Latency of tobacco smoking for head and neck cancer among HPV-positive and HPV-negative individuals

Human papillomavirus (HPV) infection and tobacco smoking are well-known risk factors for head and neck cancers (HNC). Although an effect modification between oral HPV infection and tobacco smoking may exist, evidence is lacking on how they interact temporally. We investigated the latency and life course effects of tobacco smoking on risk of HNC among HPV-positive (HPV^+ve) and negative (HPV^-ve) individuals. We used data from 631 ever-smoker participants of a hospital-based case–control study conducted in four major hospitals in Montréal, Canada. Cases (n = 320), incident, histologically confirmed, primary squamous cell carcinomas, were frequency-matched to controls (n = 311) by age and sex. Sociodemographic and behavioral factors (e.g., tobacco and alcohol use and sexual history) were collected using a structured interview applying a life grid technique. Oral exfoliated cells were used for HPV DNA detection and genotyping. Latency effects were estimated flexibly using a Bayesian relevant exposure model and further extended with a life course approach. Retrospective smoking trajectories for HPV^+ve cases and controls had similar shapes. Exposure to tobacco smoking even 40 years before diagnosis was associated with an increased HNC risk among both HPV^+ve and HPV^-ve participants. The effect of smoking before the start of sexual activity compared to afterwards was higher among HPV^+ve individuals. This pattern of association was less profound among HPV^-ve participants. Temporal interactions may exists between oral HPV infection and life course smoking trajectories in relation to HNC risk.     

Role of mucosal high‐risk human papillomavirus types in head and neck cancers in central India

Mucosal high‐risk (HR) human papillomaviruses (HPV) cause a subset of head and neck cancers (HNC). The HPV‐attributable fraction of HNC varies substantially between countries. Although HNC has a very high incidence in the Indian subcontinent, information on the contribution of HPV infection is limited. Here, we evaluated the HPV‐attributable fraction in HNC (N = 364) collected in a central region of India. HNC from three different anatomical subsites were included, namely, oral cavity (n = 252), oropharynx (n = 53) and hypopharynx/larynx (n = 59). In this retrospective study, HPV‐driven HNC were defined by presence of both viral DNA and RNA. Overexpression of p16^INK4a was also evaluated. HR‐HPV DNA was detected in 13.7% of the cases; however, only 2.7% were positive for both HPV DNA and RNA. The highest percentage of HPV DNA/RNA double positivity was found in oropharynx (9.4%), followed by larynx (1.7%) and oral cavity (1.6%) (p = 0.02). More than half of HPV DNA/RNA‐positive cases were p16^INK4a‐negative, while a considerable number of HPV RNA‐negative cases were p16^INK4a‐positive (17.9%). HPV16 was the major type associated with HNC (60.0%), although cases positive for HPV18, 35 and 56 were also detected. Our data indicate that the proportion and types of mucosal HR‐HPV associated with HNC in this central Indian region differ from those in other (developed) parts of the world. This may be explained by differences in smoking and/or sexual behaviour compared with North America and northern Europe. Moreover, we show that p16^INK4a staining appeared not to be a good surrogate marker of HPV transformation in the Indian HNC cases.     

Combined P16 and human papillomavirus testing predicts head and neck cancer survival

While its prognostic significance remains unclear, p16^INK4a protein expression is increasingly being used as a surrogate marker for oncogenic human papillomavirus (HPV) infection in head and neck squamous cell carcinomas (HNSCC). To evaluate the prognostic utility of p16 expression in HNSCC, we prospectively collected 163 primary tumor specimens from histologically confirmed HNSCC patients who were followed for up to 9.4 years. Formalin fixed tumor specimens were tested for p16 protein expression by immunohistochemistry (IHC). HPV type‐16 DNA and RNA was detected by MY09/11‐PCR and E6/E7 RT‐PCR on matched frozen tissue, respectively. P16 protein expression was detected more often in oropharyngeal tumors (53%) as compared with laryngeal (24%), hypopharyngeal (8%) or oral cavity tumors (4%; p < 0.0001). With respect to prognosis, p16‐positive oropharyngeal tumors exhibited significantly better overall survival than p16‐negative tumors (log‐rank test p = 0.04), whereas no survival benefit was observed for nonoropharyngeal tumors. However, when both p16 and HPV DNA test results were considered, concordantly positive nonoropharyngeal tumors had significantly better disease‐specific survival than concordantly negative nonoropharyngeal tumors after controlling for sex, nodal stage, tumor size, tumor subsite, primary tumor site number, smoking and drinking [adjusted hazard ratio (HR) = 0.04, 0.01–0.54]. Compared with concordantly negative nonoropharyngeal HNSCC, p16(+)/HPV16(?) nonoropharyngeal HNSCC (n = 13, 7%) demonstrated no significant improvement in disease‐specific survival when HPV16 was detected by RNA (adjusted HR = 0.83, 0.22–3.17). Our findings show that p16 IHC alone has potential as a prognostic test for oropharyngeal cancer survival, but combined p16/HPV testing is necessary to identify HPV‐associated nonoropharyngeal HNSCC with better prognosis.     

Prospective study of oral microbiome and colorectal cancer risk in low-income and African American populations

Oral microbiome may play an important role in cancer pathogenesis. However, no study has prospectively investigated the association of the oral microbiome with subsequent risk of developing colorectal cancer (CRC). We conducted a nested case–control study including 231 incident CRC cases and 462 controls within the Southern Community Cohort Study with 75% of the subjects being African-Americans. The controls were individually matched to cases based on age, ethnic group, smoking, season-of-study enrollment and recruitment method. Oral microbiota were assessed using 16S rRNA gene sequencing in pre-diagnostic mouth rinse samples. Multiple bacterial taxa showed an association with CRC risk at p <0.05. Oral pathogens Treponema denticola and Prevotella intermedia were associated with an increased risk of CRC, with odds ratios (ORs) and 95% confidence intervals (CIs) of 1.76(1.19–2.60) and 1.55(1.08–2.22), respectively, for the individuals carrying these bacteria compared to non-carriers. In the phylum Actinobacteria, Bifidobacteriaceae was more abundant among CRC patients than among controls. In the phylum Bacteroidetes, Prevotella denticola and Prevotella sp. oral taxon 300 were associated with an increased CRC risk, while Prevotella melaninogenica was associated with a decreased risk of CRC. In the phylum Firmicutes, Carnobacteriaceae, Streptococcaceae, Erysipelotrichaceae, Streptococcus, Solobacterium, Streptococcus sp. oral taxon 058 and Solobacterium moorei showed associations with a decreased risk of CRC. Most of these associations were observed among both African- and European-Americans. Most of the associations were not significant after Bonferroni correction for multiple testing, which may be conservative. Our study suggests that the oral microbiome may play a significant role in CRC etiology.     

Human papilloma viruses and p53 mutations in normal,pre-malignant and malignant oral epithelia

HPV infections have been previously observed in oral cancers, and inactivation of the p53 gene has been shown to be one of the most common genetic alterations in human tumors. We examined 179 oral specimens from 70 individuals with histologic findings of either normal mucosa (n = 6) or oral disease that ranged from mild dysplasia to invasive squamous-cell carcinoma (n = 64) to determine the occurrence of both HPV infection and p53 mutations and their relationship with several clinical factors. HPV infection was detected by PCR amplification of viral DNA, and the presence of p53 mutations was assayed using the single-strand conformation polymorphism (SSCP)-PCR technique. HPV infection was found in 31% of individuals with oral disease and was not seen in healthy individuals. Mutations in exons 5, 6, 7 or 8 of the p53 gene were detected in 37.5% of patients with oral lesions and in a biopsy from 1 healthy individual who was a heavy smoker. Approximately one-third of lesions classified as pre-malignant (dysplasia and carcinoma in situ) and 42% of invasive carcinomas contained p53 mutations. The majority of these mutations were G:T transversions located within exons 7 and 8. Tumor tissues from 6 patients with oral lesions were found both to be HPV-16-positive and to contain p53 mutations; of these, 4 were poorly differentiated carcinomas that were diagnosed as late-stage disease. In this study, p53 mutations were detected in the early stages of cancer development. © 1996 Wiley-Liss, Inc.     

Transitions in oral and gut microbiome of HPV+ oropharyngeal squamous cell carcinoma following definitive chemoradiotherapy (ROMA LA-OPSCC study)

Background Oral and gut microbiomes have emerged as potential biomarkers in cancer. We characterised the oral and gut microbiomes in a prospective observational cohort of HPV+ oropharyngeal squamous cell carcinoma (OPSCC) patients and evaluated the impact of chemoradiotherapy (CRT).Methods Saliva, oropharyngeal swabs over the tumour site and stool were collected at baseline and post-CRT. 16S RNA and shotgun metagenomic sequencing were used to generate taxonomic profiles, including relative abundance (RA), bacterial density, α-diversity and β-diversity.Results A total of 132 samples from 22 patients were analysed. Baseline saliva and swabs had similar taxonomic composition (R² = 0.006; p = 0.827). Oropharyngeal swabs and stool taxonomic composition varied significantly by stage, with increased oral RA of Fusobacterium nucleatum observed in stage III disease (p < 0.05). CRT significantly reduced the species richness and increased the RA of gut-associated taxa in oropharyngeal swabs (p < 0.05), while it had no effect in stool samples. These findings remained significant when adjusted by stage, smoking status and antibiotic use.Conclusions Baseline oral and gut microbiomes differ by stage in this HPV+ cohort. CRT caused a shift towards a gut-like microbiome composition in oropharyngeal swabs. Stage-specific features and the transitions in oral microbiome might have prognostic and therapeutic implications.Subject terms: Cancer microenvironment, Oral cancer     

The role of the antileukoprotease SLPI in smoking‐induced human papillomavirus‐independent head and neck squamous cell carcinomas

Recently, we showed that increased SLPI levels prevent human papillomavirus (HPV) infections and metastasis in smoking‐induced, non‐HPV‐driven head and neck squamous cell carcinoma (HNSCC). Here, we focus on the role of SLPI in non‐HPV‐driven HNSCC, investigating tumor tissue and non‐neoplastic mucosa from the same patients and from non‐HNSCC patients. Gene and protein expression of SLPI and gene expression of annexin 2 (a SLPI receptor), nicotine receptor (α7AChR) and arylhydrocarbon receptor (AhR) were analyzed in HNSCC patients (20 smokers; 16 nonsmokers). SLPI‐results were correlated with the patients' HPV status. Non‐neoplastic mucosa of HNSCC patients and normal mucosa from non‐HNSCC individuals (18 smokers; 20 nonsmokers) was analyzed for the same parameters. Tissue of the inferior turbinate (n = 10) was incubated with nicotine for analysis of the same genes. SLPI gene expression in tumor tissue was 109.26 ± 23.08 times higher in smokers versus nonsmokers. Non‐neoplastic mucosa of smokers showed also higher SLPI gene expression (10.49 ± 1.89‐fold non‐HNSCC; 18.02 ± 3.93‐fold HNSCC patients). Annexin 2 gene expression was also increased in smokers. SLPI data were corroborated by immunohistochemistry. A nicotine dependent correlation between SLPI and annexin 2 gene expression (r² = 0.15, p < 0.001) was shown ex vivo. Nicotine and smoking increased α7AChR and AhR gene expression. Five patients, showing no/low SLPI expression, were HPV16‐positive. A significant correlation between smoking and SLPI expression in tumors and to our knowledge for the first time in mucosa of HNSCC and non‐HNSCC patients was established. Together with the finding that all patients with HPV infection showed no/low SLPI expression, these data support our intriguing hypothesis that smoking induced upregulated SLPI prevents HPV infections.     

Tobacco smoking and alcohol drinking at diagnosis of head and neck cancer and all‐cause mortality: Results from head and neck 5000, a prospective observational cohort of people with head and neck cancer

Tobacco smoking and alcohol consumption are well‐established risk factors for head and neck cancer. The prognostic role of smoking and alcohol intake at diagnosis have been less well studied. We analysed 1,393 people prospectively enrolled into the Head and Neck 5000 study (oral cavity cancer, n=403; oropharyngeal cancer, n=660; laryngeal cancer, n=330) and followed up for a median of 3.5 years. The primary outcome was all‐cause mortality. We used Cox proportional hazard models to derive minimally adjusted (age and gender) and fully adjusted (age, gender, ethnicity, stage, comorbidity, body mass index, HPV status, treatment, education, deprivation index, income, marital status, and either smoking or alcohol use) mortality hazard ratios (HR) for the effects of smoking status and alcohol intake at diagnosis. Models were stratified by cancer site, stage and HPV status. The fully‐adjusted HR for current versus never‐smokers was 1.7 overall (95% confidence interval [CI] 1.1, 2.6). In stratified analyses, associations of smoking with mortality were observed for oropharyngeal and laryngeal cancers (fully adjusted HRs for current smokers: 1.8 (95% CI=0.9, 3.40 and 2.3 (95% CI=0.8, 6.4)). We found no evidence that people who drank hazardous to harmful amounts of alcohol at diagnosis had a higher mortality risk compared to non‐drinkers (HR=1.2 (95% CI=0.9, 1.6)). There was no strong evidence that HPV status or tumour stage modified the association of smoking with survival. Smoking status at the time of a head and neck cancer diagnosis influenced all‐cause mortality in models adjusted for important prognostic factors.     

CD56‐positive lymphocyte infiltration in relation to human papillomavirus association and prognostic significance in oropharyngeal squamous cell carcinoma

Human papillomavirus (HPV)‐related squamous cell carcinoma of the oropharynx (OSCC) are clinical and biological distinct from their HPV‐unrelated counterparts. Patients with HPV‐related OSCC display improved prognosis and therefore we investigated possible immune cell infiltrations associated with this tumor phenotype. We retrospectively analyzed a randomly selected cohort of 140 OSCC for presence of immune cells and HPV by immunohistochemistry and PCR followed by bead‐based hybridization (Luminex technology). HPV prevalence was 24.3% as determined by positive staining of p16INK4a and detection of high risk HPV‐DNA. We found significantly higher numbers of CD56 positive (CD56+) cells in tumor and surrounding microenvironment in HPV‐associated compared to HPV‐negative OSCC (t‐test: p = 0.004 and p = 0.002). For the entire cohort presence of CD56+ cells was associated with increased overall survival independent from HPV (Kaplan–Meier: p = 0.002; Cox regression: p = 0.042). Presence of CD56+ cells also correlated with a better outcome in HPV‐negative and especially in HPV‐negative OSCC with alcohol consumption ≤2 standard drinks per day (Kaplan–Meier: p = 0.05 and p = 0.003). Immunofluorescence localization of granular Granzyme B (GZMB) within CD56+ cells and coexpression of CD16 and CD56 suggests that detected CD56+ cells mainly represent cytotoxic Natural Killer (NK) cells. The fraction of potentially cytotoxic NK cells was significantly higher in HPV‐associated compared to HPV‐negative OSCC (Mann‐Whitney‐U‐Test: p = 0.011). The elevated abundance and activity of cytotoxic NK cells in OSCC with HPV driven carcinogenesis might contribute to favorable outcome in HPV‐related OSCC.     

Identification of prognostic molecular biomarkers in 157 HPV-positive and HPV-negative squamous cell carcinomas of the oropharynx

The incidence of oropharyngeal squamous cell carcinoma (OPSCC) has been increasing due to high-risk HPV infection. We explored the significance of genetic alterations in HPV-positive (HPV-P) and HPV-negative (HPV-N) OPSCC patients on long-term outcome. A total of 157 cases of primary resected OPSCC diagnosed from 1978 to 2005 were subjected to a targeted exome sequencing by MSK-IMPACT™ interrogating somatic mutations in 410 cancer-related genes. Mutational profiles were correlated to recurrence and survival outcomes. OPSCC included 47% HPV-positive (HPV-P) and 53% HPV-negative (HPV-N) tumors arising in the base of tongue (BOT, 43%), palatine tonsil (30%) and soft palate (SP, 27%). HPV negative status, SP location and smoking were associated with poorer outcome. Poorer overall survival was found in NOTCH1-mutated HPV-P (p = 0.039), and in SOX2-amplified HPV-N cases (p = 0.036). Chromosomal arm gains in 8p and 8q, and 16q loss were more common in HPV-P (p = 0.005, 0.04 and 0.01, respectively), while 9p, 18q and 21q losses were more frequent in HPV-N OPSCC (p = 0.006, 0.002 and 0.01, respectively). Novel, potentially functional JAK3, MYC and EP300 intragenic deletions were found in HPV-P, and FOXP1, CDKN2A, CCND1 and RUNX1 intragenic deletions and one FGFR3 inversion were detected in HPV-N tumors. HPV-N/TP53-wild-type OPSCC harbored recurrent mutations in NOTCH1/3/4 (39%), PIK3CA, FAT1 and TERT. In comparison to their oral and laryngeal counterparts, HPV-N OPSCC were genetically distinct. In OPSCC, HPV status, tumor subsite and smoking determine outcome. Risk-stratification can be further refined based on the mutational signature, namely, NOTCH1 and SOX2 mutation status.     

Joint effect of HPV16 with Chlamydia trachomatis and smoking on risk of cervical cancer: antagonism or misclassification (Nordic countries)

Objectives:To estimate the joint effects of infections with human papillomavirus type 16 (HPV16) and Chlamydia trachomatis and smoking on the risk of cervical cancer. To study whether the joint effects can be accounted for by misclassification in the HPV serology. Methods:A nested case–control study with incidence density sampling was conducted in three cohorts of 530,000 women, who donated serum samples to three Nordic serum banks in 1973–1994. The main outcome measure is the odds ratio (OR) of incidence rates of invasive cervical squamous cell carcinoma (SCC) among those seropositive for HPV16 and/or C. trachomatis and/or with increased levels of cotinine in serum compared to those negative for all the three exposures. Results:Two hundred eight women with SCC and 624 matched controls were identified during a mean follow-up of 5 years through linkage to the national cancer registries. Exposure to past infections and smoking was defined by presence of specific IgG antibodies to HPV16 and C. trachomatis and increased levels of serum cotinine. Observed ORs were compared to OR = 20 for HPV16 and accounting the differences for by misclassification bias. OR = 20 was elected as a gold standard on the basis of other studies with PCR-based analyses and a follow-up design. Each of the three exposures was associated with an increased risk of SCC (OR = 5.4 for HPV16, 3.4 for C. trachomatis and 1.8 for cotinine). The interaction was antagonistic (observed OR = 2.5 among those positive for all three exposures as compared to OR = 33 expected on the basis of multiplicative single effects (p = 0.047)). The antagonism could not totally be accounted for by any credible combination of sensitivity and specificity of HPV16 serology. Conclusion:HPV16, C. trachomatis, and smoking are likely to be risk factors of SCC with strong antagonistic joint effect. Non-differential misclassification in serology for HPV16 could be ruled out (but only some types of differential) as an alternative explanation for the observed antagonism.     

Human papillomavirus,smoking status and outcomes in tonsillar squamous cell carcinoma

It is now clear that the two separate entitles of tonsillar cancer, HPV induced and non‐HPV induced (smoking induced), have significantly different presenting stage and outcomes. A significant proportion of patients with human papillomavirus positive tonsillar cancer have had exposure to smoking. We examined the combined effect of human papillomavirus and smoking on the outcomes and determined whether smoking can modify the beneficial effect of human papillomavirus. A total of 403 patients from nine centers were followed up for recurrence or death for a median of 38 months. Determinants of the rate of loco‐regional recurrence, death from tonsillar cancer and overall survival were modeled using Cox regression. Smoking status was a significant predictor of overall survival (p = 0.04). There were nonstatistically significant trends favoring never smokers for loco‐regional recurrence and disease specific survival. In addition, there was no statistically significant interactions between smoking and human papillomavirus (p‐values for the interaction were 0.26 for loco‐regional recurrence, 0.97 for disease specific survival and 0.73 for overall survival). The effect of smoking on loco‐regional recurrence and disease specific survival outcomes was not statistically significant, nor was there significant evidence that the effect of smoking status on these outcomes was modified by HPV status. Irrespective of HPV status, however, smokers did have poorer overall survival than never‐smokers, presumably due to effects of smoking that are unrelated to the primary cancer.     

Smoking modifies the relationship between XRCC1 haplotypes and HPV16‐negative head and neck squamous cell carcinoma

Reports on the relationship between head and neck squamous cell carcinoma (HNSCC) and polymorphisms in X‐ray cross complementing group 1 (XRCC1) have been inconsistent. We hypothesized this may be due to not accounting for Human papillomavirus type‐16 (HPV16) and thus examined whether smoking modified the association between XRCC1 haplotypes and HNSCC risk within HPV16 serologic strata. Cases were diagnosed in Greater Boston, Massachusetts. Controls were matched to cases on age, gender and residential town. Genotyping was conducted on three XRCC1 polymorphisms (Arg194Trp, Arg280His and Arg399Gln) and serology was used to determine HPV16 exposure. Unconditional logistic regression was used to estimate odds ratios (ORs) and 95% confidence intervals (CIs), adjusting for age, sex, race, education, smoking, alcohol consumption and HPV16 serology. There was no overall association between XRCC1 polymorphisms and HNSCC risk. Smoking did not modify the association between XRCC1 polymorphisms and HNSCC risk among the HPV16 seropositive (p_interaction = 0.89) but it did for the HPV16 seronegative (p_interaction=0.04). Among the HPV16 seronegative, heavy smokers with a haplotype containing a variant allele had an increased HNSCC risk (haplotype with 399Gln: OR, 1.35; 95% CI, 0.97–1.86), whereas never/light smokers with variant alleles may have a reduced risk. In sum, the association between XRCC1 and HNSCC risk differed by HPV16 status and smoking. Among the HPV16 seronegative, heavy smokers with XRCC1 variant alleles had an increased HNSCC risk. There was no relationship between XRCC1 and HPV16‐related HNSCC, regardless of smoking. Our findings underscore the importance of accounting for HPV16 exposure even when studying susceptibility to HNSCC. © 2009 Wiley‐Liss, Inc.     

Human papillomaviruses are identified in a subgroup of sinonasal squamous cell carcinomas with favorable outcome

BACKGROUND:

The role of human papillomavirus (HPV) in the pathogenesis of squamous cell carcinomas (SCCs) of the sinonasal tract and its clinicopathological implications were evaluated.

METHODS:

All SCCs of the sinonasal tract diagnosed in the Hospital Clinic of Barcelona from 1981 to 2006 were retrospectively evaluated (N = 60). Clinical and pathological data were reviewed. HPV infection was determined and typed by amplification of HPV DNA by polymerase chain reaction using the SPF‐10 primers. p16^INK4a expression was determined by immunohistochemistry. Overall and progression‐free survival for HPV‐positive and ‐negative patients was estimated by Kaplan‐Meier analysis and by the use of a multivariate Cox proportional hazards model.

RESULTS:

HPV DNA was detected in tumor tissue of 12 of 60 (20%) patients. HPV16 was identified in 11 tumors and HPV35 in 1. Immunohistochemistry for p16^INK4a stained all HPV‐positive and no HPV‐negative tumors (P < .001). No differences were observed in terms of site and histological grade or stage at presentation between HPV‐positive and ‐negative tumors. However, HPV‐positive patients had a significantly better 5‐year progression‐free survival (62%; 95% confidence interval [CI], 23%‐86% vs 20%; 95% CI, 9%‐34%; P = .0043, log‐rank test) and overall survival (80%; 95% CI, 20%‐96% vs 31%; 95% CI, 15%‐47%; P = .036, log‐rank test) than patients with HPV‐negative tumors. In multivariate analysis, HPV‐positive tumors were associated with improved progression‐free survival (hazard ratio, 0.21; 95% CI, 0.17‐0.98; P = .012).

CONCLUSIONS:

A subgroup of sinonasal SCCs is associated with HPV infection. These tumors have a significantly better prognosis. Cancer 2009. © 2009 American Cancer Society.     

Human papillomavirus DNA load and p16INK4a expression predict for local control in patients with anal squamous cell carcinoma treated with chemoradiotherapy

As the detection rate of HPV‐DNA in anal carcinoma commonly exceeds 90%, a comparison between sole HPV‐positive and HPV‐negative cancers with respect to treatment response following chemoradiotherapy (CRT) and long‐term oncological outcome is challenging. Against this background, we aimed to assess HPV types and HPV DNA load in formalin‐fixed paraffin‐embedded tissue (FFPE) of 95 patients treated with standard CRT for anal cancer to correlate viral load (≤/> median) with local failure, distant metastases, cancer‐specific (CSS) and overall survival (OS) rates. Various clinicopathologic parameters and the immunohistochemical marker p16^INK4a were evaluated for any correlation with HPV16 DNA load and were included in uni‐ and multivariate analyses. The overall prevalence of HPV DNA was 95.8% with HPV16 monoinfection being the most commonly encountered HPV type (78.9%), followed by HPV16 and 31, 35, 39, 44, 58, 66 and 81 dual infection in 9 patients (9.5%). HPV16 DNA load was significantly associated with p16^INK4a expression (p = 0.001). Patients with HPV16 DNA load ≤ median and low p16^INK4a expression showed significantly worse local control (HPV16 DNA load: univariate p = 0.023, multivariate p = 0.042; p16^INK4a: univariate p = 0.021), and OS (HPV16 DNA load: univariate p = 0.02, multivariate p = 0.03). Moreover, a combined HPV16 DNA load and p16^INK4a variable revealed a significant correlation to decreased local failure, and increased CSS and OS (p = 0.019, p = 0.04 and p = 0.031). In conclusion, these data indicate that HPV16 DNA load and p16^INK4a expression are significant prognostic factors for local tumor control and overall survival of patients with anal SCC following CRT.     

Human papillomavirus 6 seropositivity is associated with risk of head and neck squamous cell carcinoma, independent of tobacco and alcohol use

Background: The risk of head and neck squamous cell carcinoma(HNSCC) associated with common human papillomavirus types hasnot been well defined. Methods: We conducted a case–control study of 1034 individuals(486 incident cases diagnosed with HNSCC and 548 population-basedcontrols matched to cases by age, gender, and town of residence)in Greater Boston, MA. Sera were tested for antibodies to humanpapillomavirus (HPV)6, HPV11, HPV16, and HPV18 L1. Results: HPV6 antibodies were associated with an increased riskof pharyngeal cancer [odds ratio (OR) = 1.6, 1.0–2.5],controlling for smoking, drinking, and HPV16 seropositivity.In HPV16-seronegative subjects, high HPV6 titer was associatedwith an increased risk of pharyngeal cancer (OR = 2.3, 1.1–4.8)and oral cancer (OR = 1.9, 1.0–3.6), suggesting that thecancer risk associated with HPV6 is independent of HPV16. Therewas no association between smoking and alcohol use and HPV6serostatus. Further, the risk of pharyngeal cancer associatedwith heavy smoking was different among HPV6-seronegative (OR3.1, 2.0–4.8) and HPV6-seropositive subjects (OR = 1.6,0.7–3.5), while heavy drinking also appears to conferdiffering risk among HPV6-negative (OR 2.3, 1.5–3.7) and-positive subjects (OR = 1.3, 0.6–2.9). Conclusions: There may be interactions between positive serologyand drinking and smoking, suggesting that the pathogenesis ofhuman papillomavirus in HNSCC involves complex interactionswith tobacco and alcohol exposure. Key words: epidemiology, head and neck squamous cell carcinoma, human papillomavirus, risk factors, serology Received for publication March 6, 2008. Revision received August 20, 2008. Accepted for publication August 26, 2008.     

Tobacco smoking and regression of low‐grade cervical abnormalities

The role of tobacco smoking in the multistage carcinogenesis at the cervix is not fully understood because of a paucity of prospective data. To assess the relationship between smoking and spontaneous regression of cervical precursor lesions, a total of 516 women with low‐grade squamous intraepithelial lesion (LSIL) were monitored by cytology and colposcopy every 4 months. Probability of LSIL regression within 2 years was analyzed in relation to smoking behaviors, with regression defined as at least two consecutive negative Pap smears and normal colposcopy. Women’s age, initial biopsy results, and human papillomavirus (HPV) genotypes were included in the multivariate models for adjustments. Our study subjects included 258 never‐smokers and 258 smokers (179 current and 79 former smokers). During a mean follow‐up time of 39.8 months, 320 lesions regressed to normal cytology. Probability of regression within 2 years was significantly lower in smokers than in never‐smokers (55.0%vs 68.8%, P = 0.004). The risk of LSIL persistence increased with smoking intensity and duration and with younger age at starting smoking (P = 0.003, P < 0.001, and P = 0.03, respectively). Smokers had twice as high a risk of persistent HPV infection compared to never‐smokers (odds ratio, 2.50; 95% confidence interval, 1.30–4.81; P = 0.006). In young women, passive smoking since childhood reduced probability of regression within 2 years (56.7%vs 85.9%, P < 0.001). Further adjustments for a wide range of cervical cancer risk factors did not change the findings. In conclusion, tobacco smoking may interfere with regression of cervical precursor lesions. Childhood exposure to second‐hand smoke may increase a risk of persistent cervical abnormalities among young women. (Cancer Sci 2010)     

Oral human papillomavirus (HPV) infection and HPV vaccination in an Australian cohort

Human papillomavirus (HPV)-related oropharyngeal cancer (OPC) is increasing in incidence, yet very little is known about oral HPV infection in the general population. In this Australian-based study we assess oral HPV prevalence according to HPV vaccination status. Participants of the Oral Diversity Study were Australian residents, aged 18 to 70 years, who filled out a questionnaire about lifestyle and sexual behaviour, and donated a saliva sample in 2020 to 2021. We obtained permission to access HPV vaccination status through record linkage with the Australian Immunisation Register. Saliva samples were DNA extracted, DNA quality checked and analysed for HPV. We recruited 1023 participants to the Oral Diversity Study. Nine hundred twenty-one returned a saliva sample for analysis, 911 passed the DNA quality check and were included in the study. The oral HPV prevalence was 7.2%, and was strongly associated with sexual behaviours. We identified 27 different HPV types; 53% of participants carried high-risk HPV types, with no difference between the vaccinated and the unvaccinated groups (53% both, P = .979). Two hundred thirty participants (26%) were HPV vaccinated. The oral prevalence of the nine HPV types included in the nonavalent HPV vaccine was significantly lower in the vaccinated participants compared to the unvaccinated (0.9% vs 3.4%; P = .022). These findings suggest that a sizeable minority of Australian residents harbour oral HPV infections, and many of these are high-risk subtypes. We found some evidence that HPV vaccination resulted in lower prevalence of oral HPV infections of vaccine-specific types. Larger surveys are required to confirm these findings.     

Epidemiologic associations of HPV‐positive oropharyngeal cancer and (pre)cancerous cervical lesions

Human papillomavirus (HPV)‐induced oropharyngeal squamous cell carcinoma (OPSCC) remains increasing worldwide. We aimed to investigate if the HPV‐prevalence of OPSCC in the Netherlands is rising as well, also in female patients. In addition, we evaluated the association between HPV‐positive OPSCC and suspicious Pap results of the cervix in these female patients. Patients with OPSCC treated in the period 2000–2015 at the VU University Medical Center Amsterdam, were included (n = 926). The presence of an oncogenic HPV infection was determined by p16‐immunostaining, followed by a high‐risk HPV general primer 5+/6+ DNA PCR on the p16‐immunopositive cases. A review of pathology reports in all female patients (n = 305) was undertaken to identify cytological signs of HPV‐related (pre)cancer of the cervix. In total 281 of 926 (30.3%) OPSCCs were HPV‐positive. Moreover, a significant increase in the prevalence of HPV‐positive OPSCCs was observed from 14.0% in 2000 to 48.1% in 2015 (p < 0.001). Among the female patients with an HPV‐positive OPSSC (n = 70), the results of cervical smears were available in 56 of 70 patients (80.0%). Of the female patients with HPV‐positive OPSCC, 9 of 56 (16.1%) patients had a vaginal cuff Papanicolaou (Pap) test ≥3b in their medical history compared to 7 of 168 (4.2%) in the HPV‐negative group (p = 0.003). In conclusion, a continuous increase in the HPV‐attributable fraction of OPSCC was demonstrated in the period 2000–2015 in the Amsterdam region. HPV‐positive OPSCC has a significant association with a history of suspicious Pap results of the cervix in female patients.     

The detection of circulating human papillomavirus‐specific T cells is associated with improved survival of patients with deeply infiltrating tumors

A detailed analyses of HPV‐specific immunity was performed in a large group of patients with HPV‐induced cervical cancer (CxCa) in relation to HLA‐types and prognostic factors. Patients were HLA‐typed and HPV16/18‐specific T‐cell immunity was assessed by proliferation assay and cytometric bead array using freshly isolated PBMC and by phenotypic analysis of HPV‐specific T cells. The results were analyzed in relation to known disease‐related HLA‐types (DR7, DR13, DR15/DQ06), invasion‐depth and size of tumor, lymph node (LN) status and disease free survival. In total 119 HLA‐typed patients with CxCa were analyzed. Patients expressing the HLA‐DR13 haplotype were underrepresented as compared to the Dutch population (p = 0.014), whereas HLA‐DR7 was overrepresented in patients with HPV16+ CxCa (p = 0.006). In 29 of 94 patients (31%) from whom blood could be tested, a proliferative response to HPV16/18 was detected, which was associated with increased numbers of HPV‐specific CD4+CD25+ (activated) T cells (p = 0.03) and HPV‐specific CD4+CD25+FoxP3‐positive T cells (p = 0.04). The presence of both FoxP3‐positive and negative HPV‐specific CD4+CD25+ T cells was significantly correlated (p = 0.01). Interestingly, the detection of HPV‐specific proliferation was associated with invasion depth (p = 0.020) but not with HLA type, tumor size nor LN status. Moreover, the detection of HPV‐specific immunity was associated with an improved disease free survival (p = 0.04) in patients with deeply infiltrating tumors. In conclusion, HPV‐specific proliferative T‐cell response, comprising higher percentages of HPV‐specific CD25+ and CD25+FoxP3‐positive CD4+T cells, are more frequently detected in patients with deep infiltrating CxCa tumors and associated with an improved survival.