Association of Salivary Human Papillomavirus Infectionand Oral and Oropharyngeal Cancer: A Meta-Analys

Journal of Clinical Medicine Review Association of Salivary Human Papillomavirus Infection and Oral and Oropharyngeal Cancer: A Meta-Analysis Óscar Rapado-González 1,2,3, Cristina Martínez-Reglero 4,Ángel Salgado-Barreira 4, Almudena Rodríguez-Fernández 5, Santiago Aguín-Losada 6, Luis León-Mateos 6, Laura Muinelo-Romay 2,3 , Rafael López-López 3,6,* and María Mercedes Suarez-Cunqueiro 1,3,7,* 1Department of Surgery and Medical-Surgical Specialties, Medicine and Dentistry School, Universidade de Santiago de Compostela (USC), 15782 Santiago de Compostela, Spain; oscar[email protected] 2Liquid Biopsy Analysis Unit, Translational Medical Oncology (Oncomet), Health Research Institute of Santiago (IDIS), 15706 Santiago de Compostela, Spain; [email protected] 3Centro de Investigación Biomédica en Red en Cáncer (CIBERONC), Instituto de Salud Carlos III, 28029 Madrid, Spain 4Methodology and Statistics Unit, Galicia Sur Health Research Institute (IISGS), 36312 Vigo, Spain; [email protected] (C.M.-R.); [email protected] (Á.S.-B.) 5Department of Preventive and Public Health, Universidade de Santiago de Compostela (USC), 15782 Santiago de Compostela, Spain; almudena.rodrí[email protected] 6Translational Medical Oncology (Oncomet), Health Research Institute of Santiago (IDIS), Complexo Hospitalario Universitario de Santiago de Compostela (SERGAS), 15706 Santiago de Compostela, Spain; [email protected] (S.A.-L.); [email protected] (L.L.-M.) 7Translational Medical Oncology (Oncomet), Health Research Institute of Santiago (IDIS), 15706 Santiago de Compostela, Spain *Correspondence: [email protected] (R.L.-L.); mariamercedes.suar[email protected] (M.M.S.-C.); Tel.: +34-981-95-14-70 (R.L-L.); +34-881-812-437 (M.M.S.-C.) Received: 31 March 2020; Accepted: 26 April 2020; Published: 29 April 2020   Abstract: Background. Human papillomavirus (HPV) infection has been recognized as an important risk factor in cancer. The purpose of this systematic review and meta-analysis was to determine the prevalence and effect size of association between salivary HPV DNA and the risk of developing oral and oropharyngeal cancer. Methods. A systematic literature search of PubMed, EMBASE, Web of Science, LILACS, Scopus and the Cochrane Library was performed, without language restrictions or specified start date. Pooled data were analyzed by calculating odds ratios (ORs) and 95% confidence intervals (CIs). Quality assessment was performed using the Newcastle–Ottawa Scale (NOS). Results. A total of 1672 studies were screened and 14 met inclusion criteria for the meta-analysis. The overall prevalence of salivary HPV DNA for oral and oropharyngeal carcinoma was 43.2%, and the prevalence of salivary HPV16 genotype was 27.5%. Pooled results showed a significant association between salivary HPV and oral and oropharyngeal cancer (OR =4.94; 2.82 − 8.67), oral cancer ( OR =2.58; 1.67−3.99 ) and oropharyngeal cancer ( OR =17.71; 6.42−48.84 ). Significant associations were also found between salivary HPV16 and oral and oropharyngeal cancer ( OR =10.07; 3.65−27.82 ), oral cancer ( OR =2.95; 1.23−7.08 ) and oropharyngeal cancer ( OR =38.50; 22.43−66.07 ). Conclusions. Our meta-analysis demonstrated the association between salivary HPV infection and the incidence of oral and oropharyngeal cancer indicating its value as a predictive indicator. Keywords: human papillomavirus; oral cancer; oropharyngeal cancer; saliva; meta-analysis J. Clin. Med. 2020,9, 1305; doi:10.3390/jcm9051305 www.mdpi.com/journal/jcm J. Clin. Med. 2020,9, 1305 2 of 18 1. Introduction Human papillomavirus (HPV) infection has been recognized as an important risk factor in a subset of head and neck squamous cell carcinomas, independently of traditional risk factors such as tobacco or alcohol use [ 1 , 2 ]. Globally, around 38,000 cases of head and neck cancer are attributed to the HPV infection. Of these, around 76% are cases of oropharynx cancer, 12% of oral cavity cancer and 10% of larynx cancer [ 3 ]. Currently, it is well known that HPV-status determines the molecular landscape of these tumors and their clinical evolution, with a better prognosis and response to therapy being found in HPV-positive patients [4,5]. HPVs are small, non-enveloped, close-circular, double-stranded DNA viruses of approximately 8000 base-pairs which present a specific tissue tropism infecting epithelial cells of the skin and mucosae of the anogenital and upper aero-digestive tract [ 6 ]. More than 200 different HPV types have been identified and classified into low-risk and high-risk according to their oncogenic potential. In this sense, high-risk HPV (HR-HPV) can promote the malignant transformation of HPV-infected cells through E6 and E7 viral oncoproteins, responsible for inactivating the TP53 and Rb (retinoblastoma tumor suppressor gene) [ 7 ]. A subset of 12 alpha HR-HPV (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59) has been classified as carcinogenic to humans according to the International Agency of Research in Cancer [ 8 ]. HR-HPV is considered the main cause of cervical cancer, genotypes 16 and 18 being responsible for 70% of cases [ 9 ]. In addition, several studies have also demonstrated the pathogenic role of HPV in other anogenital cancers [ 10 – 12 ] as well as in head and neck cancers [ 13 ]. Currently, HPV16 is widely recognized as an etiological factor in oropharynx tumors [ 14 ], however, not enough evidence exists regarding the HPV relationship and the anatomic subsites of head and neck squamous cell carcinoma [15]. Nowadays, a variety of molecular biological methods have been developed for the detection and genotyping of HPV at DNA, mRNA, and protein levels by polymerase chain reaction (PCR), real-time PCR, in situ hybridization, immunohistochemistry and serum antibody assays [ 16 ]. In addition, next-generation HPV sequencing approaches provide accurate information on genotype composition and pathways to better understand functional consequences [ 17 ]. Certain collection approaches present difficulties. For example, tumoral tissue biopsy is invasive and tumors may be inaccessible. For its part, the collection of oral exfoliated cells with cotton swabs or cytobrush is restricted to a specific and accessible oral area, making collection difficult for non-visual tumors and early molecular alterations. To overcome these drawbacks, the detection of HPV in oral exfoliated cells from saliva (with or without oral rinses) represents a quick and easy non-invasive alternative for oral and oropharyngeal cancer screening in high-risk populations. In this sense, several researchers have analyzed the prevalence of salivary HPV DNA from head and neck cancer, however, to our knowledge, no previous systematic review has elucidated evidence of this relationship. Therefore, the aim of the present systematic review and meta-analysis was to determine the prevalence and effect size of association between salivary HPV DNA and the risk of developing oral and oropharyngeal cancer. 2. Materials and Methods 2.1. Protocol and Registration This study was conducted according to Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines [ 18 ] and the protocol was registered with the International Prospective Register of Systematic Reviews (reference No. CRD42020161345). 2.2. Search Strategy and Study Selection The systematic literature search was performed in PubMed, EMBASE, Web of Science, LILACS, Scopus and the Cochrane Library through 9 January 2020, without language restrictions or specified start date. The following combinations of keywords and medical subject headings were used: (human papilloma virus OR HPV) AND (saliva OR oral rinses OR mouthwash) AND (oral squamous J. Clin. Med. 2020,9, 1305 3 of 18 cell carcinoma OR OSCC OR oropharyngeal squamous cell carcinoma OR OPSCC OR oral cancer OR oropharyngeal cancer). All studies were screened based on the title and abstract, and eligible manuscripts were retrieved for full-text review. Additionally, we manually searched the reference lists in each original and review article in order to avoid missing potential studies. The literature search was performed independently by two researchers (ORG and MMSC), and any disagreements were resolved by consensus. The studies selected through the search strategy and other references were managed using RefWorks software, and duplicated items were removed using the associated tools. 2.3. Eligibility Criteria We included the studies that met the following criteria: (1) case-control studies of patients with oral and/or oropharyngeal cancer and healthy controls, (2) HPV DNA prevalence determined in salivary samples (whole saliva or oral rinses), and (3) sufficient data to calculate odds ratios (ORs) with 95% confidence intervals (CIs). The exclusion criteria were as follows: (1) in vitro or animal study, (2) reviews, letters, personal opinions, book chapters, case reports, and conference abstracts, and (3) duplicate articles or suspicion of data overlap. 2.4. Protocol and Registration Two researchers (ORG and MMSC) independently assessed each eligible manuscript, extracted data using a pre-established form, and collated the data into a Microsoft Excel spreadsheet (Microsoft Corp. Redmond, WA, USA). Any disagreement among reviewers was resolved by consensus. The following information was extracted from each study: author, publication year, country, type of sample, method of collection, tumor location, sample size, HPV detection method, number of cases and HPV-positive cases, number of controls and HPV-positive controls, HPV-positive genotypes, overall HPV DNA prevalence (number of subjects testing positive for any HPV type) and type-specific HPV DNA prevalence (number of subjects testing positive for specific HPV types: HPV16 or HPV18, HR-HPV and LR-HPV). If the required data were incomplete, attempts were made to contact the authors to obtain the missing information. 2.5. Assessment of Risk Bias The Newcastle-Ottawa Scale (NOS) [ 19 ] was used to evaluate the individual quality of the selected studies by three independent researchers (ORG, ARF, and MMSC), and discrepancies were resolved by consensus. The NOS assesses the quality of non-randomized studies based on design, content and ease of use directed to the task of incorporating the quality assessments in the interpretation of meta-analytic results. This ‘star system’ consists of 8 items classified into three broad perspectives: the selection of study groups; the comparability of the groups; and the ascertainment of either the exposure or outcome of interest for case-control or cohort studies. The highest quality studies were allotted a maximum of one star for each item, except for, the item related to comparability, which was allowed the assignment of a maximum of two stars. The NOS score ranged from 0 to 9 stars and validity criteria were as follows: 8–9, high quality; 6–7, medium quality; <5 low quality. 2.6. Statistical Analysis Statistical analysis was conducted using the meta package of free R software (v.3.6.2; https: //www.r-project.org). Firstly, to evaluate the statistical model applied to the meta analytic database, heterogeneity was assessed using the Cochran’s Q statistic test-based Chi-squared test and I2 statistics. Heterogeneity was considered significant when I2 >50% and/or presence of a p<0.10 for the Cochran’s Q test. The prevalence of HPV DNA and HPV genotypes in oral and/or oropharyngeal cancer was calculated using fixed or random effects depending on the heterogeneity. The relationship between saliva HPV DNA infection and oral and/oropharyngeal cancer risk was evaluated by pooled odds ratio (OR) and 95% confidence intervals (CIs) comparing cases to controls. If significant heterogeneity was detected, the DerSimonian and Laird random-effects model was applied to calculate the pooled OR J. Clin. Med. 2020,9, 1305 4 of 18 with 95% CIs; otherwise, the Mantel–Haenszel fixed-effects model was used. Then, subgroup analyses were performed to explore the potential sources of heterogeneity among studies according to the anatomic tumor location and HPV genotypes. Additionally, publication bias was checked with Begg’s and Egger’s tests and by visual inspection in funnel plots demonstrating the relationship between the individual log ORs and their standard errors [ 20 , 21 ]. p-values of <0.05 were considered to indicate statistical significance. 3. Results 3.1. Study Selection A total of 1669 articles were identified across the six electronic databases and three additional reports from the reference lists. After removing duplicates, a total of 1542 articles were screened based on the title and abstract, and 1494 were excluded for lack of adherence to our inclusion criteria. Therefore, full-text articles were retrieved for the remaining 48 articles. After a full-text review, 34 articles were excluded for the following reasons: non case-control studies (22); controls under risk conditions (2); suspicious of data overlap (3); insufficient data (3); and reviews, letters, and meta-analysis (4). Finally, 14 articles met all the inclusion criteria and were included in the final analysis. A detailed flowchart showing the selection process is shown in Figure 1. J. Clin. Med. 2020, 9, x FOR PEER REVIEW 4 of 19 heterogeneity was detected, the DerSimonian and Laird random-effects model was applied to calculate the pooled OR with 95% CIs; otherwise, the Mantel–Haenszel fixed-effects model was used. Then, subgroup analyses were performed to explore the potential sources of heterogeneity among studies according to the anatomic tumor location and HPV genotypes. Additionally, publication bias was checked with Begg’s and Egger’s tests and by visual inspection in funnel plots demonstrating the relationship between the individual log ORs and their standard errors [20,21]. p-values of < 0.05 were considered to indicate statistical significance. 3. Results 3.1. Study Selection A total of 1669 articles were identified across the six electronic databases and three additional reports from the reference lists. After removing duplicates, a total of 1542 articles were screened based on the title and abstract, and 1494 were excluded for lack of adherence to our inclusion criteria. Therefore, full-text articles were retrieved for the remaining 48 articles. After a full-text review, 34 articles were excluded for the following reasons: non case-control studies (22); controls under risk conditions (2); suspicious of data overlap (3); insufficient data (3); and reviews, letters, and metaanalysis (4). Finally, 14 articles met all the inclusion criteria and were included in the final analysis. A detailed flowchart showing the selection process is shown in Figure 1. Figure 1. Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) flow diagram of the literature selection process, including identification, screening, eligibility and total studies included in qualitative and quantitative synthesis. Figure 1. Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) flow diagram of the literature selection process, including identification, screening, eligibility and total studies included in qualitative and quantitative synthesis. 3.2. Study Characteristics Individual characteristics of the included studies are summarized in Table 1. A total of 14 articles evaluating HPV prevalence in oral and/or oropharyngeal cancer were included in this meta-analysis, and these studies were carried out from 2005 to 2019. Study sample sizes ranged from 42 to 677 subjects. J. Clin. Med. 2020,9, 1305 5 of 18 The study units in this meta-analysis comprised a total of 2320 cases (658 from the oral cavity, 1160 from the oral cavity plus oropharynx and 502 from the oropharynx), and 5868 controls (2210 from the oral cavity, 2304 from the oral cavity plus oropharynx and 1354 from the oropharynx). As reported in Table 1, four studies were conducted in India [ 22 – 25 ], three in the USA [ 26 – 28 ], and two in Sweden [ 29 , 30 ], whereas the remaining studies were carried out in the following countries: Canada [ 31 ], France [ 32 ], Hungary [ 33 ], Pakistan [ 34 ], and Iran [ 35 ]. In terms of sampling, oral rinses and saliva ( n=7, 50% , respectively) were analyzed for HPV positivity and genotyping. The methods most used for saliva HPV-DNA determination were conventional PCR, nested PCR and quantitative PCR. However, other analytical strategies such as next generation sequencing or immunoassays were also employed for salivary HPV genotyping (Table 1). 3.3. Study Quality Assessment of risk of bias and quality was performed according to NOS (Table S1). Regarding the selection domain, adequate description about characteristics and selection criteria for cases and controls were provided by all of the included studies. Regarding the comparability domain, six out of the 14 studies matched for age and at least one additional factor. Insofar as the exposure domain, few studies reported the blinding of analyses or non-response rates. The mean NOS score in our meta-analysis was six. 3.4. Meta-Analysis 3.4.1. Salivary HPV Association with Oral and Oropharyngeal Cancer Overall, the prevalence of salivary HPV for oral and oropharyngeal carcinoma was of 43.2% ( n=1160 ) while the infection rate in the healthy control group was of 8.9% (n=2304). Salivary HPV16 was the most common type of HPV DNA positive cases (n=1116), representing 27.5% (Figure 2). J. Clin. Med. 2020, 9, x; doi: FOR PEER REVIEW www.mdpi.com/journal/jcm 3.3. Study Quality Assessment of risk of bias and quality was performed according to NOS (Table S1). Regarding the selection domain, adequate description about characteristics and selection criteria for cases and controls were provided by all of the included studies. Regarding the comparability domain, six out of the 14 studies matched for age and at least one additional factor. Insofar as the exposure domain, few studies reported the blinding of analyses or non-response rates. The mean NOS score in our meta-analysis was six. 3.4. Meta-Analysis 3.4.1. Salivary HPV Association with Oral and Oropharyngeal Cancer Overall, the prevalence of salivary HPV for oral and oropharyngeal carcinoma was of 43.2% (n = 1160) while the infection rate in the healthy control group was of 8.9% (n = 2304). Salivary HPV16 was the most common type of HPV DNA positive cases (n = 1116), representing 27.5% (Figure 2). Figure 2. Schematic drawing of salivary HPV and prevalence of oral and/or oropharyngeal cancer. Oral tissue sheds pathogen-infected cells containing different HPV DNA genotypes (HPV16, HPV18, HR-HPV, and LR-HPV) into saliva (with or without oral rinses). The prevalence of salivary HPV DNA varied according to anatomic tumor location, showing the highest infection rate in oropharyngeal carcinomas. In addition, the type-specific prevalence in saliva was also different according to the anatomic tumor location. Our meta-analysis included a total of 1160 cases and 2304 controls. The pooled analysis showed a significant association between positive salivary HPV DNA status and oral and oropharyngeal cancer with a pooled OR of 4.94 (95% CI = 2.82−8.67; p < 0.01) (Figure 3). Figure 2. Schematic drawing of salivary HPV and prevalence of oral and/or oropharyngeal cancer. Oral tissue sheds pathogen-infected cells containing different HPV DNA genotypes (HPV16, HPV18, HR-HPV, and LR-HPV) into saliva (with or without oral rinses). The prevalence of salivary HPV DNA varied according to anatomic tumor location, showing the highest infection rate in oropharyngeal carcinomas. In addition, the type-specific prevalence in saliva was also different according to the anatomic tumor location. J. Clin. Med. 2020,9, 1305 6 of 18 Table 1. Characteristics of the 14 case-control studies included in this meta-analysis. Country Tumor Location (n) Type of Sample/ Method of Collection HPV-Positive Cases (n/N) HPV-Positive Case Types HPV-Positive Controls (n/N) HPV-Positive Control Types HPV Detection Method Hansson et al.; 2005 Sweden OC (85) OPC (46) Oral rinse/7 mL of 0.9% NaCl solution for 30s 39/131 16, 18, 33, 45, 58, 59, 13, 32, 62, 10, 76 14/320 16, 67, 54, 55, 62, 87, 75, 76, RTRX9 Nested PCR (MY09/ MY11 and GP5+/6+ primers) DNA sequencing SahebJamee et al.; 2009 Iran OC (22) Oral rinse/10 mL of normal saline 9/22 16, 18, 6/11 5/20 16, 6/11 PCR (GP5+/ 6+primers for L1 region) Kulkarni et al.; 2011 India OC (34) Saliva 24/34 16, 18 255/396 16, 18 PCR (16 and 18 specific primers) Goot-Heah et al.; 2012 India OC (14) Saliva 0/14 - 0/30 - Nested PCR (MY09/11 and GP5+/6+primers for L1 region) Chen et al.; 2013 USA OC (32) OPC (52) Saliva/Oragene DNA kits (DNA Genotek) 38/84 16 1/19 16 qPCR (specific primers and probe for E6 region of HPV16) Nordfors et al.; 2014 Sweden OPC (47) Oral rinse/15 mL 50% Listerine®(Johnson and Johnson) for 30s 25/47 16, 18, 67, 6, 51 0/37 - Bead-based multiplex assay on a MagPix instrument (Luminex Corporation), GP5+/6+ primers for the L1 region and specific primers and probe for E6 region of HPV16 Khyani et al.; 2015 Pakistan OC (35) Saliva 15/35 16, 18 3/35 16 qPCR using Real-time PCR Kit HPV16/18 Real-TM Quant (Sacace Biotechnologies) Modak et al.; 2016 India OC (235) Saliva 149/235 16 193/409 16 PCR (HPV 16 specific primer) J. Clin. Med. 2020,9, 1305 7 of 18 Table 1. Cont. Country Tumor Location (n) Type of Sample/ Method of Collection HPV-Positive Cases (n/N) HPV-Positive Case Types HPV-Positive Controls (n/N) HPV-Positive Control Types HPV Detection Method Rosenthal et al.; 2017 USA OC (61) OPC (45) Oral rinse/10 mL of 0.9% NaCl solution for 30s 44/106 16, 18, * HR-HPV other 3/81 16, * HR-HPV other qPCR from the HPV L1 region (Cobas®HPV Test-Roche Diagnostics) Auguste et al.; 2017 France OC (22) OPC (41) Saliva/Oragene OG-500 kit (DNA Genotek) 21/63 16, 33, 51 80/308 16 PCR (SPF10 primer system for L1 region, INNO-LiPA®HPV Genotyping Extra; Innogenetics) Laprise et al.; 2017 Canada OC (72) OPC (183) Oral rinse/alcohol-based solution for 15–30s 125/255 16, 18, ** HPV α-9 other than HPV16, *** HPV other 61/422 16, 18, ** HPV α-9 other than HPV16, *** HPV other PCR (MY09/11 primers for HPV) and genotyping by Linear Array assay (Roche Molecular diagnostics) Hettman et al.; 2018 Hungary OPC (12) Unstimulated saliva 4/12 16, 13 2/57 13, 11 PCR (MY09/11 primers for L1 region) Nested PCR (MY09/11 and GP5+/6+primers for L1 region), sequencing for genotyping Ramesh et al.; 2018 India OC (30) Oral rinse/10mL of 0.9% normal saline 13/30 16, 18 18/60 16, 18 Nested PCR (MY09/11 primers for L1 region) Dang et al.; 2019 USA OC (16) OPC (76) Oral rinse/Original Mint Scope®mouthwash or Crest®Alcohol-free mouthwash (Proctor and Gamble) for 30s 37/92 16, NV14.4, NV69.1, NV95 1/110 18 qPCR (HPV16 E7/HPV18 E7 primers and probe) FAP-PCR from the L1 region NGS and Sanger sequencing Abbreviations: OC, oral cancer; OPC, oropharynx cancer; PCR, polymerase chain reaction; qPCR, quantitative PCR; FAP-PCR, fluorescent arbitrarily primed PCR; NGS, next-generation sequencing; * HR-HPV other: 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68; ** HPV α -9 other than HPV16: 31,33,35,52,58, and 67; *** HPV other: 6, 11, 18, 26, 34, 39, 40, 42, 44, 45, 51, 53, 54, 56, 59, 61, 62, 66, 68, 69, 70, 71, 72, 73, 81, 82, 83, 84, and 89. J. Clin. Med. 2020,9, 1305 8 of 18 Our meta-analysis included a total of 1160 cases and 2304 controls. The pooled analysis showed a significant association between positive salivary HPV DNA status and oral and oropharyngeal cancer with a pooled OR of 4.94 (95% CI =2.82−8.67; p<0.01) (Figure 3). J. Clin. Med. 2020, 9, x FOR PEER REVIEW 2 of 19 Figure 3. Forest plot for the studies on the association between salivary HPV and oral and oropharyngeal cancer. The squares indicate the ORs (odds ratios) in each study, with square sizes inversely proportional to the standard error of the OR. The diamond shape indicates the pooled ORs. Horizontal lines represent 95% CIs (confidence intervals), I2 > 50% indicates severe heterogeneity. A random-effects model was used because heterogeneity was identified among the 14 studies (I2 = 82%). Visual inspection of the funnel plot revealed a symmetrical (Egger’s test, p = 0.159; Begg’s test, p = 0.298) distribution of the studies, indicating no evidence of publication bias (Figure 4). Figure 4. Funnel plot for studies (of 14 studies) on the association between salivary HPV and oral and oropharyngeal cancer. The vertical line represents the pooled OR using random-effect meta-analysis. Two diagonal lines represent (pseudo) 95% confidence limits around the OR for each standard error on the vertical axis. In the absence of heterogeneity, 95% of the studies should lie within the funnel defined by these diagonal lines. Abbreviations: se OR, standard error of odds ratio. Figure 3. Forest plot for the studies on the association between salivary HPV and oral and oropharyngeal cancer. The squares indicate the ORs (odds ratios) in each study, with square sizes inversely proportional to the standard error of the OR. The diamond shape indicates the pooled ORs. Horizontal lines represent 95% CIs (confidence intervals), I2 >50% indicates severe heterogeneity. A random-effects model was used because heterogeneity was identified among the 14 studies ( I2 =82% ). Visual inspection of the funnel plot revealed a symmetrical (Egger’s test, p=0.159; Begg’s test, p=0.298) distribution of the studies, indicating no evidence of publication bias (Figure 4). J. Clin. Med. 2020, 9, x FOR PEER REVIEW 2 of 19 Figure 3. Forest plot for the studies on the association between salivary HPV and oral and oropharyngeal cancer. The squares indicate the ORs (odds ratios) in each study, with square sizes inversely proportional to the standard error of the OR. The diamond shape indicates the pooled ORs. Horizontal lines represent 95% CIs (confidence intervals), I2 > 50% indicates severe heterogeneity. A random-effects model was used because heterogeneity was identified among the 14 studies (I2 = 82%). Visual inspection of the funnel plot revealed a symmetrical (Egger’s test, p = 0.159; Begg’s test, p = 0.298) distribution of the studies, indicating no evidence of publication bias (Figure 4). Figure 4. Funnel plot for studies (of 14 studies) on the association between salivary HPV and oral and oropharyngeal cancer. The vertical line represents the pooled OR using random-effect meta-analysis. Two diagonal lines represent (pseudo) 95% confidence limits around the OR for each standard error on the vertical axis. In the absence of heterogeneity, 95% of the studies should lie within the funnel defined by these diagonal lines. Abbreviations: se OR, standard error of odds ratio. Figure 4. Funnel plot for studies (of 14 studies) on the association between salivary HPV and oral and oropharyngeal cancer. The vertical line represents the pooled OR using random-effect meta-analysis. Two diagonal lines represent (pseudo) 95% confidence limits around the OR for each standard error on the vertical axis. In the absence of heterogeneity, 95% of the studies should lie within the funnel defined by these diagonal lines. Abbreviations: se OR, standard error of odds ratio. J. Clin. Med. 2020,9, 1305 9 of 18 For the type-specific analysis (Figure 5), salivary HPV16 showed a significant association with a pooled OR of 10.07 (95% CI =3.65 − 27.82; p<0.01). However, salivary HPV18 did not show any significant increased risk for oral and oropharyngeal cancer with a pooled OR of 1.80 (95% CI =0.66−4.90 ). In addition, a significant association was found for salivary HR-HPV with OR of 5.94 (95% CI =2.78−12.69 ; p<0.01 ), whereas salivary LR-HPV did not show any significant increased risk with OR of 1.45 (95% CI =0.70−2.98). The respective funnel plots are represented in Figures S1–S4. J. Clin. Med. 2020, 9, x FOR PEER REVIEW 3 of 19 For the type-specific analysis (Figure 5), salivary HPV16 showed a significant association with a pooled OR of 10.07 (95% CI = 3.65−27.82; p < 0.01). However, salivary HPV18 did not show any significant increased risk for oral and oropharyngeal cancer with a pooled OR of 1.80 (95% CI = 0.66−4.90). In addition, a significant association was found for salivary HR-HPV with OR of 5.94 (95% CI = 2.78−12.69; p < 0.01), whereas salivary LR-HPV did not show any significant increased risk with OR of 1.45 (95% CI = 0.70−2.98). The respective funnel plots are represented in Figures S1–4. Figure 5. Forest plot for the studies on the association between salivary HPV and oral and oropharyngeal cancer. The squares indicate the ORs in each study, with square sizes inversely proportional to the standard error of the OR. The diamond shape indicates the pooled ORs. Figure 5. Forest plot for the studies on the association between salivary HPV and oral and oropharyngeal cancer. The squares indicate the ORs in each study, with square sizes inversely proportional to the standard error of the OR. The diamond shape indicates the pooled ORs. Horizontal lines represent 95% CIs. I2 >50% indicates severe heterogeneity. (a) HPV16, (b) HPV18, (c) HR-HPV, and (d) LR-HPV. J. Clin. Med. 2020,9, 1305 16 of 18 17. Tuna, M.; Amos, C.I. Next generation sequencing and its applications in HPV-associated cancers. Oncotarget 2017,8, 8877–8889. [CrossRef] 18. Moher, D.; Liberati, A.; Tetzlaff, J.; Altman, D.G. Preferred reporting items for systematic reviews and meta-analyses: The PRISMA Statement. PLoS Med. 2009,6, e1000097. [CrossRef] 19. Wells, G.A.; Shea, B.; O’Connell, D.; Peterson, J.; Welch, V.; Losos, M.; Tugwell, P. The Newcastle-Ottawa Scale (NOS) for Assessing the Quality of Nonrandomized Studies in Meta-Analysis. 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