Recent reports have pointed to the link between persistent inflammation, oxidative stress, and carcinogenesis; however most of the studies concerning the role of viruses in head and neck cancer (HNC) are focused mainly on one type of virus. Our present study aimed to study the relationship between Epstein–Barr virus/human papilloma virus (EBV/HPV) coinfection and glutathione peroxidase (GPx) and superoxide dismutase (SOD) level in oropharyngeal cancer. Fresh-frozen tumor tissue samples were collected from 128 patients with oropharyngeal cancer infected with EBV or HPV or with EBV/HPV coinfection. After DNA extraction, EBV and HPV DNA was detected using a polymerase chain reaction (PCR) assay. GPx and SOD activity was determined in homogenates of cancer tissue using diagnostic kits produced by Randox Laboratories. Both GPx and SOD activity was statistically lower in patients with EBV/HPV coinfection than in a single EBV or HPV infection. Analysis of GPx and SOD activity in relation to histological grading and tumor, node (TN) classification revealed that in poorly-differentiated tumors, the level of antioxidant enzymes was lower compared with well-differentiated lesions and in cases with greater tumor dimensions and lymph-node involvement, both GPx and SOD activity was decreased. Further studies are necessary to clarify the influence of interplay between EBV, HPV, and oxidative stress on malignant transformation of upper aerodigestive tract epithelial cells. Oncogenic virus-induced cancers are an important global concern nowadays. According to various reports, although all human cancers are non-communicable diseases, infections may contribute to about 13–20 % of global human cancer burden, and it is estimated that about 35 % of all infection-associated malignancies are attributable to human papilloma virus (HPV) and Epstein–Barr virus (EBV) . EBV andEBV establishes a latent infection, reactivated periodically into the lytic cycle, which plays an important role in the pathogenesis of tumors related to EBV . During HPV oncogenic potential is connected with E6 and E7 oncoprotein expression, whereas the oncogenic properties of EBV are attributed to the expression of latent genes, including LMP1 and LMP2 . HPV E6 KB), which causes disorders in the normal functioning of cells involved in the development of cancer [Oxidative stress is defined as the imbalance between the production of reactive oxygen species (ROS) and antioxidant defense, which can lead to damage of various cell components and influence cellular processes, such as proliferation or apoptosis, involved in the development of cancer . ROS actf cancer ,14.The most important defense against oxidative damage is antioxidants. The main enzymes with antioxidant activity are glutathione peroxidase (GPx), superoxide dismutase (SOD), and catalase (CAT). Superoxide dismutase is the most important antioxidant enzyme in aerobic cells, responsible for the elimination of superoxide radicals. SOD catalyzes the dismutation of hydrogen peroxide and molecular oxygen. Glutathione peroxidase (GPx) in cells is the main hydrogen peroxide-scavenging enzyme which converts this molecule to water. SOD and GPx can directly counterbalance the oxidant attack and protect the cells against DNA damage .Numerous studies have shown that both EBV and HPV infection are associated with the production of reactive oxygen species (ROS) and may activate signaling pathways mediated by oxidative stress, thus influencing migration of inflammatory cells and leading to mutations and tissue damage ,16,17.Recent reports pointed to the link between persistent inflammation, oxidative stress, and carcinogenic processes ,18,19,20The present study involved 128 patients with a diagnosed and histopathologically confirmed oropharyngeal squamous cell carcinoma (SCC), who were also infected with EBV or HPV, or coinfected with both EBV and HPV. In this group, 54 patients were EBV(+), 44 patients were HPV(+), and 30 patients were coinfected with EBV and HPV. The patients were hospitalized at the Otolaryngology Division of the Masovian Specialist Hospital in Radom, Poland. The patients had not received radiotherapy or chemotherapy before. Clinical and epidemiological characteristics of the patients are presented in The tissue samples were collected from all patients during surgery and frozen at −80 °C until analysis. Tumor, node, metastasis (TNM) classification was determined during primary diagnosis according to the criteria of the Union for International Cancer Control (UICC) . HistoloThe research was approved by the Medical University of Lublin Ethics Committee and was in accordance with good clinical practice (GCP) regulations .Fragments of the fresh frozen tumor tissue (20 mg) collected from the patients with oral squamous cell carcinoma (OSCC) were cut and homogenized in a manual homogenizer Omni TH/Omni International/Kennesewa/Georgia/USA. DNA was extracted using a protocol as described in the DNeasy Tissue Kit Handbook . Purified DNA was quantified by spectrophotometry . Isolated DNA was kept at −20 °C until the test was conducted. To verify the quality of the obtained DNA (presence of inhibitors of Polymerase Chain Reaction), a β-globin assay was performed.2, 0.2 mMdNTPs, 0.5 μM of each forward and reverse primers and 0.5 U of HotStartTaq polymerase. During each run, the samples were tested together with one negative (nuclease-free water) and one positive control .EBV DNA detection: All PCR reactions were carried out in the final volume of 25 μL using HotStartTaq DNA Polymerase . Concentrations of PCR reaction components were prepared as follows: 2.0 mM MgClIt was performed using the INNO-LiPA HPV Genotyping Extra assay . The kit is based on the amplification of a 65 bp fragment from the L1 region of the HPV genome with SPF10 primer set. PCR products are subsequently typed with the reverse hybridization assay. This kit identifies 28 HPV genotypes: HPV 6, 11, 16, 18, 26, 31, 33, 35, 39, 40, 43, 44, 45, 52, 52, 53, 54, 56, 58, 59, 66, 68, 69, 70,71, 73, 74, and 82.w/v) were prepared in 0.1 mol. l–1Tris-HCl buffer, pH = 7.4 using a laboratory MPW-120 homogenizer, and supernatants were obtained by centrifugation at 5000× g for 30 min.The tissue samples were rinsed with 0.9 % NaCl and stored at −80 °C until the analysis. Tissue homogenates (10 % The following oxidant parameters were determined in homogenates of cancer tissue: activities of superoxide dismutase (SOD) and glutathione peroxidase (GPx).SOD activity was determined using diagnostic kit RANSOD produced by RANDOX  according to Arthur and Boyne  and exprGPx activity was determined using diagnostic kit RANSEL produced by RANDOX  according to Paglia and Valentine  and exprU-test and Kruskal–Wallis test were used. Pearson’s chi-square test was used to investigate the relationship between clinical and demographical parameters. Statistical significance was defined as p  <  0.05.Descriptive statistics were used to present patient baseline characteristics. Means and standard deviations (SD) were calculated. For variables with non-normal distribution, the Mann–Whitney Both GPx and SOD values were the highest in HPV(+) patients, lower in EBV(+), and the lowest in EBV/HPV coinfection (GPx level in HPV(+) = 4.99 U/mg of protein, in EBV(+) = 4.47 U/mg of protein, in EBV/HPV coinfection = 3.75 U/mg of protein; SOD level in HPV(+) = 11.49 U/100 mg of protein, in EBV(+) = 11.06 U/100 mg of protein, in EBV/HPV coinfection = 9.77 U/100 mg of protein). There was no significant difference in the activity of GPx and SOD in patients infected with only EBV or HPV. However, the activity of both antioxidant enzymes in oropharyngeal cancer tissue in patients with EBV/HPV coinfection was statistically lower than in a single EBV or HPV infection .Comparison between GPx and SOD activity and histological grading as well as TN classification was also carried out . In poorCoinfection with two or more oncogenic viruses may induce neoplastic transformation ,7,26,27.Our previous studies revealed that HPV/EBV coinfection was found in 37.8% of patients with oropharyngeal cancer. Comparison of a single EBV infection and a coinfection, regarding different clinical and pathological features of the lesion, showed a significant relationship between histological grading and presence of EBV/HPV coinfection. In the group with EBV/HPV coinfection, the prevalence of poorly-differentiated tumors (G3) was over four-fold more frequent than in the group with EBV infection alone. . In the Jalouli et al.  reportedSome researchers suggest that coinfections with high-risk HPV and EBV are important factors in initiation of carcinogenesis in human oral epithelial cells . Both HPIt has been suggested that malignancies directly connected with several oncogenic viruses may be a result of prolonged latency following a chronic infection . The mecAs several studies revealed, both EBV and HPV can act via ROS-based mechanisms. Long-term expression of the EBNA1 causes increased ROS and a nicotinamide adenine dinucleotide phosphate (NADPH) oxidase level ,37. AccoDecreased antioxidant defense with increased oxidative stress was reported in patients with oral cavity cancer . AccordiStudies concerning oxidative stress and EBV documented that oxidative agents causing damage to DNA induce the expression of EBV lytic genes. Carcinogens and EBV lytic infection synergistically increase oxidative stress, which is an integral link between environmental factors and EBV-associated cancers .As some studies revealed, decreased antioxidant enzyme activity may also be connected with an advanced stage of malignant lesion and with the lowering level of histological differentiation of HNC tumor ,44. SrivA relationship was also reported between EBV and poor differentiation of the tumor tissue in OSCC . In expeOur study is the first original observation that implicates HPV and EBV coinfection with selected parameters of oxidative stress in pharyngeal cancer in the Polish population. A limitation of our research is, however, the small size of the group with coinfection, which makes statistical data comparing the relationship between viral coinfection and histological grading or TN classification insufficiently strong. HPV and EBV are the most commonly transmitted human viruses, but the prevalence of EBV and HPV infection is much more frequent than the incidence of malignant lesions connected with these infections. Finding factors or processes which lead to malignant transformation may be helpful in predicting which individuals exposed to these ubiquitous viruses may develop malignant tumors. Moreover, biological markers associated with viral infections and/or oxidative stress might be useful in diagnosis and treatment of cancer connected with oncogenic viruses, e.g., the treatment of EBV-positive tumors may include the use of targeted inhibitors of signaling cascades. Similarly, disorders in antioxidant enzyme status may be considered a possible biomarker and therapeutic target in cancer treatment strategy. Therefore, further studies are necessary to elucidate the influence of viral interplay, including EBV lytic reactivation induced by HPV and/or oxidative stress on malignant transformation.The present study revealed significantly decreased level of GPx and SOD activity in oropharyngeal cancer tissue with EBV/HPV coinfection compared with a single infection with EBV or HPV. In poorly-differentiated tumors, GPx and SOD activity was decreased compared with well-differentiated lesions. In cases with greater tumor dimensions (T3-T4) and lymph-node involvement (N3-N4), the level of antioxidant enzymes was lowered. Biological markers associated with viral infection and/or oxidative stress might be useful in diagnosis and treatment of cancer connected with oncogenic viruses.


Cervical cancer (CC) is one of the most common types of cancer that affect females worldwide with hundreds of thousands of women dying annually due to this disease, mainly in developing countries. Infection with human papillomavirus (HPV) is the main risk factor for this cancer. There are no public awareness and national immunization programs in most Arab countries. This study aimed to investigate the knowledge and awareness about the HPV vaccine among females in four Arab countries and their acceptance to receive the vaccine. A cross-sectional study was conducted in several Arab countries: Jordan, Qatar, the United Arab Emirates (UAE), and Iraq. Respondents that fulfilled the desired criteria and were willing to participate in the study were asked to fill out the survey. Knowledge and awareness were assessed using 13 questions. Ethical approvals were given from the four countries. A total of 3658 individuals participated in the study; however, 2804 responses were included in the analysis and more than one third of participants (n = 1007) were aged between 18 and 25 years old. This study revealed poor awareness and knowledge of the participants about HPV and its vaccine among all four countries’ participants with relatively better knowledge among participants from the UAE. Participants who are younger (18–25 years old), have a postgraduate education, have an education or career related to the medical field, or had a Pap smear in the last three years tend to have higher knowledge about the HPV vaccine compared to others. Poor knowledge and awareness findings in this study were expected, considering the lack of public education campaigns regarding the HPV virus coupled with the absence of the HPV vaccination from the national immunization schedule in three participating countries . It is recommended that there is a need to provide national educational campaigns about the HPV vaccine to the public in all Arab populations. It is the fourth most common type of cancer in females worldwide and every year several hundred thousand women die because of this disease, mainly in developing countries3.Cervical cancer (CC) is one of the greatest threats to women’s health as one woman dies every two minutes due to this disease2.One of the main risk factors for CC worldwide is infection with human papillomavirus (HPV), which is the most common sexually transmitted infection. This virus might also cause other anogenital cancers and many health problems in both genders4. The World Health Organization (WHO) recommends HPV vaccines as part of routine vaccinations in all countries5. Currently, these vaccines are highly used worldwide, and it is considered part of the national vaccination programs in 105 countries6.In 2006, the first vaccine against HPV was approved by the Food and Drug Administration (FDA) of the United States of America for the primary prevention of CC. Then, two more prophylactic vaccines against HPV were also registered. The HPV vaccination is routinely recommended for adolescents at age 11 or 12 years old and can be given as early as 97.Globally, there are several factors which might influence the slow uptake of HPV vaccines, including financial constraints, weak infrastructure for the adolescent vaccine delivery, lack of reliable data on the burden of the HPV disease, and cultural and religious sensitivities related to this topic8. These numbers are expected to increase dramatically in a few decades unless effective public health interventions are introduced9. In addition, studies conducted in Arab societies indicated that the prevalence rate of HPV was 16% in the general population and 80% in CC patients11. Moreover, there is no HPV vaccination program implemented in these countries, except in the United Arab Emirates (UAE) which has a preliminary HPV program12. In addition, there is no official CC screening program and the information related to this health issue is mainly collected from opportunistic screening tests in Arab societies15. Generally, the societies in the Arab countries are characterized by sharing relatively comparable cultures and religious conservatism7. This implies a conservative sexual behavior. Moreover, discussing these issues is relatively very low among people in the society with no formal sexual education including health sexual education in most Arab countries16. Therefore, the people in Arab countries are facing a threatening health problem of increased CC. This is mainly due to the lack of effective CC screening programs and vaccination programs as well. In addition, there is low knowledge and attitude among people relating to the HPV vaccine in some Arab societies19.In Arab countries, more than ten thousand women were diagnosed with CC (with 7365 deaths) in 2018The aim of the study was to evaluate the knowledge about the HPV infection, the awareness towards the HPV vaccination, and the perceived barriers among women in four Arab countries: Jordan, Iraq, the UAE, and Qatar. Furthermore, the aim was to assess public acceptance and willingness to receive the vaccine.The present study is a cross-sectional survey, which was intended to investigate the knowledge and awareness about HPV infection, its correlation to cervical cancer, and its vaccine among females in the Arab region.The study was conducted after obtaining ethical approval from the Institutional Review Board (IRB) of Jordan University of Science and Technology (JUST) and King Abdulla University Hospital (KAUH) in Irbid, Jordan (201/132/2020). The protocol of the study was approved by the Health Research Governance Department at the Ministry of Public Health (MoPH) as Exempt Research in Qatar. Moreover, the study was approved by the Research Ethical Committee at the University of Sharjah in the United Arab Emirates (REC-20–05-17–01) and the Scientific Research Committee at the University of Baghdad in Iraq (3311/4–2020/6/29). All methods were carried out in accordance with relevant guidelines and regulations.The study was conducted in the four Arab countries of Jordan, Qatar, the United Arab Emirates (UAE) and Iraq. Females aged ≥ 18 years from these countries were invited to participate in the study. Exclusion criteria were (a) males, (b) participants living in countries other than those included in the study, and (c) respondents less than 18 years old.The questionnaire was distributed utilizing the electronic format Google Forms. The link to the survey which was preceded by an introductory section about the study was sent via various social media platforms. The introductory section described the aim and objectives of the study and the voluntary nature of participation with a consent statement if they would like to take part in the study. Therefore, consent to participate was considered taken if the respondent signed the consent form electronically and filled out the questionnaire. Informed consent was obtained from all study participants. The questionnaire was terminated automatically if participants declined to take part or if they were males or younger than 18 years old. The participants were requested to complete the survey without consulting people, materials, textbooks, or internet web pages. All methods were approved by the IRB committee.The questionnaire utilized to collect data from the females was developed by the authors via an extensive review of the literature. The final version of the questionnaire was composed of three sections. The first section was designed to collect general demographic data about the participants such as age, gender, occupation, and level of education. The second section included 13 questions aimed to evaluate the participants’ general knowledge and awareness about HPV and its vaccine. These questions were related to the mode of transmission of virus, its correlation to CC, and the vaccine benefit, availability, target population, and side effects. Each answer scored as correct or incorrect. The respondent was given a zero for each wrong answer and one point for each correct answer, and the results were summed to give a total score out of 13. Participants who scored > 50% were considered to have good knowledge and who scored 50% and less considered as having a poor level of knowledge/awareness.The third section intended to assess the public acceptance to receive the HPV vaccine and willingness to recommend it to a child or adolescent (age between 9 and 12 years old) or friend. Another part of this section was related to concerns about the vaccine, including worries regarding side effects, efficacy, and the financial burden. The questionnaire was in the Arabic language and reviewed by the authors and then subjected to a pilot testing by 40 participants to ensure the clarity of the questions, which resulted in several minor amendments.The target sample size was estimated based on the Raosoft software sample size calculator for the minimal sample size needed for an unlimited population size using a confidence interval of 95%, a standard deviation of 0.5, and a margin of error of 5%. The required sample size was 385 participants from each study population.Data were analyzed using the Statistical Package for the Social Sciences (SPSS) version 22. Respondents’ demographic characteristics were described using descriptive statistics. Differences between categorical data were detected using a chi-square test. Continuous data was reported as median  for non-normally distributed variables. The relationship between the knowledge score and the independent categorical variables was determined using the Mann–Whitney U test for binomial variables and the Kruskal–Wallis test for multinomial variables. Variables that were significantly associated with the knowledge score were included in the multiple linear regression analysis after log transformation of the knowledge score. A statistical significant difference was considered if the p-value was less than 0.05.20.To exclude careless answers, we added a question at the end of the questionnaire if participants would recommend including their response in the analysis. This approach was utilized, as suggested by Maede et al., as a Self‐Reported Single Item (SRSI) IndicatorA total of 3658 individuals participated in the study, of which 854 were excluded from the analysis . Thus, in the present analysis, we analyzed a total of 2804 responses of which 1216 were from Jordan, 397 were from Qatar, 606 from the UAE, and 585 from Iraq. Around one third of participants  were aged between 18 and 25 years. The largest proportion of the participating females were unemployed . About half of the participating females  were studying non-medical sciences. General demographic data of the included participants are presented in Table Knowledge and awareness about HPV and its vaccine were measured using 13 questions with one point for each question. The overall median knowledge score was 2 (IQR = 5) in the whole population. Table The percentages of participants who were aware that the HPV infection will not go away on its own without treatment and that the infection causes CC were 35.2% and 43.8%, respectively. Additionally, 26.9% of participants knew that the HPV infection is a sexually transmitted disease. About a quarter of participants (25.6%) were aware that the vaccine does not protect against all types of CC. Only 21.3% of participants were aware that HPV causes some side effects such as headache and nausea, and 20.3% knew that the HPV vaccine can prevent CC and decrease the chance of having changes in the Pap smear test. Moreover, the proportion of participants who were aware that the target population for vaccination is both males and females and that taking the vaccine will not infect the recipient were 20.7% and 23.6%, respectively.The lowest median score was 1.0 (IQR = 4) for participants from Qatar while the highest median score was 3.0 (IQR = 5) for participants from the UAE.2) for the current model was 0.165, which is 16.5% of the variance.The results of the univariate analysis are presented in the supplementary material  young participants ≤ 25 years old, (b) participants from the UAE, (c) participants who have a postgraduate degree, (d) participants with education related to the medical field, (e) participants with a career related to the medical field, and (f) participants who had a Pap smear in the last three years.Among all participants, only 3.0% n = 84) reported that they had received the HPV vaccine. Moreover, 43.2% of participants reported that they are willing to receive the vaccine while 52.6% reported that they cannot decide whether they are willing to get the vaccine or not according to the information they have. Only 7.4% would recommend the HPV vaccine for a child or adolescent and 29.0% would recommend it for a friend or relative. Detailed responses are presented in Table  reportedFigure The current study investigated the level of knowledge and awareness about HPV and its vaccine among women living in four Arabic countries: Jordan, Qatar, the UAE, and Iraq. Collectively, the present findings suggested relatively poor awareness and knowledge of the participants about HPV and its vaccine in the four Arab communities with relatively better knowledge among participants from the UAE. This finding was expected considering the lack of public education campaigns regarding the HPV virus coupled with the absence of the HPV vaccination from the national immunization schedule in three of the participating countries .22.The results regarding the awareness of the relation between the HPV infection and CC showed inadequate knowledge about CC epidemiology compared to other studies conducted on Arab and non-Arab communities24. Interestingly, the Ortashi et al. study in 2013 reported that 22.0% of females in the UAE have heard about the vaccine, but our results showed a much higher percentage of 38.9% for the UAE population25.Collectively, only 26.1% of sampled participants have heard about the vaccine. This percentage is lower than what has usually been found in European countries and based on some studies even that reported in other countries with Islamic majorities such as Turkey26. The DNA of high-risk HPV was found in 99.7% of CC specimen27. Unfortunately, the present study results showed relatively low knowledge of this strong link between the HPV and CC among study participants, especially in Qatar. Furthermore, only about a quarter of the participants had heard about the HPV vaccine, which could explain the low knowledge of participants about its various aspects especially in its role to protect against CC. This is information that is quite important to communicate to the public, as it was reported that breast cancer, leukemia, and CC were the most common incident cancers in the Eastern Mediterranean Region28.Oncoviruses can enable different carcinogenesis stages, with one such virus being the HPV. Although within two years of infection most HPV infections can be cleared or deactivated, high risk HPV types remain and can progress to CC17. A result that was noted in the current study. We also found higher knowledge and awareness about the HPV and its vaccine among students in the medical field. Furthermore, younger age was associated with higher knowledge and awareness regarding HPV and its vaccine. A possible explanation is that the peak incidence of HPV is 25 years and that the vaccine is recommended to be taken routinely for ages 11–26 although it can be given to selected adults between 27 and 45 years old30.It was previously reported that being employed in the medical field was associated with higher knowledge and awareness about the HPV and its vaccine34.Despite the relatively poor overall detected knowledge regarding the HPV and its vaccine, almost half of women who responded to this survey reported their willingness to receive the vaccine, but a smaller proportion reported recommending it to a friend or a child. Refraining from recommending the vaccine to children can be understood considering the fragility of this population coupled with the low detected level of knowledge regarding the vaccine. The willingness of women to take the HPV vaccine despite their poor knowledge about it has also been reported by other researchers in both Arab and other countries17. Therefore, it is recommended that a national HPV awareness campaign should be stratified in Arab countries to gap the knowledge about the HPV vaccine.In the present study, more than half of the respondents from all participating countries reported inadequate information about CC and the HPV vaccine which was the main concern about receiving or recommending the HPV vaccine. The other main concern was about the side effects which was a similar finding to a study on public from another Arab community in BahrainTo the best of our knowledge, this is the first and largest cross-sectional study (involving 2804 participants) in the Middle East that explored knowledge about the HPV infection, its correlation to CC, and awareness about the HPV vaccine. The large sample size improved the generalizability of these findings.35. Despite this measure, the elimination of careless responses cannot be completely assured.Limitations of the present study include the inability to calculate the response rate as the survey was distributed electronically, the possibility of selection bias due to the online nature of the survey, and the possibility of careless responses. Consequently, we may have missed some of the targeted population. However, we tried to overcome this by distributing the survey among four different populations and widely used social media. In the present study, the detected careless responses based on self-reported single item was 6.4% of the total received responses. This was higher than previous research studies (2.8%) by our groupThere is limited knowledge and awareness about the HPV infection, its correlation to cervical cancer, and the HPV vaccine in Arab communities. Predominance for the willingness to take the HPV vaccine was also accompanied with concerns regarding inadequate knowledge about it.Participants who are younger (18–25 years old), have postgraduate study, have an education or career related to the medical field, and had a Pap smear in the last three years tend to have higher knowledge and awareness about the HPV vaccine compared to others.The results of this study reveal an insufficient level of knowledge about CC and the HPV vaccine; therefore, we recommend that there is an actual need to provide national educational campaigns about the HPV vaccine to the public among all Arab communities. We have highlighted some important gaps in information about the HPV infection and its vaccine, which can be addressed in the future.Supplementary Information.


As the leading human papillomavirus (HPV)-related cancer, oropharyngeal cancer places a significant burden on patients, families, and health systems. Techniques to easily and quickly test people for HPV through non-invasive means  could, in principle, help us better understand this disease, prevent it, and treat it. However, there is currently no standardized methodology for testing saliva or blood for HPV, and such testing is not a part of routine clinical practice. In this review, we discuss and compare some of the collection and testing platforms that researchers have studied to date. We also evaluate the potential strengths and limitations of these technologies for addressing unmet needs in specific clinical contexts.The burden of human papillomavirus (HPV)-related oropharyngeal cancer (OPC) has risen, now representing the most common HPV-related malignancy. For years, researchers have explored the utility of measuring HPV-related markers from mouth, throat, and blood samples, often with the aim of gathering more information about an existing HPV-related tumor in a given patient. We review the widely varying methods for collecting and testing saliva and blood samples and offer guidance for standardizing these practices. We then review an array of clinical contexts in which non-invasive testing holds the most promise for potentially addressing unmet needs. In particular, such testing could help clinicians and researchers monitor the effects of vaccination and treatment. Meanwhile, due to the currently incomplete understanding of how carrying HPV relates to infection and subsequent oncogenesis, non-invasive testing methods may not be suitable for the screening setting at this time. Human papillomavirus (HPV)-associated oropharyngeal cancer (OPC), with an estimated 12,600 new cases per year in the United States, now outpaces all other HPV-associated malignancies by incidence  . Across Unlike many other cancers, OPC is associated with a distinct, well-characterized, relatively homogeneous etiology in most cases (>70%): HPV infection . This diIn this review, we compare the methodologies that researchers have most commonly examined for sample collection and analysis of HPV among selected studies in recent literature. Among the expanse of research available on the topic of non-invasive HPV testing, we chose to focus our discussion on later studies (2017 and onward). While we chose, when possible, to discuss studies with large sample sizes, we also consider smaller studies that offer exceptional insights . We limit our scope to the most commonly studied sample collection and testing methodologies. We discuss the value that HPV testing may offer in a range of specific public health and clinical scenarios related to HPV-associated OPC.Oral rinse is a common method for collecting samples from the mouth and throat . Other aAmong oral rinse sample studies, there is substantial variation in specific approach to this collection method. Generally, participants held the collection fluid  in their mouths for 15–30 s ,17,18. TWhat is unclear, given these varying methodologies, is whether some methodologies detect more HPV due to greater sensitivity or due to lower specificity. That mystery leads us to a dilemma: what constitutes clinically meaningful HPV infection? Does the mere presence of HPV-related material in the mouth or throat mean that an individual has a potentially pathogenic infection? The fact that several studies demonstrated higher HPV rates in oral rinse samples compared to brushing and swabbing samples may indeed indicate that presence of HPV in oral rinse is not the same as infection of cells that may be collected with brushes or swabs. This question warrants further research.Future studies involving oral HPV biomarkers should provide ample detail relating to sample collection methodologies to allow for greater standardization and comparability across studies. Investigation involving head-to-head comparisons of varying oral rinse approaches is an unmet need and may help to provide insight on the question of viral presence versus persistent infection.Investigators performed HPV detection and, in some cases, quantification, using polymerase chain reaction (PCR)-based molecular analysis. Primers were designed to identify one or more of the HPV E6, E7, and L1 oncogenes. Investigators commonly used the Roche Linear Array HPV genotyping platform for non-quantitative HPV detection. Some studies incorporated reverse transcriptase (RT)-PCR, quantitative PCR (qPCR), or a combination of the two (RT-qPCR), while others made use of the more recently developed droplet digital PCR (ddPCR) method . AlthougAlthough less common than molecular testing of mouth and throat samples, there has been some exploration of HPV-directed antibodies in these samples . We addrWe observe two general approaches to serum testing: molecular analysis of circulating tumor DNA (ctDNA) and enzyme-linked immunosorbent assay (ELISA)-based analysis of HPV-targeted antibodies. In a study that compared ELISA-based detection of E6 antibodies with oral HPV16 DNA detection, nominal sensitivity of the antibody test was substantially higher (88% versus 43–51%), with similar specificity (≥98%) ,26,27. ASensibly, studies aimed at understanding the global epidemiology of HPV infection in the absence of a cancer diagnosis make use of mouth and throat testing rather than the slightly more invasive blood testing. Estimates of the prevalence of HPV genetic material in the mouth and throat in relatively unselected populations across geographies range from 1% to over 15% ,31,32,33Interestingly, one study demonstrated that the concordance of HPV presence and strain between bodily sites (mouth versus penis versus anus) was low . This fiBroad epidemiological studies using non-invasive HPV biomarker testing could reveal yet-unrealized risk factors for HPV-related disease. For example, we observe that the prevalence of mouth and throat HPV seems to be higher in individuals undergoing tonsillectomy compared to other settings . RoughlyParker and colleagues demonstrated that both mouth/throat and serum HPV-directed antibodies can offer information that correlates to some degree with timing since HPV vaccination . In theiKey issues that must be addressed to assess the utility of screening healthy individuals for the presence of HPV in saliva or serum include how results would impact clinical decision-making and whether screening could be cost-effective.Although, as expected, carrying oral HPV is associated with a greater risk for OPC (nearly 50 times the odds for a person carrying HPV16 versus someone who does not) , the incOlder non-invasive biomarker studies helped to establish the basis for our current understanding of risk factors for HPV-related oropharyngeal cancers. Knowledge gained from this scholarship could help determine which groups might be the most appropriate candidates for screening programs. Included on the lengthy list of (now well-accepted) risk factors determined by these studies to increase the risk of OPC are male sex, increased number of lifetime vaginal sex partners, and increased number of lifetime oral sex partners ,39. ThesInterestingly, the results of one more recent study, consistent with prior work, indicate that carrying oral HPV does not correlate with abnormal cytologic findings ,38. ThisAlthough the approach of using serum antibody testing as a screening tool is less common than testing orally collected specimens in the literature, there is some evidence that this strategy could be associated with higher accuracy compared with oral testing . HoweverIn all, non-invasive HPV detection would have a high bar to meet in terms of cost-effectiveness, given that a minority of patients with evidence of HPV infection will go on to develop OPC and that there are some open questions about the utility and accuracy of testing methods in this setting. Before (and if) non-invasive HPV testing could be useful in screening, a greater understanding of the implications of carrying HPV  would be needed.®  platform has demonstrated clinically meaningful utility in tracking response or outcomes to chemoradiotherapy [While quantifying HPV infection through non-invasive testing at OPC diagnosis does not seem to offer much prognostic value, there are promising signals of benefit associated with non-invasive HPV testing as part of monitoring treatment effect . The comotherapy . Individotherapy ,42. Thes® technology has also shown potential in the setting of surveillance following completion of definitive therapy. The test demonstrated a negative predictive value of 100% in anticipating disease-free status among individuals with undetectable HPV ctDNA at all post-treatment timepoints and demonstrated a 94% positive predictive value for recurrence among individuals with two consecutive positive results after completing therapy [® trial have been observed in other studies using different PCR-based technologies and other collection methods (including non-invasively collected sample from tumor brushings) [Our prior work has demonstrated that the natural course of salivary HPV-targeting antibodies is to decline over time following completion of definitive therapy for OPC . Beyond  therapy . The sec therapy . Similarushings) ,19,21. Sushings) . A numbeushings) .Non-invasive HPV testing has the potential to be of great use in certain public health and clinical contexts related to HPV-associated OPC. However, greater understanding of the pathogenic mechanisms linking HPV exposure to HPV infection and those linking HPV infection to OPC tumorigenesis are prerequisites to fully harnessing the value of non-invasive testing. For HPV testing to be useful in the screening setting, clinicians would require additional information besides the presence or absence of HPV genetic material or of immune response to HPV. Detecting potentially oncogenic infection using markers of gene expression  is a ripe area for study in the screening setting. Once we can appreciate which markers are most meaningful for predicting oncogenesis, we will be in a position to conduct head-to-head trials to understand which tissue collection and laboratory testing methodologies provide the most accurate measures of those markers. After comparative studies have provided indications of optimal collection and testing techniques, clinician researchers will then be able to work out how best to use those diagnostic tools to guide clinical decisions in the screening setting. In sum, there is currently insufficient data to support the use of non-invasive HPV testing in the screening setting. Clinical data demonstrating improved outcomes resulting from non-invasive screening would be a requirement for this strategy to prove useful. At present, the clinical implications of positive screening test results are unclear.Testing immunogenicity following vaccination can help optimize vaccine development and public health strategies aimed at vaccination. We recommend that future vaccine clinical trials incorporate immunogenicity testing to understand if some groups of patients could benefit from more or fewer doses. Trialists could also attempt longer follow-up  to gain a greater understanding of how markers of immunogenicity correlate with observed risk of HPV infection and HPV-associated malignancy.Monitoring HPV levels during definitive therapy could, in the future, help personalize treatment. We could hypothesize, for example, that individuals whose HPV markers drop precipitously early on in definitive therapy might be candidates for de-escalation . Inversely, those with persistently elevated HPV markers might benefit from a more extended course of definitive therapy or novel adjuvant regimens. Incorporating non-invasive HPV testing as part of routine monitoring in clinical trials studying definitive therapy can help us uncover such fresh approaches. Incorporating non-invasive HPV testing into follow-up after definitive therapy in these trials could offer insight into how such testing could help detect recurrences earlier. Even only with currently available data, we believe that there is a role for plasma HPV ctDNA platforms as an adjunct to traditional response metrics  in the context of monitoring response and surveilling for recurrence. Although research to date has focused on treatment with curative intent, future studies should examine the utility of monitoring HPV in the recurrent/metastatic setting. Especially as our understanding of how HPV causes OPC improves, non-invasive HPV testing promises to offer innovations in the field of HPV-related OPC.


The dominant paradigm for HPV carcinogenesis includes integration into the host genome followed by expression of E6 and E7 (E6/E7). We explored an alternative carcinogenic pathway characterized by episomal E2, E4, and E5 (E2/E4/E5) expression. Half of HPV positive cervical and pharyngeal cancers comprised a subtype with increase in expression of E2/E4/E5, as well as association with lack of integration into the host genome. Models of the E2/E4/E5 carcinogenesis show p53 dependent enhanced proliferation in vitro, as well as increased susceptibility to induction of cancer in vivo. Whole genomic expression analysis of the E2/E4/E5 pharyngeal cancer subtype is defined by activation of the fibroblast growth factor receptor (FGFR) pathway and this subtype is susceptible to combination FGFR and mTOR inhibition, with implications for targeted therapy. PIK3CA mutations in comparison to other solid tumors, and the PIK3CA-AKT-mTOR pathway is the most frequent dysregulated signaling pathway in HNSCC, including >80% of all HPV negative and HPV positive cases [Human papillomavirus (HPV) is a ~7.9 kb, non-enveloped, double-stranded, circular DNA virus that has a specific tropism for squamous epithelium, and persistent infection with oncogenic HPV subtypes is associated with development of cancers of the cervix, oropharynx, vagina, vulva, penis, and nasal cavity. HPV is responsible for 4.5% of all cancers worldwide  . High-riAfter infection, HPV viral DNA may remain in a latent infective state in affected basal epithelial cells that can transit to a productive neoplastic infection, followed by transformation over many years to invasive cancer. A critical step in progression to cancer is thought to be the integration of HPV DNA sequence into the host genome , associaIn this study, we validated the oncogenic potential of a non-integrated, E2/E4/E5 expressing subtype in HPV positive HNSCC as well as in cervical carcinoma. To provide functional insights into the HPV16 E2/E4/E5 subtype, we mimicked HPV16 E2/E4/E5 carcinogenesis using in vitro and in vivo models, and performed single sample gene set enrichment analysis (ssGSEA) to map key downstream networks and identify novel therapeutic targets for HPV positive HNSCC. This approach defines a novel mechanism of HPV carcinogenesis driven by non-integrated, E2/E4/E5 expression, and will provide opportunities for novel, effective therapies for these tumors.n = 55, 79.71%), HPV33 , and HPV35  genomes independently, and calculated read counts of each gene  in TCGA HNSCC dataset , but this was not significant (P = 0.41) .HPV gene transcripts include complex alternative splicing arrangements with inclusion of multiple genes in a single transcript , 18. WheP < 0.05; Fig. To define the effects of E2/E4/E5 further, we conditionally expressed the E2/E4/E5 genes using a doxycycline inducible cytokeratin 5 promoter—reverse tetracycline transactivator (cK5-rtTA) system as described . We isolP < 0.05; Fig. A hallmark of HPV mediated carcinogenesis is inhibition of p53 and Rb through the actions of E6 and E7, respectively. The definition of a clinical HPV cancer phenotype that preferentially works through E2/E4/E5 expression implies that these genes may also affect key cell cycle components and DNA damage response mediators like p53, and prior data indicate that HPV16 E2 binds p53 , 22. To 137Cs and collected after 24 h at the same time with control cells to monitor cell cycle changes , and cells in the S-phase increased significantly (P < 0.01), which indicated that a G1-arrest was partially blocked by E2/E4/E5   Fig. . These s02) Fig. .Fig. 4HPTo identify gene sets that correlated with each HPV gene expression subtype, we analyzed RNAseq RSEM in TCGA HPV positive HNSCC dataset and our JHH HPV positive OPSCC dataset using ssGSEA  and idenT308, and synergized with rapamycin in decrease in pFGFR1, FGFR1, pFGFR3, FGFR3, pFRS2, FRS2, pAKTT308 and pS6  of the alphapapillomavirus genus primarily contains HPV16, HPV33, HPV35. These HPVs account for more than 60% of cervical cancers , 30 and In order to detect the effect of only downregulating expression of E2/E4/E5 or E6/E7 respectively, we upregulated E6/E7 or E2/E4/E5 at the same time. E6/E7 are oncoproteins and have been attributed to tumorigenesis for decades –35. The To further explore the potential pathways and downstream molecules in E2/E4/E5 subtype, we employed ssGSEA to define significant gene sets in E2/E4/E5 subtype, which includes REACTOME signaling by FGFR3 mutants, REACTOME FGFR ligand binding and activation, and REACTOME FGFR4 ligand binding and activation. FGFRs, a class of RTK, dimerize and undergo transphosphorylation of the kinase domain upon ligand binding, leading to the recruitment of adapter proteins and initiating downstream signaling. Because of the ability of FGFR signaling to induce cell proliferation, migration and survival, FGFRs are readily co-opted by cancer cells . FGFR siPrior studies attempted to identify subtypes in HNSCC treated HPV as a discrete variable (positive vs. negative). In one study, Zhang et al.  identifiIn conclusion, we found that concurrent expression of HPV16 E2/E4/E5 genes represents an alternative pathway to HPV-related carcinogenesis and defined an alternative mechanism which may provide an opportunity for a novel understanding of HPV carcinogenesis and may allow for rational therapeutic design to interrupt networks involved in cancer development.https://github.com/favorov/viruses-in-sequencing) [Both HPV positive HNSCC and CESC datasets from TCGA, as well as an independent primary HPV positive OPSCC dataset from JHH, were used in this study. Methods for sequencing and data processing of RNA using the RNAseq protocol have been previously described for TCGA , 6 and Juencing) . Samples2.Colon cancer cell line HCT116 p53+/+ was purchased directly from the American Type Culture Collection . Colon cancer cell line HCT116 p53−/− was a gift from Vogelstein’s lab. Cervical cancer cell line Caski and SiHa, and HPV positive HNSCC cell lines UM-SCC-047, UPCI-SCC-090, UM-SCC-104, and 93-VU-147T were gifts from Sidransky’s lab. HPV positive HNSCC cell line UD-SCC-2, and HPV negative HNSCC cell line Detroit 562 and CAL27 were gifts from Gutkind’s lab. HCT116 p53+/+, HCT116 p53−/−, SiHa, UM-SCC-047, UPCI-SCC-090, UM-SCC-104, UD-SCC-2, 93-VU-147T, Detroit 562, and CAL27 were grown in DMEM . Caski was grown in RPMI-1640 Medium (Sigma-Aldrich). All media were supplemented with 10% FBS (Sigma-Aldrich) and 1% penicillin, streptomycin (Sigma-Aldrich), and cells were cultivated at 37 °C with 5% CO−11 M cholera (Sigma-Aldrich) at 37 °C with 5% CO2.Primary oral keratinocytes isolation and culture was performed as described . PrimaryThe cK5-rtTA transgenic FVB/N mice have been previously described . cK5-rtTt test was performed to analyze normally distributed data. P < 0.05 was treated as statistical significance. Chou Talalay combination index were calculated using CompuSyn software to examine synergistic effects (combination index score < 0.5).Statistical analyses were performed using R/Bioconductor version 3.3.2. The Fisher’s exact test was used to explore the association between mutations and CNAs, and two subtypes, and the association between number of mice with papillomas and two groups. The Log-rank test was applied to explore the association of groups and disease-free survivals. The two-sided Student Supplementary DataFigure S1Figure S2Figure S3Figure S4Figure S5Figure S6Figure S7Figure S8Figure S9Table S1Table S2Table S3Table S4Table S5


Lactobacillus sp. enrichment coincided with the depletion of L. iners and Shuttleworthia, which dominate under normal physiological conditions. Changes in the diversity of microbial species in HPV+H groups influence the capacity of new community members to produce or consume metabolites. In particular, the functionalities of four metabolic enzymes were predicted to be associated with the microbiota, including acylphosphatase, prolyl aminopeptidase, prolyl-tRNA synthetase, and threonyl-tRNA synthetase. Such metabolic changes may influence systemic health effects in women at risk of developing cervical cancer. Overall, even assuming the limitation of the power due to the small sample number, our study adds to current knowledge by suggesting how microbial taxonomic and metabolic shifts induced by HPV infections may influence the maintenance of microbial homeostasis and indicate that HPV+H infections may alter the ecological balance of the cervicovaginal microbiota, resulting in higher bacterial diversity.In this study, we evaluate the association between vaginal and cervical human papillomavirus infections high-risk types (HPV+H), negative controls (HPV−), the bacterial biota, and urinary metabolites via integration of metagenomics, metabolomics, and bioinformatics analysis. We recently proposed that testing urine as a biofluid could be a non-invasive method for the detection of cervical HPV+H infections by evaluating the association between cervical HPV types and a total of 24 urinary metabolites identified in the samples. As a follow-up study, we expanded the analysis by pairing the urine metabolome data with vaginal and cervical microbiota in selected samples from 19 Puerto Rican women diagnosed with HPV+H infections and HPV− controls, using a novel comprehensive framework, Model-based Integration of Metabolite Observations and Species Abundances 2 (MIMOSA2). This approach enabled us to estimate the functional activities of the cervicovaginal microbiome associated with HPV+H infections. Our results suggest that HPV+H infections could induce changes in physicochemical properties of the genital tract through which niche partitioning may occur. As a result,  Lactobacillus spp. [Sneathia spp. were considered cervical biomarkers for neoplasia [Atopobium vaginae and Gardnerella vaginalis in patients with CIN3 lesions with HPV+H, low-risk HPV, or both co-infections [Sneathia sanguinegens, Anaerococcus tetradius, and Peptostreptococcus anaerobius [Oribacterium, Lachnobacterium, and Thermus were associated with HPV 16, while a composition of Motilibacter associated with HPV 52, and a composition of Litorilinea and Paludibaculum with a concomitant paucity of L. iners with HPV 58 [Candida sp, and bacterial vaginosis (characterized by a reduction of Lactobacillus and overgrowth of anaerobic bacteria) [Discoveries resulting from human microbiome research have demonstrated that microbes play a role in the susceptibility to HPV and neoplasia ,2,3. Onclus spp. ,13. In Meoplasia , while ifections . Vaginalaerobius . Anotherh HPV 58 . Lastly,acteria) . Most ofThe integration of multi-omics techniques such as metagenomics and metabolomics is becoming a very promising approach for the understanding of the complex bacterial interactions with the host and their contributions to systemic health. Given that many metabolites and metabolic pathways are relatively conserved across species, the coupling of two omics approaches provides insights into the association of the human microbiome with health status and risk of disease development . Much woMost of the studies associating the microbiome and systemic health were conducted via assessment of interactions between the gut microbiome and gut metabolome . A populAs a follow-up study, in the current manuscript, we extend our previous analysis aimed at assessing the possible association of urine metabolome with cervicovaginal microbiota in 19 selected Puerto Rican women. A novel comprehensive framework, the Model-based Integration of Metabolite Observations and Species Abundances 2  , was usep-value = 0.026) compared to HPV− patients  and exclusively HPV+H types consisting of n = 11 patients. Our previous metabolomic analysis showed an altered urine metabolome in the HPV+H group compared to HPV− [p-value < 0.1  (VAG) and cervical (CERV)) regarding HPV genotyping into HPV− infections  acylphosphatase (K01512), responsible for the synthesis of acetate from acylphosphate; (2) prolyl aminopeptidase (K01259), responsible for the release of N-terminal proline from a peptide; and (3) prolyl-tRNA synthetase (K01881), responsible for the synthesis of tRNAPro and degradation of proline  activity and positive contributor to the degradation of threonine by threonyl-tRNA synthetase, while no other contributions to identified enzymes were found  and one negative (Arcanobacterium) predictors associated with the synthesis of cystathionine and succinate by cystathionine gamma-synthase . The specific role of this enzyme is still not completely elucidated, and limited information is available about its function. Nevertheless, acylphosphatase is known to participate in glycolysis and pyrimidine biosynthesis [Lactobacillus sp. and Shuttleworthia in HPV+H groups, could lead to the development of cervical cancer. It is recognized that the healthy vaginal microbiome is enriched with hy women . Moreoveynthesis . Moreoveynthesis . Thus, tShuttleworthia and L. iners and an increase in Lactobacillus sp. in HPV+H groups also contributed positively to the activity of prolyl aminopeptidase (K01259), which is one of the critical enzymes that control the cell cycle division. This enzyme is still not considered as the target for antitumor therapies. However, recently, it was reported that inhibition of prolyl aminopeptidase could be a targeted treatment strategy in myeloma [Shuttleworthia, L. iners, and Lactobacillus sp. was positively associated with the degradation of proline and synthesis of tRNAPro by prolyl-tRNA synthetase (K01881), while changes in the composition of Shuttleworthia, Lactobacillus sp., Atopobium vaginae, Megasphaera, and Gardnerella positively predicted the degradation of threonine and the synthesis of tRNAThr by threonyl-tRNA synthetase (K01868) . Atopobium vaginae is a marker for bacterial vaginosis [A. vaginae were reported to be associated with the metabolism of l-threonine [A healthy cervicovaginal environment consists mostly of low-proliferating or quiescent cells. The taxonomic shift likely induced by HPV infections might reprogram cellular metabolism, provoking a “new” metabolic requirement to support uncontrollable cellular proliferation and enhancing protein synthesis. A reduction of populations of  myeloma . Moreove(K01868) . In addiaginosis  and otheaginosis ,43,44. Taginosis ,45, and hreonine . Prolyl-hreonine . Therefohreonine  or parashreonine . Recent hreonine , while ohreonine . Megasphaera was the only one negative contributor to the synthesis of acetate from acetyl-CoA associated with the activity of acetyl-CoA hydrolase (K01067), an enzyme that participates in pyruvate metabolism catalyzing the hydrolysis of acetyl-CoA. The negative contribution may indicate an antagonistic relationship between microbes and competition for the substrate in the same niche [Megasphaera could be associated with the change of pyruvate carbon flux towards the release of lactate, rather than conversion to acetyl-CoA. In this case, acetyl-CoA could be further metabolized to CoA and acetate and eventually lead to the malignant transformation of the cervical cells [In both HPV+H groups, me niche . As a real cells . Shuttleworthia and Arcanobacterium, could predict the production of cystathionine and succinate from cysteine by cystathionine gamma-synthase (K01739), either positively (Shuttleworthia) or negatively (Arcanobacterium). These data suggest that Shuttleworthia and Arcanobacterium were competing for cysteine. We speculate that this competition is required to support a physiological, metabolic balance between anabolic and catabolic reactions in the cervicovaginal environment of HPV− groups. In contrast to HPV+H groups, in HPV− groups, we identified that only two low-dominant members of the microbial community, Overall, the cervicovaginal microbiota seems to regulate the differential synthesis and degradation of amino acids with possible roles in the inflammatory and cell-cycle outcomes, suggesting that these biochemical gradients may result in niche partitioning and physiological outcomes in this infectious context. Indeed, species sharing the same cervical habitat (both dominant and rare taxa) have similar biochemical needs and may use resources in somewhat different ways to avoid direct competition, thus devising a certain niche partition, with more direct competition among the low dominant taxa. These results suggest that metabolite-driven niche partitioning may be an important factor in the maintenance of high microbial diversity in HPV+H infected cervix, although additional studies using cervical lavage metabolomics will add more information as to the specific metabolisms of cervical bacteria during HPV infections. We also cannot discard the relationship between metabolites and microbiota not detected in our study due to low power. Our study provides only a partial view of the metabolite-driven niche partitioning and potential metabolic interactions among these microbes and urine level metabolites in different HPV infections. Metabolite diversity must contribute to niche differentiation and support diversity due to substrate preferences among heterotrophs. However, given that this data relates urine-based metabolites with HPV infections status and concomitant cervical microbiota, it calls for more niche directed studies to be conducted. Indeed, we anticipate that the addition of other samples such as cervical lavages and an increase in the sample number would be required to support general conclusions about ecological niche partitioning in the cervix considering HPV status and metabolic profiling. The functional characterization of vaginal and cervical HPV+H, HPV−, microbiota, and urinary metabolome via integration of multi-omics approaches showed specific differences in microbiome/metabolome associations. We identified species that can predict the production or degradation of key metabolites, either positively or negatively, contributing to the pool of circulating urinary metabolites. However, since MIMOSA2 is a tool for hypothesis generation, further validation studies would be needed to confirm whether the taxa and genes in question are modifying the metabolites according to the predictions. We also recognize that other confounding effects, such as inter-individual variation and diet, can be a limitation when analyzing serum and urine metabolomic levels. Moreover, further investigations are required using larger patient cohorts, including samples with cervical neoplasia or cancer and application of different analytical platforms to validate our finding or identify the other metabolites/microbiota relationships that are beyond the scope of this study. Nonetheless, our findings that HPV+H and HPV− cervicovaginal microbiomes and urinary metabolomes were functionally separated through niche partitioning offers additional information on the complex systemic interactions of the microbiome in human health. Taken together, analysis of functional coupling of cervical microbiome/host urine metabolome predicts the occurrence of specific metabolic abnormalities that could lead to cervical cancer development.We selected 19 samples  corresponding to 8 HPV− and 11 HPV+H (HPV low risk were not considered) from the analyses of 62 patient samples from women coming for gynecology evaluation at the University of Puerto Rico and San Juan City clinics (San Juan Metropolitan area). Samples were obtained under the Ethics Committees of the UPR-Medical Sciences Campus IRB (Protocol ref. 1050114), described in Godoy-Vitorino et al. (2018a). Cervical and vaginal samples were collected by the ob/gyn after informed consent procedures, and HIPAA forms were signed in accordance with the Declaration of Helsinki. Besides swabs, urine biofluid was self-collected at the time of gynecology evaluation. All samples were stored at −80 °C and processed for further genomic DNA and metabolite extractions. HPV genotyping was done with Labo Bio-Medical Products  as previously described . The sel16S rRNA genes were analyzed using the Quantitative Insights into Microbial Ecology (QIIME) v1.9 pipeline . The Gren = 6). The percent of relative standard deviation (%RSD) of 2-fluobiphenyl peak abundance in the QC samples (n = 6) was calculated using GCMS Labsolution data analysis software (Shimadzu). Thus, %RSD for peak abundances of QC samples accounted for 4.6%, which demonstrates good reproducibility of the method. To mitigate systematic bias, we performed the randomization of the sample analysis order. Blanks and QC samples were spaced evenly among the injections to monitor instrument stability. Quality control during data processing was previously described in detail [Two hundred μL of liquefied urine samples were collected and used for metabolite extraction, derivatization, and fractionation by gas chromatography-mass spectrometry (GC-MS) , as described before . Mass spn detail . In our n detail . PrelimiIntegration of metagenomics and metabolomics data were performed using Model-based Integration of Metabolite Observations and Species Abundances 2 (MIMOSA2), freely available at borensteinlab.com/software_MIMOSA2.html . MIMOSA2p-value < 0.1. Given that MIMOSA2 is prone to false negatives because its model is approximate, it requires a linear/monotonic relationship between metabolite abundances and inferred metabolic effects of the microbiome based on genes or taxa. Therefore, developers of MIMOSA2 suggest using a weak statistical threshold  to capture more relationships of possible interest, where the data might be only partially described by the approximate model but might still suggest some underlying biological signal. Greengenes OTUs taxonomic abundances of VAG and CERV microbiomes and a host metabolome previously identified in urine  were useShuttleworthia in the Greengenes database and in our manuscript, has been proposed to be BVAB1 [Metabolic capabilities of microbial taxa and variation in microbiome composition paired to both metabolite measurements and information from the KEGG database were used to estimate the metabolic capabilities of individual species identified in VAG and CERV microbiota by metagenomics. The heat map depicts microbial features that may underlie differences in microbial metabolite concentrations between similar communities. It was constructed using values that represent the fraction of the variation (VarShare) in each metabolite explained by the taxon in question, according to the overall community model . To notebe BVAB1 .


N = 168) responded to open-ended questions regarding their thinking about the HPV vaccine. Data were analyzed and interpreted using qualitative thematic analysis. Three domains of themes emerged from responses: (1) characteristics of HPV vaccination, (2) knowledge-related themes, and (3) beliefs-related themes. Prevention of HPV and cancer was the most referenced characteristic of HPV vaccination followed by HPV vaccine rates and HPV vaccine efficacy. Student inquiries were mostly centered on HPV vaccine composition, administration, duration and how the vaccine interacts with the body. Some responses indicated a desire for more information about HPV not specific to the HPV vaccine. Overall, adolescent attitudes were positive towards the HPV vaccine. This study highlights specific questions adolescents have about the vaccine that can be used to tailor future HPV educational efforts, empowering adolescents with the knowledge to be more active students in the decision-making process. In addition, the potential for adolescents to serve as community advocates for the vaccine should be considered for future interventions.Despite educational efforts, Tennessee human papillomavirus (HPV) vaccination rates are 43%, among the lowest in the United States. This study examined how adolescents think about the HPV vaccine to identify patterns and misconceptions to enhance educational efforts. Adolescents (ages 11–12) ( Healthy People 2020 levels of 80% and prevent HPV-associated cancers .” Similarly, student AC003 mentioned that “I find that HPV can spread very easily and fast that is why we need HPV vaccine.” Two student responses pointed toward barriers to HPV vaccination. Student DE010 stated “If your busy and really wanna [sic] get a vaccine you won’t have time to get a shot” while student WS006 highlighted that, “Everyone may not be able to get one.” Student BR010 took on the role of advocate when they responded “Parents you need to let your children get a vaccine shot because it’s not bad”.Forty student responses were related to HPV vaccination rates. Twenty-four student responses explicitly stated whether they would get the HPV vaccine. There were more responses that expressed they would get the HPV vaccine  desired HPV vaccination knowledge and (2) desired HPV knowledge. Desired HPV vaccination knowledge was organized into four subthemes to further elucidate specific types of HPV vaccine knowledge wanted by the students.Ninety-seven student responses indicated a desire for more HPV vaccination knowledge. Forty-six responses indicated a desire to know more about the HPV vaccine administration process. Student DT003 asked, “Can our parents be in there with us?” while student LW011 stated, “The downside is you could get scared because you will be like wonder if its hurts wonder if the needle is sharp.” Thirty-three student responses indicated a desire to know more about HPV composition. For example, student HO015 asked, “What are all the ingredients in the vaccine?” Student DE005 similarly asked, “What kind of chemicals are in the vaccine?” Twelve student responses indicated a desire to know more about how the vaccine interacts with the body upon administration. Student SD012 claimed “I need to know about the vaccine and what it will do to me.” Student CR001 asked, “How does it work?” Six student responses indicated a desire to know more about HPV vaccine durability. Student PE007 argued that “You should know if you get the shot how long will it last” while student WS005 asked, “Is it going to last for a lifetime or a month?”sic] skin to skin contact?” Similarly, student CR004 stated, “I want to find out how HPV get in your body and why so many people get it?”Forty-five student responses indicated a desire to gain more information related to HPV overall and not specific to HPV vaccination. For example, student WH009 asked, “What, why, how do you have HPV?” Student RC002 asked “How does it spread to .”One hundred forty student responses described the nature of students’ overall orientation towards the HPV Vaccine. One hundred twenty student responses indicated that there was no concern around the HPV vaccine and that the HPV vaccine is good and helpful. Student BR004 explicitly claimed that “it’s helpful.” Student BR006 argued that “It’s great and doctors should continue to develop it,” and student RC015 stated that, “They are good so there are no worrisomes .” indicating that students were implicitly aware of the individual’s right to choose whether or not to vaccinate and that many individuals may choose not to vaccinate. One approach that future educational efforts might take is to explicitly highlight this dilemma by facilitating conversations around reasons why people choose either to vaccinate or not. More research, however, is needed to determine the impact this would have on adolescents’ perceptions of the HPV vaccine—whether it fuels adolescents’ excitement or exacerbates adolescents’ worries and how either outcome influences adolescents’ stance on whether or not to self-vaccinate.The results of this qualitative study indicated adolescents were capable of engaging in complex thinking about HPV and the HPV vaccine. Students were able to comprehend basic information about HPV, such as HPV can cause cancers to form in the body and getting the HPV vaccine reduces the spread of HPV in the general population while also minimizing their own risk of developing an HPV related cancer. In this way, they were able to understand they are not only protecting themselves when they are vaccinated, but also protecting members of their community. The findings suggest adolescents may be motivated by educational efforts that facilitate deep thinking around the population implications of the HPV vaccine and the repercussions that choosing to vaccinate or not to vaccinate might have on their community. It is interesting to note, however, that students expressed this understanding as either an excitement or a worry depending on whether they focused their thinking on individuals who choose to vaccinate or individuals who choose Students also expressed many worries about the vaccine that appear to be driven by unanswered questions. While many of these same worries have been identified in previous studies ,28,29, tOur data also revealed a tendency for adolescents to take on the role of “advocate.” Students often made declarative stances that indicated a need to advocate for the HPV vaccine to parents and other adolescents, revealing that adolescents are aware of controversies related to either HPV vaccination specifically or vaccinations more generally. It is unclear as to whether this push towards advocacy is driven by student excitements about the preventative power of the HPV vaccine or student worries about the consequences of vaccine refusers. More research is needed to better understand these driving forces and the potential for training adolescents to advocate for HPV vaccination during their own vaccination decision-making process.In a similar line of thinking, students expressed awareness of structural barriers that limit vaccination access. This may be a result of a large portion of our sample coming from underserved communities where they are more likely to be exposed to the effects of structural violence from an early age. For instance, when WS006 pointed out that “everyone may not be able to get one,” they underscore these structural barriers to vaccine access that carry implications not only for an individual but also for an entire community. This, coupled with the tendency of students to take on the role of advocate, suggests that adolescents from underrepresented populations in particular may respond well to framing HPV conversations through a social justice lens. Future educational efforts should intentionally design programs that acknowledge and address structural barriers to vaccination access for adolescents regardless of socioeconomic status and highlight programs, such as Vaccines for All, that aim to minimize these barriers. By doing this, these future efforts can serve as an introduction to the relationship between healthcare and policy while also introducing the HPV vaccine to adolescents.While our study contributes to the overall understanding on how early adolescents think about the HPV vaccine, it is clear that students still desire more information about the vaccine. Negative stances towards the HPV vaccine stem from common vaccine misconceptions. Future educational efforts should focus on instilling a deeper and fuller understanding of the HPV vaccine specifically and vaccines in general in this population. Our results suggest that adolescents are eager to learn more about their health. This has significant implications for the potential role of adolescents in the vaccine decision-making process. Pediatricians may find success in patient uptake of the HPV vaccine by explicitly framing the HPV vaccine as a cancer-fighting measure during their patient-clinician and parent-clinician conversations while also paying special attention to structural barriers that may plague their patient and the patient’s family. Seeing as the adolescents in our study responded well to framing the HPV vaccine as a cancer-prevention measure, future studies should explore how parents of HPV vaccine eligible adolescents respond to this same framing. By providing adolescents with an opportunity to critically think about the HPV vaccine in a classroom setting we are equipping them with knowledge that can be used to better participate in their own medical decision-making processes while also gaining valuable insight into the ways they perceive the vaccine, which can then be used to better tailor future interventions.While our study is one of the few focused on understanding the thinking of adolescents related to HPV vaccination, there were several limitations to note. Working with adolescents had some challenges especially when collecting written response data. Responses were sometimes illegible to the researchers or off-topic. Researchers conducting similar studies should consider coupling our method with student interviews to resolve confusion that may arise from solely using written responses or including closed-ended options to gather information from the adolescent perspective. Four questions were used to ask about the HPV vaccine, but many student responses were regarding HPV itself. This discrepancy may have been a result of facilitators failing to emphasize this distinction and/or the wording of our questions. Further, because the data in our study were a part of a larger cancer educational intervention, this study was not designed with analysis in mind, rather it is a by-product of these cancer educational activities and emergence of insight into adolescent understanding of HPV vaccination. Our study did not consider descriptive characteristics, such as social and demographic factors, when exploring adolescent understanding of the HPV vaccine. For example, sex or gender may matter and is unknown for students in our study. There has been research to suggest that females hear about the vaccine from a much younger age than their male counterparts, and this can contribute to the lower vaccination rates seen in males . While tIn conclusion, adolescents (ages 11–12) are capable of complex thinking around HPV and HPV vaccination and express a desire for more knowledge on a variety of topics related to the vaccine, including vaccine composition, the body’s response to the vaccine, and immunization duration.Although this was a small study, our results make notable contributions to the field as a gap in the literature exists around adolescent thought patterns regarding HPV and the HPV vaccine. Understanding how adolescents think about the HPV vaccine helps researchers to better tailor future efforts. Specific worries and concerns identified in this study should be incorporated into future health educational efforts to provide adolescents with a strong enough knowledge base to become more active participants in their medical decision-making processes.


The incidence of oropharyngeal squamous cell carcinoma (OPSCC) in the US is rapidly increasing, driven largely by the epidemic of human papillomavirus (HPV)-mediated OPSCC. Although survival for patients with HPV mediated OPSCC (HPV+ OPSCC) is generally better than that of patients with non-virally mediated OPSCC, this effect is not uniform. We hypothesized that tobacco exposure remains a critical modifier of survival for HPV+ OPSCC patients.p < 0.05).We conducted a retrospective analysis of 611 OPSCC patients with concordant p16 and HPV testing treated at a single institute (2002–2013). Survival analysis was performed using Kaplan-Meier analysis and Cox regression. Recursive partitioning analysis (RPA) was used to define tobacco exposure associated with survival . RPA identified 30 pack-years (PY) as a threshold at which survival became significantly worse in HPV+ patients. OS and disease-free survival (DFS) for HPV+ > 30 PY patients didn’t differ significantly from HPV- patients . HPV+ > 30 PY patients had substantially lower 5-year OS when compared to their ≤30 PYs counterparts: 78.4% vs 91.6%; p = 0.03, 76% vs 88.3%; p = 0.07, and 52.3% vs 74%; p = 0.05, for stages I, II, and III , respectively.Tobacco exposure impacted overall survival (OS) for HPV+ patients on univariate and multivariate analysis (Tobacco exposure can eliminate the survival benefit associated with HPV+ status in OPSCC patients. Until this effect can be clearly quantified using prospective datasets, de-escalation of treatment for HPV + OPSCC smokers should be avoided. Tobacco remains a critical driver of survival and treatment response in HPV+ OPCClinical trials are investigating treatment deintensification in advanced HPV+ OPCCurrent efforts to consider tobacco exposure in this context should be encouragedThe human papillomavirus (HPV) has transformed the landscape of cancer diagnosis and treatment. Oropharyngeal squamous cell carcinoma (OPSCC), a disease traditionally associated with tobacco and alcohol exposure, is now overwhelmingly a disease associated with HPV –4. This In 2010, Ang et al. identified an interaction between HPV status and tobacco exposure in OPSCC patients and defined “low-risk” and “intermediate-risk” tumors based on differential survival . TobaccoThe recent changes to the AJCC staging system for OPSCC create an urgent need to better define the potential impact of tobacco exposure on clinical outcomes for HPV+ OPSCC in order to generate appropriate pre-treatment risk stratification and provide appropriate patient counseling. Furthermore, given continued efforts at treatment de-escalation for HPV+ OPSCC tumors, it is critical to determine whether a subset of patients may be at risk for inappropriate de-escalation. We hypothesized that tobacco exposure has a significant negative impact on HPV+ OPSCC patient survival and sought to test this hypothesis in a large single-institution cohort of patients with robust tumor biological data (concordant p16/HPV testing status) who received radiation-based treatment.Following approval by the University of Texas MD Anderson Cancer Center institutional review board, we reviewed 1171 patients with a primary diagnosis of OPSCC who underwent definitive non-surgical treatment between 2002 and 2013. This end date was chosen to allow for a minimum follow up period of 5 years. Patients with recurrent disease, previous oncologic treatment, and/or lost to follow-up were excluded from the analysis. Demographics, tobacco usage, and clinical-pathologic history were comprehensively reviewed through the institutional electronic medical record. Cancer staging was conducted according to the American Joint Commission on Cancer Staging Manual (7th and 8th Editions). Tobacco exposure was obtained from the medical record in the form of PY defined as: 1 PY = 1 pack of cigarettes/day for 1 year. Supplementary Figure Tumors were tested for HPV by use of the in situ hybridization (ISH)-catalyzed signal amplification method for biotinylated probes and for p16 protein expression via immunohistochemistry (IHC) consistent with clinical practice at our institution. Only patients with concordant p16 and HPV testing results were included in this analysis .The treatment strategy for individual patients was determined by discussion at the institutional multidisciplinary tumor conference. Surgically treated patients were excluded from this analysis. All patients were treated using intensity-modulated radiotherapy (IMRT) using previously described protocols , 11. GenWe used the Charlson Comorbidity Index (CCI) to assess pre-treatment patient comorbidity status . The CCIp < 0.05). For multivariable analysis, we tested the prognostic impact of the AJCC staging system and tobacco exposure in HPV+ patients compared with a baseline model of standard clinical variables. The baseline model included age, sex, AJCC 7th edition, chemotherapy sequence, and total EBRT dose. We then compared the alternative models using Bayesian information criteria [The outcomes of interest included: ‘Loco-regional control (LRC)’ defined as time from date of completion of treatment to date of diagnosis of local and/or regional recurrence; ‘Freedom from distant metastasis (FDM)’ defined as time from date of completion of treatment to date of diagnosis of malignant metastasis to distant body organ; ‘Disease-free survival (DFS)’ defined as time from date of completion of treatment to date of diagnosis of loco-regional and/or distant recurrence ; and ‘Overall survival (OS)’ defined as time from date of completion of treatment until death or last recorded follow-up. Chi-square tests were used to compare the categorical variables  between the p16+/HPV+ (abbreviated to HPV+) versus p16−/HPV- (abbreviated to HPV-) cohorts. Survival analysis was performed using Kaplan-Meier analysis (log-rank test). Uni- and multi-variable survival analyses were performed using Cox regression. Recursive partitioning analysis (RPA) was used to quantify a threshold for tobacco exposure significantly associated with overall survival (criteria  with thep < 0.0001). Subsite distribution was comparable between the cohorts. One hundred twenty six patients (21%) had at least one pretreatment comorbid condition. The most common comorbid condition was diabetes mellitus , followed by cardiovascular conditions  and respiratory conditions . There was no statistically significant difference in CCI score between the HPV+ and HPV- cohorts  were HPV+ and half of these patients reported 0 PY history of tobacco exposure whereas only 20% of the HPV- patients were non-smokers. Approximately one-third of HPV+ smokers reported heavy exposure (i.e. > 30 PY history) compared with approximately one-half of HPV- smokers. The details of tobacco exposure in PY for both cohorts are shown in Supplementary Figure p = 0.0009; DFS 84.7% vs 70.2%, p = 0.004; LRC 89.8% vs 81.2%, p = 0.040; FDM 91.7% vs 88.3%, p = 0.44) . Subsequent RPA identified 30 PY as the cut-off threshold for differential OS risk; the resulting binary risk groups (PY ≤30 and PY > 30) were then integrated into the final analysis. Smokers with > 30 PY of exposure were shown to have more than double the hazard of death in both the HPV+  and HPV-  cohorts as compared to patients with ≤30 PY of exposure. The following clinical variables were also significantly associated with OS in the HPV+ cohort: T-classification, AJCC stage (8th edition), CCI, chemotherapy sequence and total radiation dose. In the HPV- cohort, T-classification was the only additional clinical variable with significant association with OS  was significantly associated with OS in both the HPV+ and HPV- cohorts (p = 0.0001) and 5-year DFS  compared to patients with tobacco exposure ≤30 PY , 5-year FDM did not demonstrate significant differences between the two subsets  . Smoking status at diagnosis did not significantly correlate to OS or DFS in HPV+ patients on univariable analysis. When patients in the HPV+ cohort with tobacco exposure > 30 PY were stratified into current and former smokers (n = 43 each), OS and DFS Kaplan-Meier survival probability estimates at 5 years still did not statistically differ between the two subgroups.Overall survival and DFS Kaplan-Meier survival probability estimates at 5 years did not statistically differ for the HPV+ > 30 PY and HPV- cohorts  HPV+ patients into low (≤30 PY) and high (> 30 PY) tobacco exposure Fig. . PatientOn multivariable analysis, AJCC 8th edition markedly improved the OS model performance over the baseline model when added instead of the 7th edition (i.e. BIC decreased by 11). Further addition of the tobacco risk grouping achieved the best OS model performance . Uni- and multi-variable analysis for DFS showed no correlation between tobacco exposure and DFS in either the HPV+ or HPV- cohorts. While chemotherapy sequence, total EBRT dose, T- and N-classifications, and AJCC stage (8th edition) were associated with DFS in the HPV+ cohort on univariable analysis, only AJCC stage (8th edition) retained significant association with DFS when combined into a multivariable model. On the other hand, DFS in HPV- OPSCC subset was significantly associated with age at diagnosis and T-classification , and total EBRT dose  . For HPV- patients none of the examined variables were significantly associated with non-cancer mortality (likely due to relatively lower sample size).Given the potential impact of heavy tobacco exposure on non-cancer related mortality, we performed an additional analysis of this datapoint in the context of our patient cohort. Among patients with PY ≥ 30, 66 and 59% had no pre-treatment comorbidities (CCI = 0) in the HPV+ and HPV- cohorts, respectively. Distribution of comorbidities within the HPV+ cohort were as follows. Among patients with tobacco exposure < 30 PY, 1% had respiratory comorbidities, 8% had endocrine comorbidities and 4% had cardiovascular comorbidities. Among patients with tobacco exposure ≥30 pack-years 9% had respiratory comorbidities, 10% had endocrine comorbidities and 7% had cardiovascular comorbidities. Distribution of comorbidities within the HPV- cohort were as follows. Among patients with tobacco exposure < 30 PY, 0% had respiratory comorbidities, 5% had endocrine comorbidities and 5% had cardiovascular comorbidities. Among patients with tobacco exposure ≥30 PY 6% had respiratory comorbidities, 11% had endocrine comorbidities and 15% had cardiovascular comorbidities. For HPV+ patients higher CCI score (≥1) and higher PY history (≥30) were independently associated with higher non-cancer related mortality, both with more than triple the hazard  stage in terms of overall survival outcomes. However, statistical significance was only reached in stage I, probably because of smaller numbers of patients with more advanced disease (i.e. ~ 60% of the whole cohort was categorized as stage I). Nonetheless, heavy smokers with stage I or II disease had 5-year outcomes that were approximating or -in some cases- even worse than outcomes of patients with lower smoking index and more advanced disease stage. This observation must be considered in the inclusion criteria for future dose de-escalation studies in early stage HPV+ OPSCC. In agreement with these results, a recent study by Vawda et al.  has demoIn the meantime, the putative impact of smoking on cancer-specific mortality in HPV+ OPSCC cannot be easily interpreted given the known adverse effects of smoking on general comorbidity and interaction with co-existing risk factors  , 37. OurAlthough large, this is a single institution patient cohort and as such our findings must be validated in additional patient cohorts. It is also important to note, that our outcomes for HPV- patients in this series are dramatically better than historical data even from our own institution , 2, 6. TIn addition, the tobacco exposure range for the HPV+ cohort is substantially skewed toward the lower range of exposure , namely decreased treatment response and a high rate of loco-regional failure. We previously showed that > 90% of recurrence/progression occurs loco-regionally in a patient cohort with significant tobacco exposure . The facBased on this dataset, we strongly recommend development of a multi-institutional cooperative group focused on characterizing and quantifying the relative impact of tobacco exposure on HPV + OPSCC clinical outcomes. Until such time that definitive national datasets can be generated, we recommend strong consideration of tobacco exposure in the context of ongoing institutional and cooperative group trials aimed at de-escalation regimens for HPV+ OPSCC along the same lines as the phase II/III PATHOS and the recently completed NRG HN002 randomized controlled trials , 48. FurTobacco remains a critical driver of survival and treatment response in patients with HPV associated OPSCC receiving radiation treatment. HPV associated OPSCC in smokers should be considered a distinct entity after validation of this dataset in multi-institutional and prospective settings.Additional file 1: Supplementary Figure 1. CONSORT flow diagram of selection process of patients for this study .Additional file 2: Supplementary Figure 2. Histogram of tobacco exposure in human papillomavirus-mediated (HPV+) and HPV- groups.Additional file 3: Supplementary Figure 3 Parts A-B. Impact of human papillomavirus (HPV) status on survival. Kaplan-Meier plots for clinical outcomes for the entire patient cohort stratified by HPV status: (A) Loco-regional control; and (B) Freedom from distant metastasis.Additional file 4: Supplementary Figure 4 Parts A-B. Impact of tobacco exposure on survival. Kaplan-Meier plots for clinical outcomes for the HPV+ oropharyngeal cancer group stratified by extent of tobacco exposure: (A) Loco-regional control; and (B) Freedom from distant metastasis.Additional file 5: Supplementary Table 1. HPV and tobacco impact on overall survival. Uni- and multi-variable analyses and corresponding hazard ratios and 95% confidence intervals (CI) for clinical variables associated with overall survival for the entire cohort stratified by human papillomavirus (HPV) status. *For AJCC stage (7th edition) analysis; stages I-III were collectively compared against stage IV (the majority class) given the imbalanced distribution of patients among AJCC 7th stages.Additional file 6: Supplementary Table 2. HPV and tobacco impact on disease free survival. Uni- and multi-variable analyses and corresponding hazard ratios and 95% confidence intervals (CI) for clinical variables associated with disease-free survival for the entire patients cohort stratified by HPV status. *For AJCC stage (7th edition) analysis; stages I-III were collectively compared against stage IV (the majority class) given the imbalanced distribution of patients among AJCC 7th stages.