Human Papillomavirus P16 Research Articles

Molecular subclassification determined by human papillomavirus and epidermal growth factor receptor status is associated with the prognosis of oropharyngeal squamous cell carcinoma

Human papillomavirus (HPV) infection is an indicator of good response to chemoradiotherapy in oropharyngeal squamous cell carcinoma (OPSCC), and epidermal growth factor receptor (EGFR) is a molecular-therapeutic target in head and neck squamous cell carcinoma. Here we investigated the prevalence and prognostic significance of HPV infection and EGFR alteration in OPSCC. We analyzed the presence of high-risk HPV using in situ hybridization, protein expressions of p16 and EGFR using immunohistochemistry, and the EGFR gene copy number gain using chromogenic in situ hybridization (CISH) in 105 cases of OPSCC. The biopsy specimens before chemoradiotherapy were used for these analyses. HPV infection and p16 protein overexpression were detected in 53.3% and 52.4% of the OPSCCs, and each factor was associated with better overall survival (P = .0026 and P = .0026) and nonkeratinizing histology (P = .0002 and P = .0004), respectively. EGFR gene copy number gain (high polysomy or amplification) was detected in 12.4% of the OPSCCs and was correlated with EGFR protein overexpression (P = .0667) and worse overall survival (P < .0001). HPV infection and EGFR gene copy number gain (EGFR CISH positive) were mutually exclusive. The HPV-negative/EGFR CISH-positive OPSCCs had significantly worse overall survival than did the HPV-positive/EGFR CISH-negative OPSCCs and HPV-negative/EGFR CISH-negative OPSCCs (P < .0001 and P < .0001, respectively). The EGFR CISH-negative OPSCCs had favorable prognosis irrespective of HPV infection. Our results suggest that EGFR gene copy number gain-positive tumors represent an HPV-negative, aggressive subgroup of OPSCCs. The molecular subclassification of OPSCCs based on HPV infection and EGFR status may serve as important information for appropriate therapeutic strategy.

Prognostic Relevance of HPV Infection and p16 Overexpression in Squamous Cell Anal Cancer

Human papillomavirus (HPV) DNA and p16 status have both been reported as prognostic factors in anal cancer, but the prognostic relevance of combined detection and particularly HPV-/p16+ and HPV+/p16- signatures is unknown. We evaluated combined HPV DNA and p16 status as a prognostic factor of treatment response in anal cancer. 106 patients treated with radiochemotherapy (RCT+5-FU/MMC) with available paraffin-embedded tumor tissue specimens were evaluated regarding local control (LC) and overall survival (OS) at 5 years. In addition to HPV DNA/p16 status, the influence of age, gender, previous surgery, initial recurrence, T stage, N status, and tumor localization was analyzed. 63 patients were HPV+/p16+, 9 were HPV+/p16-, 11 were HPV-/p16+, and 23 were HPV-/p16-. In univariate analysis, LC was significantly better in patients with T1/2 stage, female gender, and HPV/p16 status. HPV+/p16+ was associated with significantly better LC (88.1%; 95% confidence interval [CI]: 78.89-97.31) compared with HPV-/p16+ (63.6%; 95% CI: 35.18-92.02; P=.021) and especially HPV-/p16- (55.8%; 95% CI: 33.46-78.14; P=.002) but not with HPV+/p16- (77.8%; 95% CI: 50.56-105.04; P=.270). OS was influenced by T stage and LC. HPV+/p16+ patients showed a trend toward better OS compared with HPV-/p16- patients (HPV+/p16+: 81.1%; 95% CI: 70.12-92.08 vs HPV-/p16-: 68.8%; 95%CI: 47.44-90.16; P=.138). On multivariate analysis, T3/4 stage and HPV/p16 status (HPV-/p16+, HPV-/p16- vs HPV+/p16+) predicted poorer LC (T3/4: 50.3% vs T1/2: 86.6%, hazard ratio [HR] 0.22; 95% CI: 0.09-0.53; P<.001; HPV+/p16+ vs HPV-/p16+: HR 4.73; 95% CI: 1.33-16.82; P=.016, and HPV+/p16+ vs HPV-/p16-: HR 6.40; 95% CI: 2.23-18.35; P<.001), whereas local relapse dramatically influenced OS. Our data suggest that HPV/p16 signature determines prognosis. HPV+/p16+ patients had the best prognosis, and HPV-/p16+ and HPV-/p16- patients showed the worst outcome and therefore require therapy optimization, particularly given that LC is the most important factor for OS.

Utility of Human Papillomavirus Capsid Protein L1 and p16 in the Assessment and Accurate Classification of Anal Squamous Intraepithelial Lesions.

Morphologic evaluation of anal dysplasia remains problematic, especially in cases with limited biopsy samples or obscuring inflammation. Studies in cervical neoplasia have shown that human papillomavirus (HPV) L1 capsid production is highest in low-grade squamous intraepithelial lesions (LSILs) and progressively decreases in high-grade squamous intraepithelial lesions (HSILs). The combined utility of HPV L1 and p16 expression in assessing anal squamous intraepithelial lesions (SILs) has never been analyzed and forms the basis of this study. In total, 145 anal lesions were reviewed and immunohistochemically stained for HPV L1 and p16. p16 expression was recorded as negative, patchy/focal, or diffuse. For analytical purposes, condylomas were evaluated separately from rest of the LSILs. There were 34 (23%) condylomas, 64 (44%) LSILs, and 47 (32%) HSILs. HPV L1 was significantly associated with condylomas (68%) and LSILs (52%) compared with HSILs (9%; P < .0001). Diffuse p16 staining was present only in HSILs (P < .0001), whereas a patchy/focal p16 staining pattern was observed in both LSILs and condylomas. HPV L1 and diffuse p16 expression is mutually exclusive in most anal SILs and helps separate LSIL and HSIL cases. Application of both HPV L1 and p16 can not only facilitate accurate grading but also contribute to risk assessment in anal neoplasia.

Human papillomavirus DNA and p16INK4a expression in hypopharyngeal cancer and in relation to clinical outcome, in Stockholm, Sweden

Hypopharyngeal cancer is a subset of head neck squamous cell carcinoma (HNSCC) with particularly poor prognosis. Human papillomavirus (HPV) is a risk factor for some HNSCC, and its presence is of prognostic value for certain subsites. However, its influence on survival in hypopharyngeal cancer has not been thoroughly investigated. Here we examine HPV DNA and p16(INK4a) (p16) overexpression in relation to clinical outcome. Hypopharyngeal tumour biopsies from 82 patients diagnosed 2008-2013 were examined for presence of HPV DNA by a bead-based multiplex assay and for p16 expression by immunohistochemistry, and the obtained data compared to that acquired previously from 109 patients diagnosed 2000-2007 at the same clinic. A survival analysis was then performed on 142 patients (from both studies) treated with curative intent and a 3-year follow-up time. Of the tumour biopsies 3/82 (3.7%) were HPV16 DNA and p16 positive, while 12/82 (14.6%) were p16 positive, equivalent to that in the previous study. Overall 3-year survival was significantly more favourable for patients with HPV16 DNA and p16 positive tumours as compared to survival of the other patients (86% vs. 31%, p=0.0185). A similar but not statistically significant trend was found for disease specific survival. HPV DNA and p16 positive hypopharyngeal cancer was rare and had not increased, but had a better clinical outcome as compared to other HPV-unrelated hypopharyngeal cancer. In addition, p16 overexpression was not a suitable surrogate marker for presence of HPV or for prediction of survival in this type of cancer.

Genomic alterations in head and neck squamous cell carcinoma determined by cancer gene-targeted sequencing

To determine genomic alterations in head and neck squamous cell carcinoma (HNSCC) using formalin-fixed, paraffin-embedded (FFPE) tumors obtained through routine clinical practice, selected cancer-related genes were evaluated and compared with alterations seen in frozen tumors obtained through research studies. DNA samples obtained from 252 FFPE HNSCC were analyzed using next-generation sequencing-based (NGS) clinical assay to determine sequence and copy number variations in 236 cancer-related genes plus 47 introns from 19 genes frequently rearranged in cancer. Human papillomavirus (HPV) status was determined by presence of the HPV DNA sequence in all samples and corroborated with high-risk HPV in situ hybridization (ISH) and p16 immunohistochemical (IHC) staining in a subset of tumors. Sequencing data from 399 frozen tumors in The Cancer Genome Atlas and University of Chicago public datasets were analyzed for comparison. Among 252 FFPE HNSCC, 84 (33%) were HPV positive and 168 (67%) were HPV negative by sequencing. A subset of 40 tumors with HPV ISH and p16 IHC results showed complete concordance with NGS-derived HPV status. The most common genes with genomic alterations were PIK3CA and PTEN in HPV-positive tumors and TP53 and CDKN2A/B in HPV-negative tumors. In the pathway analysis, the PI3K pathway in HPV-positive tumors and DNA repair-p53 and cell cycle pathways in HPV-negative tumors were frequently altered. The HPV-positive oropharynx and HPV-positive nasal cavity/paranasal sinus carcinoma shared similar mutational profiles. The genomic profile of FFPE HNSCC tumors obtained through routine clinical practice is comparable with frozen tumors studied in research setting, demonstrating the feasibility of comprehensive genomic profiling in a clinical setting. However, the clinical significance of these genomic alterations requires further investigation through application of these genomic profiles as integral biomarkers in clinical trials.

HPV-negative squamous cell carcinoma of the anal canal is unresponsive to standard treatment and frequently carries disruptive mutations in TP53

Background:Human papillomavirus (HPV), p16 expression, and TP53 mutations are known prognostic factors in head and neck squamous cell carcinoma, but their role in squamous cell carcinoma of the anal canal (SCCAC) is less well established. The objective of this study was to determine the prognostic significance of tumour HPV status, p16 and p53 expression, and mutations in TP53 in patients with SCCAC receiving (chemo)radiotherapy.Methods:Human papillomavirus DNA was determined using an INNO-LiPA-based assay in tumour tissue of 107 patients with locally advanced SCCAC. Patients were treated with radiotherapy, with or without concurrent chemotherapy consisting of a fluoropyrimidine and mitomycin C. Expression of p16 and p53 was determined using immunohistochemistry. Exons 2–11 of TP53 in tumour tissue were sequenced.Results:DNA of high-risk HPV types was detected in 93 out of 107 tumours (87%), all of which overexpressed p16 (HPV+/p16+). Of 14 HPV-negative (HPV−) tumours (13%), 10 (9%) were p16-negative (HPV−/p16−) and 4 (4%) overexpressed p16 (HPV−/p16+). Patients with HPV−/p16− disease had inferior 3-year locoregional control (LRC) (15%) compared with patients with HPV+/p16+ tumours (82%, P<0.001) and HPV−/p16+ tumours (75%, P=0.078). Similarly, 3-year overall survival (OS) was 35% (HPV−/p16−) vs 87% (HPV+/p16+, P<0.001) and 75% (HPV−/p16+, P=0.219). Disruptive mutations in TP53 were found in 80% of HPV−/p16− tumours vs 6% of HPV+/p16+ tumours (P<0.001). In multivariate analysis, HPV−/p16− status was an independent predictor of inferior LRC and OS.Conclusions:HPV− tumours are frequently TP53 mutated. HPV−/p16− status is a strong predictor for reduced LRC and OS, and alternative treatment strategies for patients with HPV−/p16− disease need to be explored.

High-grade acute organ toxicity and p16(INK4A) expression as positive prognostic factors in primary radio(chemo)therapy for patients with head and neck squamous cell carcinoma.

Superior treatment response and survival for patients with human papilloma virus (HPV)-positive head and neck cancer (HNSCC) are documented in clinical studies. However, the relevance of high-grade acute organ toxicity (HGAOT), which has also been correlated with improved prognosis, has attracted scant attention in HPV-positive HNSCC patients. Hence we tested the hypothesis that both parameters, HPV and HGAOT, are positive prognostic factors in patients with HNSCC treated with definite radiotherapy (RT) or radiochemotherapy (RCT). Pretreatment tumor tissue and clinical records were available from 233 patients receiving definite RT (62 patients) or RCT (171 patients). HPV infection was analysed by means of HPV DNA detection or p16(INK4A) expression; HGAOT was defined as the occurrence of acute organ toxicity >grade 2 according to the Common Toxicity Criteria. Both variables were correlated with overall survival (OS) using Cox proportional hazards regression. Positivity for HPV DNA (44 samples, 18.9 %) and p16(INK4A) expression (102 samples, 43.8 %) were significantly correlated (p < 0.01), and HGAOT occurred in 77 (33 %) patients. Overall, the 5-year OS was 23 %; stratified for p16(INK4A) expression and HGAOT, OS rates were 47 %, 42 %, 20 % and 10 % for patients with p16(INK4A) expression and HGAOT, patients with HGAOT only, patients with p16(INK4A) expression only, and patients without p16(INK4A) expression or HGAOT, respectively. After multivariate testing p16(INK4A) expression (p = 0.003) and HGAOT (p < 0.001) were significantly associated with OS. P16(INK4A) expression and HGAOT are independent prognostic factors for OS of patients with HNSCC, whereas p16(INK4A) expression is particularly important for patients without HGAOT.

Intensified intensity-modulated radiotherapy in anal cancer with prevalent HPV p16 positivity.

To investigate the toxicity and response of intensity-modulated radiotherapy schedule intensified with a simultaneous integrated boost in anal canal cancer. From March 2009 to March 2014, we retrospectively analyzed 41 consecutive patients treated with intensity-modulated radiotherapy (IMRT) and concurrent chemotherapy for anal canal squamous cell carcinoma at our center. Radiotherapy was delivered via simultaneous integrated boost (SIB) technique by helical tomotherapy, and doses were adapted to two clinical target volumes according to the tumor-node-metastasis (TNM) stage: 50.6 Gy and 41.4 Gy in 23 fractions in T1N0, 52.8 Gy and 43.2 Gy in 24 fractions in T2N0, and 55 Gy and 45 Gy in 25 fractions in all patients with N positive and/or ≥ T3, respectively, to planning target volumes 1 and 2. The most common chemotherapy regimen was 5-fluorouracil and mitomycin-based. Human papilloma virus (HPV) p16 expression was performed by immunohistochemistry and evaluated in the majority of patients. Acute and late toxicity was scored according to CTCAe v 3.0 and RTOG scales. The median follow-up was 30 mo (range: 12-71). Median age was 63 years (range 32-84). The stage of disease was: stage I in 2 patients, stage II in 13 patients, stage IIIA in 12 patients, and stage IIIB in 14 patients, respectively. Two patients were known to be HIV positive (4.9%). HPV p16 expression status was positive in 29/34 (85.3%) patients. The 4-year progression-free survival and overall survival in HPV-positive patients were 78% and 92%, respectively. Acute grade 3 skin and gastrointestinal toxicities were reported in 5% and 7.3% of patients, respectively; patients' compliance to the treatment was good due to a low occurrence of severe acute toxicity, although treatment interruptions due to toxicity were required in 7.3% of patients. At 6 mo from end of treatment, 36/40 (90%) patients obtained complete response; during follow-up, 5 (13.8%) patients presented with disease progression (local or systemic). In our experience, intensified SIB-IMRT with chemotherapy is very feasible in clinical practice, with excellent results in terms of overall survival and local control.

Human papillomavirus in head and neck squamous cell carcinoma of unknown primary is a common event and a strong predictor of survival.

BackgroundThe purpose of this study was to examine the prevalence of human papillomavirus (HPV) in patients with head and neck squamous cell carcinoma of unknown primary (CUP).MethodsAll patients diagnosed with and treated for CUP between January 1, 2000, and June 1, 2011, at two Danish medical centers were included. All patients received a thorough diagnostic work-up, including FDG-PET, before being diagnosed as CUP. We determined the HPV status in all patients using a combination of HPV DNA PCR and p16 stain. In addition, clinical information on the study patients was retrieved from clinical records.ResultsOf the identified 60 patients with CUP, 13 were shown to be positive for HPV DNA, amounting to 22% of the study population. In addition, we were able to show a clear disease-free and overall-survival benefit in the HPV-positive group, with a hazard ratio of 0.16 (95% CI: 0.038–0.67) for over-all survival. This survival benefit was also apparent when adjusted for advanced age in a multivariate Cox regression analysis.ConclusionA fairly large percentage of CUP cases are HPV-related, and because this is related to both the location and prognosis, we recommend HPV testing as part of the diagnostic work-up.

Risk stratification of early stage oral tongue cancers based on HPV status and p16 immunoexpression.

Recent epidemiological data have implicated human papilloma virus (HPV) infection in the pathogenesis of head and neck cancers, especially oropharyngeal cancers. Although, HPV has been detected in varied amounts in persons with oral dysplasia, leukoplakias and malignancies, its involvement in oral tongue carcinogenesis remains ambiguous. HPV DNA prevalence was assessed by PCR with formalin fixed paraffin embedded sections (n=167) of oral tongue squamous cell carcinoma patients and the physical status of the HPV16 DNA was assessed by qPCR. Immunohistochemistry was conducted for p16 evaluation. We found the HPV prevalence in tongue cancers to be 51.2%, HPV 16 being present in 85.2% of the positive cases. A notable finding was a very poor concordance between HPV 16 DNA and p16 IHC findings (kappa<0.2). Further molecular classification of patients based on HPV16 DNA prevalence and p16 overexpression showed that patients with tumours showing p16 overexpression had increased hazard of death (HR=2.395; p=0.005) and disease recurrence (HR=2.581; p=0.002) irrespective of their HPV 16 DNA status. Our study has brought out several key facets which can potentially redefine our understanding of tongue cancer tumorigenesis. It has emphatically shown p16 overexpression to be a single important prognostic variable in defining a high risk group and depicting a poorer prognosis, thus highlighting the need for its routine assessment in tongue cancers. Another significant finding was a very poor concordance between p16 expression and HPV infection suggesting that p16 expression should possibly not be used as a surrogate marker for HPV infection in tongue cancers. Interestingly, the prognostic significance of p16 overexpression is different from that reported in oropharyngeal cancers. The mechanism of HPV independent p16 over expression in oral tongue cancers is possibly a distinct entity and needs to be further studied.

A high and increasing HPV prevalence in tonsillar cancers in Eastern Denmark, 2000-2010: the largest registry-based study to date.

The aim was to explore whether the incidence of tonsillar squamous cell carcinomas (TSCCs) increased in Eastern Denmark, 2000-2010, and whether human papillomavirus (HPV) could explain the increase, and to assess the association of HPV prevalence with gender, age, and origin (i.e., the certainty of tonsillar tumor origin). We applied HPV DNA PCR and p16 immunohistochemistry to all TSCCs registered in the Danish Head and Neck Cancer Group (DAHANCA) and in the Danish Pathology Data Bank (n = 632). Pathologists reviewed and subdivided the tumors into two groups: specified and nonspecified TSCCs. Approximately 10% of HPV-positive tumors was genotyped by amplicon next-generation sequencing. The overall crude incidence of TSCCs increased significantly (2.7% per year) and was explained by an increasing incidence of HPV-positive TSCCs (4.9% per year). The overall HPV prevalence was 58%, with HPV16 being the predominant HPV type. In multivariate analysis, the HPV prevalence was associated with age (<55 vs. >60 years) (OR, 1.72; 95% CI 1.13-2.63) and origin (nonspecified vs. specified TSCCs) (OR, 0.15; 95% CI 0.11-0.22). The association of HPV prevalence with origin increased over time in specified TSCCs (OR per year, 1.10; 95% CI 1.01-1.19), whereas no change over time was observed among nonspecified TSCCs (OR per year, 0.99; 95% CI 0.90-1.08). In conclusion, the observed increase in the number of HPV-positive TSCCs can explain the increasing number of TSCCs in Eastern Denmark, 2000-2010. HPV prevalence was associated with younger age (<55 years) and a high certainty of tonsillar tumor origin.

The prognostic value of HPV status and p16 expression in patients with carcinoma of the anal canal.

BackgroundIn anal cancer studies, the detection frequency of high-risk HPV (human papillomavirus) is variable, depending on the method used. There are limited data reporting results of different HPV detection techniques in the same clinical series, and very few correlating results with clinical outcome.ObjectivesTo evaluate tumor expression of p16/HPV16 using three different methods, and to determine their association with clinical outcome in patients with anal canal squamous cell carcinomas (SCC).DesignThis retrospective study included patients with anal canal SCC treated with definitive radiotherapy or chemoradiotherapy at a single institution between 1992 and 2005. Formalin-fixed paraffin–embedded tumor samples from 53 of the 89 (60%) patient pre-treatment biopsies were adequate for tissue microarray construction. HPV status was determined using: p16 expression by conventional immunohistochemistry (IHC) and quantitative IHC (AQUA), HPV genotype analysis by chromogenic in situ hybridization (CISH) and HPV linear array sub-typing. Expression status was correlated with clinical outcome.Results80% (28/35) of patient tumors had high p16 expression using conventional IHC. HPV16 CISH was positive in 81% (34/42) of tumors, and 78% (28/36) of tumors were HPV subtype 16. HPV16 CISH correlated with p16 evaluated by conventional IHC (correlation coefficient 0.46; p = 0.01) and by p16 AQUA score (correlation coefficient 0.49; p = 0.001). A subset of cases (15%) had very high p16 quantitative IHC scores (>244) and were associated with a higher incidence of local or distant recurrence (p = 0.04).ConclusionsThe vast majority (80%) of anal canal SCC in our series were positive for HPV16/p16, regardless of the testing method used. The exploratory analysis of automated quantitative IHC scoring was the only technique to define a subset of patients with a worse prognosis by p16 expression status on univariate analysis. Further exploration of the molecular mechanisms of treatment resistance in association with very high p16 expression is warranted.

Detection of human papillomavirus (HPV) in patients with squamous cell carcinoma and the clinical characteristics of HPV-positive cases.

The association between human papillomavirus (HPV) and squamous cell carcinoma (SCC) has been reported; however, the prevalence of HPV infection varies, and the clinical characteristics of HPV-positive cases remain unknown. To elucidate the frequency of HPV infection in a series of Japanese patients with SCC and to identify the characteristics of HPV-positive cases. We evaluated 38 patients with SCC treated at our department. HPV typing was performed using SCC samples from different body sites. Immunohistochemical staining for HPV proteins and p16(INK) (4a) was performed, in addition to polymerase chain reaction and in situ hybridization. The clinical characteristics of the HPV-positive cases were clarified. Two genital lesions were positive for HPV type 16. Both cases showed basaloid features histopathologically, and were considered to have SCC that had arisen from bowenoid papulosis. p16(INK) (4a) expression was observed in 11 cases, including the two HPV-positive cases. The present study indicates that the prevalence of HPV is not high and that bowenoid papulosis is an HPV-associated precancerous lesion. Further investigation is necessary to assess the relationship between HPV infection and SCC.

Evaluation of Lymph Node Metastasis in Oropharyngeal Squamous Cell Carcinoma

Objectives: (1) Examine the association between histopathologic variables and neck metastasis. (2) Analyze the effect of human papillomavirus (HPV) status on lymph node (LN) metastasis. Methods: Medical records of 93 patients who underwent transoral robotic surgery (TORS) with concurrent neck dissection for oropharyngeal squamous cell carcinoma (SCC) between 2008 and 2013 were reviewed. Results: At the time of presentation, 60 (64.5%) patients had tonsil cancer, 14 (15%) had base of tongue, and 19 (21%) had carcinoma of unknown primary. High risk types of HPV and p16 positivity were 74.4% and 85.4%, respectively. Mean primary tumor size was 2.3 cm (range, 0.3-5.1 cm). Nodal status based on pathologic examination were N0 in 7 (7.5%) patients, N1 in 12 (12.9%), N2a in 25 (26.9%), N2b in 37 (39.8), N2c in 5 (5.4%), and N3 in 7 (7.5%). Average positive LN number was 2.56 (range, 0-37). Extracapsular spread (ECS) was identified in 29.1% of all patients. Primary tumor size had no effect on positive LN size, number or ECS. HPV positivity (rs = 0.25, P = .021), p16 overexpression (rs = 0.36, P = .001), and lymphovascular invasion (rs = 0.34, P = .001) were significantly associated with increased positive LN number. Positive LN number correlates with ECS (rs = 0.28, P =.009). Conclusions: HPV positivity, p16 overexpression, and lymphovascular invasion increase the risk of cervical lymph node metastasis. Increased primary tumor size does not necessarily mean advanced neck disease.

Human Papillomavirus (HPV) Infection in Squamous Cell Carcinomas Arising From the Oropharynx: Detection of HPV DNA and p16 Immunohistochemistry as Diagnostic and Prognostic Indicators—A Pilot Study

Human papillomavirus (HPV) 16 infection is associated with oropharyngeal carcinogenesis and is likely the cause of the reported increase in disease incidence. We evaluated the prevalence of HPV infection and the reliability of different diagnostic tools using primary tumor samples from a cohort of 50 patients. Formalin-fixed paraffin-embedded (FFPE) tumor samples were collected from all 50 consecutive primary oropharyngeal SCC patients who were enrolled in the study; fresh tumor samples were available in 22 cases. NucliSENS EasyQ HPVv1 was used for RNA, and Digene Hybrid Capture-2(HC2) was used for DNA detection. p16 Expression was evaluated by immunohistochemistry in FPPE specimens. Based on the DNA detection assay on FFPE samples, the frequency of high-risk HPV infection was 32%. The agreement rate between HPV RNA and HPV DNA detection in fresh samples was 100%. The agreement rate between p16 immunohistochemistry (IHC) and the detection of HPV DNA in the FFPE samples was fair but not excellent (κ = 0.618). HPV DNA detection was highly significant, as measured by disease-specific survival and determined using a Wilcoxon test (P=.001). p16 IHC also exhibited a prognostic value but with a lower statistical significance (P=.0475). The detection of HPV DNA, but not p16 IHC, was also significantly correlated with locoregional control (P=.0461). Diagnostic methods based on the detection of HPV nucleic acids appear to be more reliable and objective because they do not require reading by a trained histopathologist. Furthermore, the detection of HPV DNA exhibits an improved correlation with survival, and therefore appears definitely more reliable than p16 IHC for routine use in clinical practice.

A comprehensive evaluation of human papillomavirus positive status and p16INK4a overexpression as a prognostic biomarker in head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) patients with human papillomavirus (HPV) infection have better prognosis than those without HPV infection. Although p16INK4a expression is used as a surrogate marker for HPV infection, there is controversy as to whether p16INK4a reliably indicates HPV infection. Here, to evaluate the accuracy of p16INK4a expression for determining HPV infection and the prognostic value of HPV infection and p16INK4a expression for HNSCC survival, especially oropharyngeal squamous cell carcinoma (OPSCC) survival, 150 fresh-frozen HNSCC samples were analyzed for HPV DNA, E6/E7 mRNA and p16INK4a expression by polymerase chain reaction and immunohistochemistry. p16INK4a expression was scored from 0 to 4 according to the percentage of p16INK4a-positive cells, with overexpression defined as >40% positive cells. Of the 150 tumor samples tested, 10 tumors were nasopharyngeal, 53 oropharyngeal, 39 hypopharyngeal, 24 laryngeal and 24 were located in the oral cavity. HPV DNA was detected in 47 (31.3%) samples, but only 21 also exhibited HPV mRNA expression. Inter-rater agreement was low between p16INK4a expression and HPV DNA presence and between p16INK4a expression and HPV mRNA expression, but was good between the combination of HPV DNA status and p16INK4a overexpression and HPV mRNA expression. Three-year recurrence-free survival was significantly higher for OPSCC patients who were HPV DNA-positive than for OPSCC patients who were HPV DNA-negative (P=0.008) and for OPSCC patients over-expressing p16INK4a than for without overexpressing p16INK4a (P=0.034). Multivariate analysis revealed that T1-3 stage and the combination of HPV DNA positivity and p16INK4a overexpression predicted significantly better recurrence-free survival. This combination is a more accurate marker for active HPV infection in HNSCC than HPV DNA status or general p16INK4a-positive status alone and offers a useful and reliable method for detecting and determining the prognosis of HPV-related HNSCC.

Human papillomavirus genotyping and p16 expression as prognostic factors for patients with American Joint Committee on Cancer stages I to III carcinoma of the anal canal.

Carcinomas of the anal canal are strongly associated with the human papillomavirus (HPV). Expression of p16 is used as a surrogate marker of HPV infection. In a retrospective study, we evaluated HPV genotyping and p16 expression as prognostic markers of overall survival (OS) and disease-specific survival (DSS) in patients diagnosed with American Joint Committee on Cancer (AJCC) stages I to III carcinoma of the anal canal. HPV genotyping polymerase chain reaction (high-risk subtypes 16, 18, 31, 33, 45, 52, and 58) and immunohistochemical expression of p16 were analyzed by using paraffin-embedded tumor biopsies from 143 anal carcinomas. The patients were treated with combined chemoradiotherapy or radiotherapy alone. HPV16 was detected in 81.0% of the tumors, followed by HPV33 (5.1%), HPV18 (2.2%), and HPV58 (0.7%). p16 positivity was found in 92.9% of the tumors. In univariable survival analysis, HPV positivity was significantly correlated with improved OS (74% v 52%; P=.036) and DSS (84% v 52%; P=.002), and p16 positivity was significantly correlated with improved OS (76% v 30%; P<.001) and DSS (85% v 30%; P<.001). In multivariable COX analysis that included HPV status, p16 status, sex, T stage, N stage, and treatment, p16 positivity remained an independent prognostic factor for OS (hazard ratio [HR], 0.07; 95% CI, 0.01 to 0.61; P=.016) and DSS (HR, 0.07; 95% CI, 0.01 to 0.53; P=.011). p16 positivity is an independent prognostic factor for OS and DSS in patients with AJCC stages I to III carcinoma of the anal canal.

Relationship between HPV and the biomarkers annexin A1 and p53 in oropharyngeal cancer.

BackgroundHuman papillomavirus (HPV) is often present in oropharyngeal cancers. Head and neck tumors have been examined for other molecular markers including p53 and annexin A1 (ANXA1). Here, we investigated the prevalence of HPV and its relationship with p53 and ANXA1 in patients with oropharyngeal cancer.MethodsWe have analyzed tumor and adjacent mucosa from 22 patients with squamous cell carcinoma of the oropharynx in addition to samples of the oropharyngeal epithelium in subjects without cancer. We evaluated the presence of the HPV (subtypes 16/18 and 31/33) by chromogenic in situ hybridization. Additionally, we used immunofluorescence to examine the expression of p16, p53, ANXA1 and the phosphorylation of the ANXA1 residues Ser27 (ANXA1-SER) and Tyr21 (ANXA1-TYR).ResultsWe have detected the presence of HPV genome in 59% of the 22 tumors. Of those, 92% were also positive for p16 immunostaining. Furthermore, we demonstrated a reduction in the expression of p53 in HPV + compared to HPV- tumors. Also, a reduction was observed in the expression of ANXA1 in tumors compared to epithelium from the margins and from controls. We also noted a reduction in ANXA1-TYR in tumors. However, the expression of both ANXA1 and ANXA1-SER were elevated in the margins of the HPV + versus HPV- tumors.ConclusionsOur results confirm a high prevalence of HPV in oropharyngeal cancer and a reduction in p53 expression in HPV + tumors. We observed a hypoexpression of ANXA1 and ANXA1-TYR in oropharyngeal cancer. The increase in ANXA1-SER in the margins of HPV + tumors suggests that the epithelium in these cases had been activated by an infectious agent. Those findings indicate that ANXA1 and its phosphorylated forms can play important roles in the response to HPV infection and the carcinogenesis of the oropharynx.

Relationship between HPV infection/p16 expression and radiotherapy prognosis in oropharyngeal squamous cell carcinoma

Objective To investigate the relationship between human papillomavirus (HPV) infection/p16 expression and radiotherapy prognosis in oropharyngeal squamous cell carcinoma (OSCC) and the prognostic value of p16 in OSCC patients treated with radiotherapy.Methods Tissue samples were collected from 42 patients newly diagnosed with OSCC in our hospital from January 1999 to December 2008.PCR was performed to detect HPV DNA,and p16 expression was measured by immunohistochemistry.The chi-square test was used to compare the local/regional control rate (CR) between HPV (+)/p16 (+) patients and HPV (-)/p16 (-) patients after radical radiotherapy and evaluate the association between HPV infection and p16 expression; the Kaplan-Meier method was used to calculate overall survival (OS),and the log-rank test was used for survival difference analysis.Results The follow-up rate was 100％.The HPV infection rate was 19％,and the positive rate of p16 was 43％.In patients who received radical radiotherapy,the local CR for HPV (+) patients was 100％,versus 54％ for HPV (-) patients (P =0.026) ;the local CR for p16(+) patients was 92％,versus 44％ for p16(-) patients (P =0.006) ;the locoregional CR for p16(+) patients was 69％,versus 22％ for p16(-) patients (P =0.009).For high-risk patients,HPV infection was significantly associated with p16 expression (P =0.000).The 3-year OS rates for p16(+) and p16 (-) patients were 9 1％ and 2 6 ％,respectively (P =0.001).Conclusions The p16 expression is closely associated with HPV infection in OSCC patients,and it is expected to become one of the prognostic markers in OSCC patients treated with radiotherapy. Key words: Oropharyngeal neoplasms/radiotherapy; Human papillomavirus; p16 ; Prognosis

Lobular Endocervical Glandular Hyperplasia Is a Neoplastic Entity With Frequent Activating GNAS Mutations

To clarify the significance of GNAS mutations in cervical tumorigenesis, we performed mutational analyses in a total of 154 lesions and in 22 normal tissues of the uterine cervix. Activating GNAS mutations were found in 8 of the 19 lobular endocervical glandular hyperplasias (LEGH; 42%) and 4 of the 79 endocervical-type mucinous adenocarcinomas (5%) but were never seen in the normal endocervical tissue, minimal deviation adenocarcinomas, endometrioid adenocarcinomas, or squamous cell carcinomas. We further examined the presence of human papillomavirus (HPV) DNA and p16 expression to probe the relationship between GNAS mutations and HPV infection in LEGHs and carcinomas. All the GNAS-mutated LEGHs were negative for HPV DNA and p16 expression, whereas all the GNAS-mutated adenocarcinomas were positive for HPV DNA and/or p16 expression, implicating GNAS mutations in the development of LEGH and a minor subset of HPV-related cervical adenocarcinomas. Additional mutational analyses of LEGH identified KRAS and STK11 mutations in 1 and 2 cases, respectively. The GNAS, KRAS, and STK11 mutations were mutually exclusive; thus, a total of 11 LEGHs (58%) had 1 of these genetic alterations. Although LEGH has been regarded as a metaplastic lesion, the frequent presence of genetic alterations suggests a neoplastic nature.