Prevalence of HPV infection in racial–ethnic subgroups of head and neck cancer patients

Carcinogenesis, Feb 2017

The landscape of human papillomavirus (HPV) infection in racial/ethnic subgroups of head and neck cancer (HNC) patients has not been evaluated carefully. In this study, a meta-analysis examined the prevalence of HPV in HNC patients of African ancestry. Additionally, a pooled analysis of subject-level data was also performed to investigate HPV prevalence and patterns of p16 (CDNK2A) expression amongst different racial groups. Eighteen publications (N = 798 Black HNC patients) were examined in the meta-analysis, and the pooled analysis included 29 datasets comprised of 3129 HNC patients of diverse racial/ethnic background. The meta-analysis revealed that the prevalence of HPV16 was higher among Blacks with oropharyngeal cancer than Blacks with non-oropharyngeal cancer. However, there was great heterogeneity observed among studies (Q test P < 0.0001). In the pooled analysis, after adjusting for each study, year of diagnosis, age, gender and smoking status, the prevalence of HPV16,18 in oropharyngeal cancer patients was highest in Whites (61.1%), followed by 58.0% in Blacks and 25.2% in Asians (P < 0.0001). There was no statistically significant difference in HPV16,18 prevalence in non-oropharyngeal cancer by race (P = 0.682). With regard to the pattern of HPV16,18 status and p16 expression, White patients had the highest proportion of HPV16,18+/p16+ oropharyngeal cancer (52.3%), while Asians and Blacks had significantly lower proportions (23.0 and 22.6%, respectively) [P < 0.0001]. Our findings suggest that the pattern of HPV16,18 status and p16 expression in oropharyngeal cancer appears to differ by race and this may contribute to survival disparities.

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Prevalence of HPV infection in racial–ethnic subgroups of head and neck cancer patients

Carcinogenesis Prevalence of HPV infection in racial-ethnic subgroups of head and neck cancer patients Camille Ragin 12 Jeffrey C.Liu 18 Gieira Jones Olubunmi Shoyele 17 Bukola Sowunmi Rachel Kennet Denise Gibbs 12 Elizabeth Blackman 12 Michael Esan Margaret S.Brandwei 16 Karthik Devaraja n 15 Francesco Bussu 20 Rebecca Chernock 19 Chih-Yen Chien 13 Marc A.Cohen 10 Samir El-Mofty 19 Mikio Suzuki Gypsyamber D'Souza 7 Pauline Funchain 9 Charis Eng 9 Susanne M.Gollin 3 Angela Hong 5 Yuh-S Jung Maximilian Krüger 1 James Lewis Jr Patrizia Morbin Santo Landolf o 11 Massimo Rittà 11 Jos Straetmans 0 Krisztina Szark a Ruth Tachezy 2 Francis P.Worden 4 Deborah Nelson Samuel Gathere 6 12 Emanuela Taiol 12 0 lDye,partment of Otorhinolaryngology-Head and Neck Surgery, GROW Institute, Maastricht University Medical Centre , Maastricht, The Netherl 1 aD, epartment of Oral and Maxillofacial Surgery - Plastic Surgery, University Medical Center of the Johannes Gutenberg-University , Mainz, Germa 2 rDye,partment of Immunology, Institute of Hematology and Blood Transfusion National Reference Laboratory for Papillomaviruses , Prague , Czech Republic 3 SDAe,partment of Human Genetics, Graduate School of Public Health, University of Pittsburgh , Pittsburgh, PA 15261, U , USA 4 Department of Internal Medicine, Division of Hematology-Oncology, University of Michigan , Ann Arbor, MI 48109 , USA 5 SCAen,tral Clinical School, The University of Sydney , Sydney, NSW, Austral 6 Non Communicable Diseases Research Programme, Kenya Medical Research Institute , Nairobi , Kenya 7 Department of Epidemiology, Johns Hopkins Bloomberg School of Public Health , Baltimore, MD 21205 , USA 8 Departments of Population Health Science and Policy, of Thoracic Surgery, and Institute For Translational Epidemiology, Icahn School of Medicine at Mount Sinai , New York, NY 10029 , USA 9 Genomic Medicine Institute, Cleveland Clinic Lerner Research Institute , Cleveland, OH 44195, U , USA 10 nD,epartment of Surgery, Head and Neck Service, Memorial Sloan Kettering Cancer Center , New York, NY 10065, U , USA 11 aDleyp,artment of Sciences of Public Health and Pediatrics, University of Turin , Turin, It a 12 Dnedpsa,rtment of Medical Microbiology, Faculty of Medicine, University of Debrecen , Debrecen, Hunga 13 ,Department of Otolaryngology, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine , Kaohsiung, Taiwa 14 D, epartment of Molecular Medicine, Unit of Pathology, University of Pavia, and à IRCCS Policlinico S. Matteo Foundation , Pavia, It 15 aD, epartment of Otolaryngology, Research Institute and Hospital, National Cancer Center , Gyeonggi-do, Kore 16 SDAe,partment of Pathology and Anatomical Sciences, SUNY at the University at Buffalo , Buffalo, NY 14214-3005 , US 17 ,Department of Pathology and Laboratory Medicine, Western Connecticut Health Network, Danbury Hospital , Danbury, CT 06810, U , USA 18 D,epartment of Otolaryngology, Fox Chase Cancer Center, Temple University , Philadelphia, PA 19111 , USA 19 Dye,partment of Pathology, Microbiology, and Immunology, Vanderbilt University , Nashville, TN 37232 , US 20 I,nstitute of Otolaryngology, Università Cattolica del Sacro Cuore, Policlinico Agostino Gemelli , Rome, It - The landscape of human papillomavirus (HPV) infection in racial/ethnic subgroups of head and neck cancer (HNC) patients has not been evaluated carefully. In this study, a meta-analysis examined the prevalence of HPV in HNC patients of African ancestry. Additionally, a pooled analysis of subject-level data was also performed to investigate HPV prevalence and patterns of p16 (CDNK2A) expression amongst different racial groups. Eighteen publicationNs =( 798 Black HNC patients) were examined in the meta-analysis, and the pooled analysis included 29 datasets comprised of 3129 HNC patients of diverse racial/ethnic background. The meta-analysis revealed that the prevalence of HPV16 was higher among Blacks with oropharyngeal cancer than Blacks with non-oropharyngeal cancer. However, there was great heterogeneity observed among studies (Q testP < 0.0001). In the pooled analysis, after adjusting for each study, year of diagnosis, age, gender and smoking status, the prevalence of HPV16,18 in oropharyngeal cancer patients was highest in Whites (61.1%), followed by 58.0% in Blacks and 25.2% in Asians P( < 0.0001). There was no statistically significant difference in HPV16,18 prevalence in nonoropharyngeal cancer by raceP( = 0.682). With regard to the pattern of HPV16,18 status and p16 expression, White patients had the highest proportion of HPV16,18+/p16+ oropharyngeal cancer (52.3%), while Asians and Blacks had significantly lower proportions (23.0 and 22.6%, respectively)P[ < 0.0001]. Our findings suggest that the pattern of HPV16,18 status and p16 expression in oropharyngeal cancer appears to differ by race and this may contribute to survival disparities. Abbreviations FF FT FFPE HPV HNC HR PCR Introduction fresh frozen fresh tissue formalin-fixed paraffin-embedded human papillomavirus head and neck cancer hazard ratios polymerase chain reaction of HNC patients by examining the published literature. We co-n ducted a meta-analysis examining the prevalence of HPV in the Black population. We also performed a pooled analysis of cases reporting HNC and HPV status using subject-level data from the published literature to investigate HPV segregation and pr-eva lence amongst different ethnic groups. Materials and methods This study was approved by the Fox Chase Cancer Center Institutional Review Committee. Head and neck cancer (HNC) is the sixth most common can- Literature review and data collection cer in the world, accounting for approximately 4% of all cancer A PubMed search was conducted (from inception to December 2014) using cases (1). In 2012, there were an estimated 599 637 new cases of the search terms, [‘human papillomavirus’ (All Fields) OR ‘HPV’ (All Fields)] cancer of the oral cavity, larynx and oropharynx, and 324 794 AND [‘squamous cell carcinoma’ (All Fields) OR ‘cancer’ (All Fields)] AND deaths attributed to the disease worldwid1e).(Although tobacco [‘oropharyngeal’ (All Fields) OR ‘oropharynx’ (All Fields) OR ‘head and neck’ and alcohol use are the primary risk factors for developing HNC, (All Fields) OR ‘tonsil’ (All Fields)]. All abstracts and full text of articles human papillomavirus (HPV) is also an established risk factor from the PubMed search were reviewed independently by two reviewers. for cancers arising in the oropharynx2,(3). Recently, HPV has When there was a discrepancy between reviewers, a third reviewer eva-lu also been reported to be associated with a subset of oral -cav parteesdetnhcee aorftHicPlVe(isn) tHoNrCestoislsvueetshferodmiscpraetpiaennctys. dAilalgsntousdeideswtihtahtstqeusatmedoufosr the ity cancers 4(,5), but an etiological role has not been clearly cell carcinoma of the head and neck (oral cavity, oropharynx, larynx and demonstrated. hypopharynx) were eligible for inclusion in this analysis. The bibliog-ra A recent review and meta-analysis from our group of HNC phies of several review articles were also examined in order to identify survival in relation to HPV demonstrated a survival advantage additional publications that might have been missed by our PubMed for all HPV-positive patients6(), but the survival advantage was search (11–15). This review identified 291 original articles that qualified only significant for patients with cancer of the oropharynx. conditionally for the analysis. Studies that used serology methods to Compared to patients with HPV-negative oropharyngeal cancer, detect HPV antibodies were excluded from the analysis, as this method the risk of death and risk of recurrence for patients with HPV- does not identify which tissue is infected by HPV. Studies that primarily positive oropharyngeal cancer was reduced by ~28% and ~49%, eevxacleuptaitoendoHfPstVuidnielsipwchaenrceeirtswwaesriempeoxscsliubdleedtofrdoismtitnhgiusiashnallipycsiasn,cweirthdatthae respectively. In the USA, a clear disparity in HNC survival has from the other head and neck subsites. In addition, case reports and st-ud been reported between Black and White patients, particularly for ies that included only HPV-positive HNC tumors/patients were excluded. oropharyngeal cancers. Poor survival rates for Black Americans Additional exclusion criteria includes studies of HNC patients who were compared to White Americans have been observed 7(), and some co-infected with other diseases, such as HIV; studies in which the cancer studies have suggested that this disparity may be explained at tissues were sampled via cytobrushing and not biopsy or surgery; studies least partially by a difference in prevalence of HPV infection that classified HNC as HPV-related or HPV non-related tumors based on (8–10). Comparisons of HPV prevalence in cancer of the oral ca-v tumor site without directly testing that tissue for HPV; studies in which ity and larynx between various racial/ethnic populations have fewer than 80% of the eligible cases were tested for HPV; and studies that been reported in a recent meta-analysis11(). However, a sum- selected patient samples non-randomly, but applied pre-defined criteria mary of HPV prevalence for Black patients was only reported fmoertpaasttiaesnist oinncllyu,psoiosniti(vee.gl.,ypmapthiennotdsewsiotnhluy,naddifvfaenrecnedtiasttaegdecoanrclyin,poamtaieonntlsy, for oral cavity cancer in this stud1y1(). Furthermore, an assess- who underwent a specific treatment regimen, studies where smoking and ment of attributed survival differences for oropharyngeal cancer drinking patient tissues were matched with nonsmoker and nondrinker between racial/ethnic populations was not conducted. patient tissues, etc.). For overlapping studies, the publication with the The goal of this study was to develop a more complete pe-r largest population and/or more complete information was included in spective of the landscape of HPV infection in ethnic subgroups this analysis. After accounting for these inclusion and exclusion criteria, 140 articles with data for all racial/ethnic populations were eligible forcavity, oropharynx or larynx. Patients with hypopharyngeal cancers were inclusion in this study. Of these, only 18 articles presented data that could grouped with the patients with cancers of the larynx. Patients with me-tas be abstracted and were included in the meta-analysis of Black cancer tases or unknown primaries were excluded from this analysis. In total, patients. All 140 articles were eligible for inclusion in the pooled analysis. there were 29 datasets including a total of 3129 HNC cases. A flow diagram of study selection is illustratedFingure 1. Statistical analysis The Meta-proportion of any HPV and HPV16 only was calculated for all From each of the 18 articles that included data from Black HNC patients HNC subsites combined as well as separately for oropharynx and non-o-ro (818 cases), information on the number of patients, HPV prevalence, pharynx data. All statistical analyses were performed using Intercooled HPV genotype, tumor subsite, mean age, year of cancer diagnosis, ge-o STATA SE (version 10) software (StataCorp. LP, College Station, TX). Metagraphic location of the study, tissue source, HPV test methodology and analyses of the proportion of HPV-positive HNC were performed using the HPV-infected cancer site were extracted and tabulated. All data were metaprop command in STATA. HPV proportions were calculated for each abstracted independently by two reviewers and cross-referenced to -con individual study and the reported confidence intervals were based on firm that there were no data entry errors. Three studies that included dataClopper–Pearson exact binomial procedures1(9). Pooled proportions of the for fewer than 10 Black patients16(–18) were therefore excluded from the multiple studies were estimated using a random effects model. The Metameta-analysis, leaving 15 studies including 798 cases. prevalence estimates were calculated by multiplying the Meta-proportion and confidence interval values by 100. ThQe-statistics were used to test Pooled analysis for heterogeneity between the studies included in the meta-analyses. The All investigators from the 140 studies were invited to submit their subject- I2 metric was also calculated to quantify variation between stud2ie0s). ( level data for this pooled analysis; data from 22 studies were obtained. Large between-study variation was observed when t hI2evalues were ≥50% The remaining study investigators either did not respond or did not wish while moderate between-study heterogeneity was denoted bI2y values to participate. Common data elements included in the pooled analysis between 25 and 50%. Evidence of publication bias or small study effects were HPV test method, HPV status, HPV genotype, DNA source, geographic (P < 0.05) was assessed using the Egger’s test2(1). location of the study, age at diagnosis, gender, race/ethnicity, p16 status, For the pooled analysis, unequal variance in age was observed tobacco and alcohol use, clinical variables (such as tumor site, histology between categories of race. Therefore, a square root transformation of and stage) and survival variables (such as vital status and follow-up time). age at diagnosis was performed. Adjusted HPV prevalence and 95% con-fi Seven additional articles reported demographic, clinical, HPV results, dence intervals for each racial/ethnic group was calculated from logistic tobacco, alcohol and survival data in the publications, which enabled regression estimates for HPV-positive status, adjusting for study, year of us to create pseudo-datasets for inclusion in the pooled analysis. All diagnosis, square root of age, sex, history of alcohol drinking and sm-ok patients included in this analysis were diagnosed with cancers of the oral ing history. The adjusted prevalence refers to the average HPV prevalence while averaging the values of the covariates in the regression model. The Asia (17%, 5/29) and a single study in Australia. Most of the logistic coefficients and standard errors are provided in Supplementary studies (65%, 19/29) involved patients diagnosed with ca-n Materials, available aCtarcinogenesis Online. A Likelihood Ratio chi-square cers at both oropharyngeal and non-oropharyngeal sites (oral test was performed to evaluate differences between the adjusted pre-va cavity, larynx, hypopharynx and non-oropharyngeal sites not lceonmcpearaecctohredminegantosqraucaereanrodoatnoafnaagleyasitsdoifavganroisainscbeet(AwNeOenVAr)awciaasl ugsreoduptso otherwise specified). The remaining studies included patients (P values for pairwise comparisons were Bonferroni adjusted)P. values < diagnosed with oropharyngeal cancers only. Formalin-fixed 0.05 were considered statistically significant. Mean age at diagnosis for paraffin-embedded (FFPE) tissues were examined in 66% each stratum was back transformed and reported. Follow-up time for of studies to test for the presence of HPV, rather than Fresh overall survival refers to the interval between date of diagnosis and theFrozen (FF) or Fresh Tissue (FT). All except for four studies used date of last contact (if the patient was alive) or date of death. Hazard ratiosPCR methodology to detect HPV DNA, using either consensus (HR) were calculated and adjusted for each study and other confounders or type-specific primers, and of these, five also evaluated HPV for risk of death or risk of disease progression (i.e., disease persistence, status using DNAin situ hybridization combined with PCR. Two recurrence and/or metastasis). HR < 1.0 represents an overall survival-ben studies detected HPV RNA using only RT-PCR and the other efit and HR > 1.0 represents poor overall survival. two detected both HPV RNA and DNA using RT-PCR and PCR. CDKN2A (p16) expression was evaluated in 16 studies using Results immunohistochemistry. With regard to race/ethnicity, the pooled dataset was diverse with patients representing African, Description of studies: meta-analysis African American, Asian and White populations. There was Table 1summarizes all published studies from which data were one study that included Aboriginal Australian patients. These available to estimate HPV (any HPV or HPV16) prevalence in patients were combined with the African and African American Black populations. Study size ranged from 13 to 161 patients.The patients and classified as Black. There were 82 patients cl-as majority (13/15, 87%) of studies included polymerase chain rea-c sified as other race (for 63 patients race was unknown and tion (PCR)-based methods to test for the presence of HPV DNA. 19 patients included Pacific Islander, Middle Eastern, Indian, For all site strata (all head and neck, oropharynx and non-oro Hispanic or other not otherwise specified). These patients were pharynx), large heterogeneity was observed between the studies grouped and classified as other race. Follow-up time was ava-il (Q testP-value range from 0.000 to 0.048I;2 values range from 62.1 able for 19 studies and ranged from 0.03 to 244.5 months with to 94.6%). Nevertheless, as expected, the prevalence of any HPV a mean follow-up of 41.7  months and a median follow-up of or HPV16 was higher among oropharyngeal cancer patients (any 30.6 months. HPV: 31.5%, 95% CI = 17.7–47.1; HPV16: 45.7%, 95% CI = 25.5–66.6) in comparison to non-oropharyngeal cancer patients (any HPV: Prevalence of HPV16 and HPV18 according to race 14.5%, 95% CI  =  1.4–36.0; HPV16: 1.1%, 95% CI  =  0.0–6.0). There and head and neck subsite was no evidence of publication bias or small study effect. The The prevalence of HPV16 and/or HPV18 (HPV16,18) stratified by reasons for underlying heterogeneity were explored by str-ati race was calculated for all HNCs, oropharyngeal cancers only and fying the dataset according to geographic region (Sub-Saharan non-oropharyngeal cancers only after adjusting for study, year Africa versus USA) as well as HPV test methods (ISH versus PCR/ of diagnosis, age, gender, alcohol drinking, and smoking status RT-PCR). Large heterogeneity remained when stratified by HPV (Table 3, and Supplementary Table 2, available atCarcinogenesis test method (data not shown). When stratified by geographic Online, which summarizes the logistic coefficients and stan-d region (see Supplementary Table  1, available aCtarcinogenesis ard errors). As expected, the overall mean age for HPV-positive Online), large heterogeneity was still observed except when patients diagnosed with oropharyngeal cancers was lower than data were limited to HPV16 infections only. For all head and the mean age of HPV-positive patients diagnosed with nonneck subsites combined, the meta-prevalence of HPV16 in oropharyngeal cancers irrespective of whether the patient -car patients from Sub-Saharan AfricaN(  = 4 studies) was 1.0% (95% ried HPV16 or HPV18 in their tumor. The mean age at diagnosis CI = 0.0–3.9), Q testP-value was 0.129,I2 was 47.0%. Large hetero- was 56.3  years for HPV16,18+ oropharyngeal cancer patients, geneity was still observed between the remaining eight studies and 60.1 years for HPV16,18+ non-oropharyngeal HNC patients that included patients from the USAQ(testP <0.0001, I2 = 89%). (P < 0.0001). There was no statistically significant difference in Further stratification of the Sub-Saharan Africa studies acc-ord the mean age at diagnosis of HPV16,18+ oropharyngeal cancer ing to head and neck subsite resulted in a meta-prevalence of patients according to race. However, for non-oropharyngeal HPV16 in non-oropharyngeal cancers at 0.1% (95% CI = 0.0–1.8, HNC patients, a Bonferronipost hoc test shows that Asians were Q testP value = 0.768,I2 = 0.0%). The only study in the USA that statistically significantly older compared to Whites (HPV16,18: reported HPV16 data for non-oropharyngeal cancer showed Asians, 64.1 years versus Whites, 54.9 yearsP, = 0.038). a higher prevalence (13.6%, 95% CI  =  1.9–31.7) than that of As expected, the prevalence of HPV16,18 was higher in or-o patients in Sub-Saharan Africa. There were no studies in Sub- pharyngeal cancer tissues compared to non-oropharyngeal Saharan Africa that reported data for HPV16 in oropharyngeal cancer tissues (HPV16,18: 48.7% versus 18.2%). HPV16 was the cancer patients and the large heterogeneity remained for the predominant genotype carried in all patient tissues, 46.6% of USA studies that reported HPV16 data in Black oropharyngeal oropharyngeal cancer patients and 13.4% of non-oropharyngeal cancer patients. HNC patients were positive for this genotype. In contrast, only approximately 1–2% of patients carried HPV18, irrespective of Description of studies: pooled analysis whether the cancer was diagnosed in the oropharynx or at a There were a total of 3129 patients included in this analysis non-oropharyngeal head and neck site. (Table  2). Variations among the 29 studies were noted with For oropharyngeal cancers, there was a statistically sig-nifi regard to study size, the geographic region where the study cant difference in the prevalence of HPV16,18 according to race. was conducted, tumor site and the tissue source. Studies v-ar White patients had the highest prevalence of HPV16,18+ cancers ied in size from 15 to 489 patients and were conducted mostly followed by Blacks then Asians, however, only the prevalence in in Europe (48%, 14/29 studies), followed by the USA (31%, 9/29), Asian patients was statistically significantly lower (61.1 versus Study All head and neck Van Rensburget al. (22) Gillisonet al. (23) Boy et al. (24) Agrawalet al. (25) Lewis et al. (26) Jalouliet al. (27) Jiron et al. (28) Stephen et al. (29) Babiker et al. (30) Isayeva et al. (31)a Ndiaye et al. (32) Salazaret al. (33) Worsham et al. (10) Isayeva et al. (34)a Liu et al. (35)a Total P value,Q test I2 test P value, Egger’s test Oropharynx Lewis et al. (26) Jiron et al. (28) Isayeva et al. (31)a Salazaret al. (33) Worsham et al. (10) Liu et al. (35)a Total P value,Q test I2 test P value, Egger’s test Non-oropharynx Van Rensburget al. (22) Boy et al. (24) Jalouliet al. (27) Jiron et al. (28) Ndiaye et al. (32) Isayeva et al. (34)a Total P value,Q test I2 test P value, Egger’s test ISH PCR PCR ISH ISH, PCR PCR PCR qRT-PCR PCR qRT-PCR PCR PCR, RT-PCR q-PCR qRT-PCR PCR ISH, PCR PCR qRT-PCR PCR, RT-PCR q-PCR PCR ISH PCR PCR PCR PCR qRT-PCR NR, not reported; X, HPV genotype unknown. aStudies included in the pooled analysis. 58.0% and 25.2%, respectively;P <0.0001). A similar pattern was patients had lower proportions of tumors with HPV16,18+/p16+ observed for the prevalence of HPV16 infections. However, for cancers (23.0 and 22.6%, respectively). In addition, Black patients HPV18, Black patients had the highest prevalence (14.8%) co-m had a higher proportion of cancers that were HPV16,18+, pared to Asians (1.6%) and Whites (1.1%) and this difference was but p16− compared to Asian and White patients (31.1 versus statistically significanPt  =(  0.0025). For the non-oropharyngeal 10.5% and 4.7%, respectively). The proportion of patients with cancer patients, there was no statistically significant differenceHPV16,18−/p16− disease also differed significantly by race. Asian in HPV16 and/or 18 prevalence according to race. patients had the highest proportion of HPV16,18−/p16− cancers, in contrast to Black and White patients (66.8 versus 37.7% and Expression of p16 and HPV16,18 DNA according to 29.6%, respectively). race in oropharynx cancer patients When the oropharyngeal cancer patients were stratified Twelve studies (1397 patients) presented with both HPV16,18 according to smoking history, the pattern of combined HPV16,18 and p16 data. Among oropharyngeal cancer patients, the pa-t and p16 status according to race co-segregated with the f-rac tern of combined HPV16,18 and p16 status differed according to tion of patients that were ever smokersFi(gure 2B). Among never race, and this difference was statistically significaFnitg u(re 2A, smokers (Figure  2C), as expected, patients with HPV16,18+/ P < 0.0001). White patients had the highest proportion of cancers p16+ cancers comprised the predominant fraction among that were HPV16,18+/p16+ (52.3%). In contrast, Asian and Black Asian, Black and White patients. However, White patients still N Race/ ethnicity W AS had the highest proportion, and Asian patients had the lo-w patients, but not for White patients. For non-Whites, the risk of est (White: 80.1%, Black: 62.5% Asian: 39.6%,P  <  0.0001). Even death was statistically significantly increased for patients with among never smokers, Asians continued to have the largest p-ro p16-negative oropharyngeal cancers, irrespective of HPV16,18 portion of patients with HPV16,18−/p16− cancers (37.4%), which status (HPV16,18+/p16−: HR = 2.95, 95% CI = 1.60–5.42, HPV16,18−/ was almost equal to the proportion of HPV16,18+/p16+ cancers p16−: HR  =  3.11, 95% CI  =  1.97–4.92 versus HPV16,18−/p16+: (39.6%) observed in this subgroup. HR  =  0.69, 95% CI  =  0.24–2.01). In contrast, the risk of death for White patients with p16+ cancers was dependent upon HPV16,18 Predictors of overall survival for oropharyngeal status. White patients with HPV16,18-/p16+ oropharyngeal c-an cancer patients according to race cers had an increased risk of death (HR = 2.91, 95% CI = 1.72–4.92) Independent predictors of overall survival for oropharyngeal -can in comparison to White patients with HPV16,18+/p16+ or-o cer patients were age at diagnosis, smoking history, late stage (III/ pharyngeal cancers. IV) at diagnosis, and combined HPV16,18 and p16 statusT(able 4). The risk of disease persistence, recurrence or metastasis based Patients with HPV16,18−/p16+ cancers had an increased risk of on HPV16,18/p16 status differed between White and non-White death compared to patients with HPV16,18+/p16+ oropharyngeal oropharyngeal cancer patients and is presented iTnable 5. White cancers. There was also an even greater increased risk of death patients that did not have HPV16,18+/p16+ disease had an increased for patients with p16− cancers irrespective of HPV status. When risk of disease persistence and/or recurrence in comparison to stratified according to smoking history, among never smokers, patients diagnosed with HPV16,18+/p16+ disease. In contrast nonHPV16,18−/p16− patients were the only group with a statistically white patients with HPV16,18−/p16− were the only subgroup with a significantly increased risk of death compared to HPV16,18+/p16+ greater risk of disease persistence and/or recurrence in comparison patients (Hazard Ratio[HR]: 2.70, 95% Confidence Interval [CI] to HPV16,18+/p16+ disease (HR = 2.70, 95% CI = 1.52–4.82).The risk of 1.12–6.51). Patients with HPV16,18+/p16− or HPV16,18−/p16+ oro- metastasis was only associated with non-White patients carrying pharyngeal cancers also had an increased risk of death compared HPV16,18−/p16− oropharyngeal cancers. to patients with HPV16,18+/p16+ oropharyngeal cancers, but the hazard ratios were not statistically significant. When stratified according to race, non-White patients differed in comparison to Discussion White patients regarding risk of death based on HPV16,18/p16 This study expands on our prior reported meta-analysis of HPV status.Table 4 shows that p16 status rather than HPV DNA s-ta and HNC (6). In that study, we showed that the presence of HPV tus appeared to be a predictor of overall survival for non-White infection, specifically in the oropharynx had a significant effect b e c 8 n ,1 e 6 l 1 a V v P re H p ) ean SD m ± um 632 131 915 58 367 um 432 110 229 44 15 6 9 8 um 200 21 86 14 21 1 2 N 1 1 N N N in ag ra s n lc ru u to ow ex fo A o V r n s e O e k a h w t N raq dun dn ee n A u t ea /e H d ck ,y ec d eh a y a te t 9 b d r c r u r i o 50 sa st e d f h e e 1 w h t r o p lu is c s a s s a o e e u v d t n r a P ag fo lu t e i c s r b e c n e l a v e r p 8 1 V P H ) e D it S s n b a ± a r t s b e e c c n a e r l o a t ev )I g pr C n i d 6 % r 1 5 o PV (9 c c H % a H ) D d S n n a a ± d a e h y b d e i f i t 8 1 V P a v e r p t s u j d A . 3   e l b a T f o n H a a u e s 24 0 0 0 s m ra  = .9 .2 .5 eck 81+ (ey  ise   0±   0±   0± n V ae d .9 .7 .5 d P g r e s n a w f16ecePonVH 61eP+VHma(sreeyaag   ifsste28=odu   ..096485±   ..065565±   ..054765±   ..035255±   ..085065±   ifsste25=odu   ..086365±   ..075865±   ..024665±   ..904955±   ..304965±   ifsste21=odu   ..005056±   ..640225±   ..290525± —  . .500556± ffsrrteaaodnm ,iifssragoodn ipsrtcaoguhn fplrtece18yPV iffsrteceeebn l d e N N r e b b um 634 158 .123 58 973 um 433 120 317 44 1 4 9 2 2 N 1 1 x N b um 201 38 06 14 8 r e r e n y r a h p x n y C n k te cre l a r lHN isaA lcaB ihW thO taoT proh isanA lckaB itehWcrtehO ltaoT -rooon isanA lckaB itehWcrtehO ltaoT teagd jsteddu irtehn ikognm i-saquh l A O N aA bA cOdS ¥C on disease-free survival and overall survival. Since the time of We performed a pooled analysis of published HPV and HNC that publication, HPV-positive squamous cell carcinoma of the data in racial/ethnic subgroups in order to obtain a broader-per oropharynx has been well described and reported as a distinct spective. HPV status was obtained predominantly by PCR on clinical entity. Oropharyngeal cancer patients are often non- FFPE tissues. Evaluation of HPV16, HPV18 and HPV16,18 prev-a smokers, male, younger and White compared to traditional lence by subsite and race yielded multiple findings. First, it is substance abuse-related (tobacco and alcohol) HNC. A  d-ra clear that HPV, specifically HPV16 or HPV18 within the oroph-ar matic increase in oropharyngeal cancer prevalence has been ynx is most common in Whites (61%). There is a similar yet identified over the last decade2,(61,62). The number of cases of lower rate of HPV16,18+ disease in Blacks (58%) and a significant oropharyngeal cancer exceeded the number of cervical cancer difference in the rate of HPV16,18+ disease in Asians (25%). This cases in 2010 in the United States, and the number of HPV+ highlights the major HPV prevalence difference between Whites oropharyngeal cancer is expected to exceed the incidence of and Asians. This finding is curious, since the prevalence of HPV cervical cancer by 20202(). In addition, the more favorable ou-t in Black patients has been reported to be statistically sig-nifi come of HPV+ oropharyngeal cancer is well-documented and cantly lower than what has been reported for White patients in has been confirmed in multiple studies 6(3,64). These tumors the literature61(,65). However, our pooled analysis reflects data appear to be HPV-related, and a hallmark of favorable tumors from multiple institutions which is more reliable than a single is p16 positivity. study. The observed differences in HPV prevalence between For unclear reasons, the prevalence and favorable outcome of Asians and Whites is also interesting and is not consistent with HPV+ oropharyngeal cancer is seen mostly in Whites. Variations the previously reported meta-analysis1(1). This inconsistency in the prevalence of HPV have been noted previously in studies might be explained by differences in the type of Asian populaof Black patients with oropharyngeal cancer, where some report tions included in our study. This pooled analysis only included lower prevalence and others report a prevalence that is higher Asians from Taiwan (China) and Japan while the previously puband/or comparable to White oropharyngeal cancer patients lished meta-analysis included Asian populations from China (9,10,35). In the first part of this study, the meta-analysis of p-ub and Korea. Significantly higher HPV prevalence was observed lished HPV prevalence and HNC in Black patients echoes these in Korean patients compared to Chinese patients and could findings. Consistent with what is expected when comparing HPV explain the higher prevalence of HPV+ oropharyngeal cancer in prevalence in oropharyngeal and non-oropharyngeal cancer s-ub Asians in that review1(1). sites, we show that for Black patients, cancers in the oropharynx An unexpected finding was the higher prevalence of HPV18 have a higher prevalence of HPV16 (45.7%), than non-orophary-n amongst Blacks. While HPV18 is rarely reported at either -oro geal sites (14.5%). There was large heterogeneity between the pharyngeal (1.1%) or non-oropharyngeal cancer sites (1.5%) in studies included in our meta-analysis. It is possible that dif-fer Whites, HPV18 is nearly 15 times more frequently detected in ences in the HPV detection methods used in different studies Black oropharyngeal cancer patients. This major difference was may have influenced HPV positivity rates. For example, DNA ISH unexpected. It is unclear if this is due to a higher rate of HPV18 assays lack sensitivity and in general, PCR may lack specificity infection in HNC in Blacks or a lower rate of HPV16+ orophar-yn for transcriptionally active virus. Nevertheless, we observed thatgeal cancer in Blacks, thereby unmasking HPV18. the meta-prevalence of HPV16 among Black patients is similar to To better characterize oropharyngeal cancers, we evaluated the prevalence reported in our pooled analysis (i.e., higher in the by both HPV and p16 status. Canonical HPV oropharyngeal c-an oropharynx and lower in non-oropharyngeal sites). cer is characterized by a HPV+/p16+ signature and p16 status ) 1 3 7   ) 9 4 1   ) 1 0 4   =   n ( e t i h W n o N ) 5 7 4   =   n ( e t i h ) 4 3 1   =   n ( W ) 6 4 7   =   n ( ) 0 8 8 ) ) sr )91 .08 .18 )39 e . 4 4 . k 5 2 3 2 o – – – – s ) 7 9 6 0 cea srm .100 .(02 .(01 .(04 .(09 llr eev (fe .72 .41 .99 .74 A N R 1 2 3 1 ) ) ) 8 7 2 sr .2 .3 .4 e 1 1 1 – – – s k ) 0 5 6 eca som .001 .(50 .(40 .(01 llr rev (fe .80 .78 .23 A E R 0 0 — 1  (llsrec=aAN .)(f001eR ...()–650173780 ...()–050145580 ...()–800415750 ...()–901144521 .)(f001eR ...()–341283591 .()f001eR ...()–800126001 .()f001eR ...()–504140170 .()f001eR ...()–518627702 .()f001eR ...()–118992781 ...()–224564623 ...()–237942013 ,rrrteeeyaaaoogu q s : d + + − − e isso re r rek re stu /p16 /p16 /p16 /p16 lcdu k e pryxaohn eca itehW isanA lckaB rtehO iteaaggdn ikgonm sreevomN srevEkom llcooh irreevdnN irrevEkdn ex leaM leeFam teag III//0 IIII/V /pst61PaV ,1618P+VH ,1618P−VH ,1618P+VH ,1618P−VH iisrtevaaon r S O R A S aC Table  5. Risk of disease progression for oropharyngeal cancer p-a HPV mRNA. For each of the studies included in the pooled ana-ly tients according to HPV/p16 status and race sis, we did not have detailed information on the cutoffs used to DHiRs,e9a5s%e pCeIrasistence and/or recurrence cdyetfionpelap1sm6)s.tTahtiussi(si.ae.l,sforaactliiomnitoaftpi1o6neoxfproeusrsisotnudiny,nauscltehiisvedrestuasil may have provided more accurate correlations of p16 expression White Non-White and outcome according to race. HPV/p16 status N = 475 N = 401 The reasons for this difference in patterns of HPV/p16 in oropharyngeal cancer are unclear. While smoking status has HPV16,18+/p16+ Ref (1.00) Ref (1.00) predicted p16 status 6(7), even amongst never smokers in HPV16,18−/p16+ 2.33 (1.22–4.45) 0.52 (0.12–2.27) this study, the prevalence of HPV+/p16+ disease is lower in HPV16,18+/p16− 3.62 (2.21–5.95) 1.44 (0.58–3.61) non-Whites. Possible explanations include genetic and en-vi HPV16,18−/p16− 3.23 (2.14–4.88) 2.70 (1.52–4.82) ronmental causes. The development of HPV+ oropharyngeal Metastasis HR, 95% CaI cancer has been associated with differences in sexual beh-av HHHPPPVVV111666,,,111888+−+///ppp111666−++ 21R..e68f14(1((00.0..94009)––76..5910)) 10R..e08f81(1((00.0..43085)––21..4828)) iboerhapvaitotrerpnasttaenrdnsmaamriojunagnsta Wushei6t(8e)s. vDeifrfseursenntoianl-Wsehxiuteasl haanvde HPV16,18−/p16− 2.08 (0.88–4.91) 1.94 (1.26–2.99) not been studied well. While the number of oral sex partners has been identified in the risk of developing HPV+ orophary-n aAdjusted for year of diagnosis, square root age, sex, race, stage, smoking, geal cancer 6(8). The percentage difference in ever oral sex alcohol and study. partners in individuals 45–60  years old between Whites and Blacks appears modest (about 15% difference in prevalence) has been reported previously as the best prognostic marker from a few major studies (69,70), but this remains an area of for this disease 6(3,66). Oropharyngeal cancer that develops in active research. Other potential explanations are genetic- dif White nonsmokers is mostly likely to be HPV-associated. Our ferences between races and differences in the host response study confirmed this finding; nearly 80% of White nonsmo-k to HPV infection, which merit further investigation. Intratypic ers were HPV+/p16+ (Figure 2C). As p16 loss is associated with variation of HPV16 is associated with geographical distri-bu smoking (67), amongst ever smokers, a much higher incidence tion and may contribute to differences in outcome7 1(–76). For of p16− disease was reported in all races. Although appro-xi example, African and Asian-American intratypic variants of mately 45% of ever smokers continue to be HPV+/p16+, only HPV16 show higher transforming potential in tumors of the half that frequency of HPV+/p16+ is reported in non-Whites. anogenital tract. Therefore, in HNC, differential infection by Amongst Blacks and especially Asians, HPV−/p16+ disease com- HPV variants between races may also be an important area for prises the majority of oropharyngeal disease, in distinction to investigation. Whites, where HPV+/p16+ disease is the predominant disease. At this time, we do not have sufficient understanding to While it is not surprising that patients with HPV−/p16+ o-ro offer a clear recommendation as to how to reduce orophary-n pharyngeal cancer have a higher risk of death compared to geal HPV infection or the risk of developing HPV+ orophary-n patients with HPV+/p16+ oropharyngeal cancer, it was int-er geal cancer. This appears to be a problem of environment esting to note that among non-Whites, the risk of death for and biology, without a reversible modifiable factor to reduce patients with HPV−/p16+ oropharyngeal cancer was not d-if risk. We hope that greater adoption of HPV vaccination will ferent from patients diagnosed with HPV+/p16+ orophary-n alter the incidence curve within about 20  years. Our study geal cancer (HPV16,18−/p16+ HR: 0.69, 0.24–2.01). Unlike Whites has examined HPV and HNC, with a focus on oropharyngeal (HPV16,18−/p16+ HR: 2.91, 1.72–4.92), the survival benefit among cancer. This study demonstrates that while HPV-related -oro non-Whites appears to be attributed to p16 status rather than pharyngeal cancer (HPV+/p16+) represents the majority cause HPV. In Whites, the survival benefit appears to be attributed to among White patients, Blacks and Asians have lower rates. HPV status rather than p16 status. However, it is possible that Because HPV-related oropharyngeal cancer has a more favo-ra HPV16,18−/p16+ oropharyngeal cancers in non-Whites may be ble outcome regardless of race, the differential HPV prevalence attributed to other high-risk HPV types. Further investigation amongst Blacks and Asians is expected to cause a significant of the possible role of high-risk HPV types other than HPV16,18 outcome disparity in oropharyngeal cancer treatment. Further in non-White oropharyngeal cancer patients is needed. Overall, studies specifically examining racial differences in HPV+ o-ro our findings suggest that the difference in HPV/p16 patterns pharyngeal cancer are needed to corroborate these findings. according to race may impact survival differently. Given the However, this comprehensive pooled analysis of the published multifactorial cause of racial survival disparities, such as poor literature strongly supports a prevalence disparity in HPV+ socioeconomic status and poor access to care, the effect of HPV/ oropharyngeal cancer that would predict an outcome/survival p16 patterns on racial disparities in survival is not easily ide-nti disparity. fied and further investigations are needed. A limitation of this study is the use of publications as the Supplementary material source of patient data. Unlike database data, like SEER or The National Cancer Database, published data represent a sa-m Supplementary data are available aCtarcinogenesis online. pling of the true population. A major assumption of our pooled analysis is that the landscape of the published literature is-rep Funding resentative of the population as a whole. Given the dramatic Supported by the American Cancer Society (RSG-14-033-01-CPPB differences noted in survival here between Whites and non- to C.R.) and in part by National Cancer Institute (P30 CA006927) Whites, we feel it is highly unlikely that an error in sampling of and Commonwealth of Pennsylvania. This work was also su-p the literature can explain these differences. A  high fraction of ported in part by The Lagrange Project – CRT Foundation/ISI cells with expression of p16 in both the nucleus and cytoplasm Foundation, Turin, Italy to M.R. is the only good correlation with prognosis and with high-risk Conflict of Interest Statement: None declared. 47. Tachezy , R. et al. ( 2009 ) Demographic and risk factors in patients with 61 . Fakhry , C. et al. ( 2015 ) Oropharyngeal cancer survivorship in Denmark, head and neck tumors . J. Med . Virol., 81 , 878 - 887 . 1977 - 2012 . Oral Oncol., 51 , 982 - 984 . 48. D'Souza , G. et  al. 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Ragin, Camille, Liu, Jeffrey C., Jones, Gieira, Shoyele, Olubunmi, Sowunmi, Bukola, Kennett, Rachel, Gibbs, Denise, Blackman, Elizabeth, Esan, Michael, Brandwein, Margaret S., Devarajan, Karthik, Bussu, Francesco, Chernock, Rebecca, Chien, Chih-Yen, Cohen, Marc A., El-Mofty, Samir, Suzuki, Mikio, D’Souza, Gypsyamber, Funchain, Pauline, Eng, Charis, Gollin, Susanne M., Hong, Angela, Jung, Yuh-S, Krüger, Maximilian, Lewis, James, Morbini, Patrizia, Landolfo, Santo, Rittà, Massimo, Straetmans, Jos, Szarka, Krisztina, Tachezy, Ruth, Worden, Francis P., Nelson, Deborah, Gathere, Samuel, Taioli, Emanuela. Prevalence of HPV infection in racial–ethnic subgroups of head and neck cancer patients, Carcinogenesis, 2017, 218-229, DOI: 10.1093/carcin/bgw203