-infectious-diseases","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"infd","sideBox":"Learn more about [BMC Infectious Diseases](http://bmcinfectdis.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/infd","title":"BMC Infectious Diseases","twitterHandle":"#bmcinfectdis","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"HPV DNA PCR, p16INK4a IHC, HPV mRNA, oropharyngeal carcinoma, HPV-AHEAD","lastPublishedDoi":"10.21203/rs.3.rs-1008778/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1008778/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBACKGROUND\u003c/p\u003e\u003cp\u003eThe incidence of high-risk human papillomavirus (hrHPV)-driven head and neck squamous cell carcinoma, in particular oropharyngeal cancers (OPC), is increasing in high-resource countries. Patients with HPV-induced cancer respond better to treatment and consequently have lower case-fatality rates than patients with HPV-unrelated OPC. These considerations highlight the importance of reliable and accurate markers to diagnose truly HPV-induced OPC.\u0026nbsp;\u003c/p\u003e\u003cp\u003eMETHODS\u003c/p\u003e\u003cp\u003eThe accuracy of three possible test strategies, i.e. a) hrHPV DNA PCR (DNA), b) p16\u003csup\u003eINK4a\u003c/sup\u003e immunohistochemistry (IHC) (p16), and c) the combination of both tests (considering joint DNA and p16 positivity as positivity criterion), was analysed in tissue samples from 99 Belgian OPC patients enrolled in the HPV-AHEAD study. Presence of HPV E6*I mRNA (mRNA) was considered as the reference, indicating HPV etiology. \u003c/p\u003e\u003cp\u003eRESULTS\u003c/p\u003e\u003cp\u003eNinety-nine OPC patients were included, for which the positivity rates were 36.4%, 34.0% and 28.9% for DNA, p16 and mRNA, respectively. Ninety-five OPC patients had valid test results for all three tests (DNA, p16 and mRNA). Using mRNA status as the reference, DNA testing showed 100% (28/28) sensitivity, and 92.5% (62/67) specificity for the detection of HPV-driven cancer. p16 was 96.4% (27/28) sensitive and equally specific (92.5%; 62/67). The sensitivity and specificity of combined p16+DNA testing was 96.4% (27/28) and 97.0% (65/67), respectively. In this series, p16 alone and combined p16+DNA missed 1 in 28 HPV driven cancers, but p16 alone misclassified 5 in 67 non-HPV driven as positive, whereas combined testing would misclassify only 2 in 67. \u003c/p\u003e\u003cp\u003eCONCLUSIONS\u003c/p\u003e\u003cp\u003eSingle hrHPV DNA PCR and p16\u003csup\u003eINK4a\u003c/sup\u003e IHC are highly sensitive but less specific than using combined testing to diagnose HPV-driven OPC patients. Since HPV-driven OPC cancer can be treated less aggressively, combined testing can reduce treatment sequelae at the expense of a small number of patients that may receive less effective therapy.\u003c/p\u003e","manuscriptTitle":"Accuracy of High-risk HPV DNA PCR, p16INK4a Immunohistochemistry or the Combination of Both to Diagnose HPV-driven Oropharyngeal Cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-11-22 16:46:27","doi":"10.21203/rs.3.rs-1008778/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2021-12-14T13:58:24+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2021-11-25T14:32:27+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"1416bf36-db18-4933-b29e-af041ef4c6d4","date":"2021-11-22T12:35:00+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2021-11-18T18:57:56+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2021-11-18T18:50:22+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2021-11-18T18:07:22+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2021-11-18T18:03:13+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Infectious Diseases","date":"2021-10-22T17:11:06+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-infectious-diseases","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"infd","sideBox":"Learn more about [BMC Infectious Diseases](http://bmcinfectdis.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/infd","title":"BMC Infectious Diseases","twitterHandle":"#bmcinfectdis","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"1b5c2498-d522-4615-8f86-188e26772a32","owner":[],"postedDate":"November 22nd, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":8660879,"name":"Infectious Diseases"}],"tags":[],"updatedAt":"2022-07-08T06:59:13+00:00","versionOfRecord":[],"versionCreatedAt":"2021-11-22 16:46:27","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-1008778","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1008778","identity":"rs-1008778","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}