Background. HPV is a positive prognostic factor in HNSCC. We studied the prevalence and prognostic impact of HPV on survival parameters and treatment toxicity in patients with locally advanced HNSCC treated with concomitant chemoradiation therapy. Methods. Data on efficacy and toxicity were available for 560 patients. HPV was detected by PCR. Analysis was performed using Kaplan-Meier survival curves, Fisher's test for categorical data, and log-rank statistics for failure times. Results. Median follow-up was 4.7 years. DNA extraction was successful in 255 cases. HPV prevalence was 68.6%, and 53.3% for HPV 16. For HPV+ and HPV−, median LRC was 8.9 and 2.2 years (P = 0.0002), median DFS was 8.9 and 2.1 years (P = 0.0014), and median OS was 8.9 and 3.1 years (P = 0.0002). Survival was different based on HPV genotype, stage, treatment period, and chemotherapy regimen. COX adjusted analysis for T, N, age, and treatment remained significant (P = 0.004). Conclusions. Oropharyngeal cancer is increasingly linked to HPV. This study confirms that HPV status is associated with improved prognosis among H&N cancer patients receiving CRT and should be a stratification factor for clinical trials including H&N cases. Toxicity of CRT is not modified for the HPV population. Tobacco and alcohol consumption has long been known as the major risk factor for HNSCC. However, HPV has recently been recognised to play a role in the pathogenesis of a subset of clinically and molecularly distinct HNSCC, most often located in the oropharynx and associated with wild-type p53 and downregulation of cyclin D and retinoblastoma protein pRb –5, and i HPV prevalence in HNSCC has been increasing significantly in the past few decades , 7; it iMost of the available data is derived from small randomised trials with different treatment options or small heterogeneous cohorts; moreover, data were often collected retrospectively. Even though one prospective clinical trial concluded with the same prognostic advantage , other fThe present series comprises cases from patients participating in an ongoing tumor bank of patients treated for HNSCC at CHUM Hôpital-Notre-Dame since 1998. Eligibility criteria included locally advanced HNSCC and treatment with primary chemoradiation and with a minimal followup of three years. Surgical treatment preceding chemoradiation was the main exclusion criterion. Data were collected prospectively from a regular assessment of outcome variables such as response rates, local or regional recurrences, and survival rates by means of regular clinical and radiological evaluations. All patients had histological confirmation of SCC based on histological features in hematoxylin and eosin-stained tissue sections diagnosed by a pathologist experienced in head and neck pathology. Staging was performed according to the TNM classification system from clinical and radiological assessment.2 q 3 weeks × 3 or Carboplatin 70 mg/m2 d1-4 + 5-FU 600 mg/m2 d1-4 q 3 weeks × 3 or Cisplatin 6 mg/m2 daily or Carboplatin 25 mg/m2 daily) were included in this analysis. Patients were secondarily selected based on the availability of tumor samples  and concurrent chemotherapy  to control for DNA integrity and for the absence of competing inhibitors. Prior to HPV detection, samples underwent PCR for detection of Samples were then tested for the presence of HPV DNA using the Roche Linear Array detection method (LA-HPV) (primers 450 bp).Samples that tested negative for HPV-DNA with the LA-HPV technique were tested using the GP5+/GP6+ PCR detection method (primers 150 bp).β-globin with the LA-HPV detection method, and also negative for HPV DNA using the GP5+/GP6+ technique, were also tested for the presence of β-globin using PCO3/PCO4 probes (110 bp) to ensure that negative results were not caused by excessive DNA fragmentation due to extended preservation in paraffin.Samples that tested negative for both HPV-DNA and μL lysis buffer  containing 0.2 mg/mL proteinase K for 2 hours at 55°C. The mixture was then heated at 96°C for 5 minutes in order to inactivate proteinase K. Optic density was calculated for the supernate after having centrifuged the mixture at 12000 G for 20 minutes. Tubes were then stored at −4°C.Paraffin-embedded samples were heated to 72°C and washed with xylene for two minutes, four times. The samples were then submitted to four one-minute washes with ethanol 100% and four one-minute washes with ethanol 95%. Remaining tissue was then incubated in 200 β-globin gene fragment was performed by the use of PCO4 and GH2O primers to control for target DNA integrity.Amplification of a β-globin using the PCO4/GH2O method.PCR products were separated by electrophoresis on a 2% ethidium-stained agarose gel and visualised on a UV transilluminator. Samples generating a visible 268-base pair band were judged suitable for detection of HPV DNA using the LA-HPV method , which iμL with 5 μL of sample material and 95 μL of kit working master mix , uracil-N-glycosylase, dATP, dCTP, dGTP, dUTP, dTTP, and biotinylated PGMY primers and β-globin primers GH2O and PCO4). Test tubes were incubated in a TC 9700 thermal cycler set at maximum ramp speed for 2 minutes at 50°C and 9 minutes at 95°C, followed by 40 cycles of 30 seconds at 95°C, 1 minute at 55°C, and 1 minute at 72°C, with a final extension at 72°C (ramp set at 50%) for 5 minutes.As previously described  PCR was Negative and positive controls were included for each reaction.Amplicons were denatured in 0.4 N NaOH and hybridise to an immobilised probe array containing probes for 37 HPV genotypes (according to the protocol provided by Roche Molecular Systems). Following the hybridization reaction, Linear Array HPV Genotyping Strips were stringently washed to remove unbound material, and positive hybridization reactions were detected by streptavidin-horseradish peroxidase-mediated color precipitation on the membrane at the probe line.The probe for detection of HPV 52 amplicons was a cross-reactive probe that also hybridised with types 33, 35, and 58; samples positive with the HPV 52 probe and containing at least one of those types were thus also tested with a real-time PCR assay specific for HPV 52 (see below). Only samples positive for HPV 52 with real-time PCR were considered HPV 52 positive.μL reaction mixtures contained 10 mM tris-HCl; pH 8.0; 50 mM KCl; a 200 μM concentration of (each) ATP, dGTP, and dCTP; 400 μM dUTP; 0.05 μM of TaqMan probe 52-TM (CGTGCAGGGTCCGGGGTC); 0.3 pmol each of primers 52JA-3 (GAACACAGTGTAGCTAACGCACG) and 52JA-4 (GCATGACGTTACACTTGGGTCA) (targeting the E6 gene); 2.0 mM MgCl2; and 5 units of AmpliTaq gold DNA polymerase. Capillaries were placed in a LightCycler system  and amplified at 95°C for 10 minutes, followed by 50 cycles at 95°C for 15 seconds and 60°C for 60 seconds. Ten copies of an HPV 52-expressing plasmid in 500 ng of cellular DNA served as a weak positive control.As described previously , 20 μL rμL containing 50 mM KCl, 10 mM Tris HCl (pH 8.3), 200 μM each deoxynucleoside triphosphate, 3.5 mM MgCl2, 1 unit of thermostable DNA polymerase , and 50 pmol each of the GP5+ (5′-TTTGTTACTGTGGTAGATACTAC-3′) and GP6+ (3′-CTTATACTAAATGTCAAATAAAAAG-5′) primers. Samples were denatured for 4 minutes at 94°C and then underwent 40 cycles of amplification with a PCR processor . Each cycle consisted of a denaturation step (1 minute at 94°C), a primer annealing step (2 minutes at 40°C), and a chain elongation step (1.5 minute at 72°C). Complete extension of amplified DNA was ensured by prolongation of the final elongation step by 4 minutes [The primers used for HPV PCR were a single pair of consensus GP5+/GP6+ (150 bp), as previously described . PCR was minutes . Negative and positive controls were included for each reaction.PCR products were layered on 1.5% agarose gel and transferred onto positively charged nylon membranes  by diffusion blotting in 0.5 N NaOH-0.6 M NaCl.DNA purification by gel extraction was done for samples that tested weakly positive.A BLAST search was performed to assign sequences to known HPV types.μL of elution buffer  to the central membrane of the column followed by centrifugation for 1 minute.Gel extraction was performed with the QIAquick Gel Extraction Kit , according to the supplied protocol. Briefly, DNA fragments were excised from the agarose gel with a clean, sharp scalpel. Gel slices were weighed in a colorless tube, and 3 volumes of buffer QG were added to 1 volume of gel. Gel was then dissolved via incubation at 50°C for 10 minutes. DNA was bound to the supplied column by centrifugation for 1 minute followed by discarding of flow-through. 0.5 of buffer QG was added to each sample, and flow-through discarded once more after centrifugation for 1 minute. 0.75 mL of buffer PE was added to the column and flow-through discarded after centrifugation for further washing. Columns were centrifuged again at 17,900 ×g and DNA was eluded by addition of 50 Locoregional control (LRC) was defined as time elapsed between initial diagnosis and development of recurrent locoregional disease. Overall survival (OS) was defined as time elapsed between diagnosis and death from any cause, and disease-free survival was defined as time from initial diagnosis to tumour recurrence.Statistical analysis was performed using Fisher's test for categorical data and Kaplan-Meier's curves and log-rank statistics for disease-free survival, overall survival and locoregional control according to HPV status (and HPV genotype), treatment period, chemotherapy regimen, TNM stage, tumour site, and patient age. Multivariate analysis using COX models was used to adjust for imbalances in the aforementioned prognostic factors between groups. Fisher's exact test was also performed to determine the difference in acute toxicities  according to HPV status. Smoking status was excluded from our analyses because the data were inconsistently recorded in our database.This protocol was approved by our institution's ethics committee.Prospective data on efficacy and toxicity was available for 560 patients treated with concomitant CRT. All patients had histological confirmation of SCC based on histological features in hematoxilin and eosin-stained tissue sections diagnosed by a pathologist experienced in head and neck pathology. From these 560 patients, 270 fixed and paraffin-embedded specimens were collected. Sufficient tissue for DNA extraction was present in 255 samples.n = 203) were male and 20.39%  (n = 52) female. Patient characteristics are listed in Of these 255 patients, 79.61%   to ensure that DNA suitable for detection with the LA-HPV method was present, and to control for the absence of competing inhibitors. 24 samples tested positive for β-globin. Prior to HPV DNA detection, 26 samples underwent PCR for detection of Combining the results of HPV DNA detection with the LA-HPV and GP5+/GP6+ detection methods, 175 samples (68.63%) tested positive for HPV DNA. HPV 16 was identified in 138 samples (78.86%).n = 1), HPV 16 and HPV 11 (n = 1), and HPV 33 and HPV 35 (n = 1). The other genotypes detected were HPV 18 (n = 4), HPV 33 (n = 3), HPV 35 (n = 3), HPV 26 (n = 1), and HPV 58 (n = 1).255 samples were tested for the presence of HPV DNA using the LA-HPV detection method. Of these, 127 samples tested positive for the presence of HPV DNA. HPV 16 was the only genotype in 109 samples; coinfection was found in 6 samples. Among theses, coinfection with HPV 16 and HPV 18 was present in 3 cases; the others were co-infected with HPV 16 and HPV 84 , to ensure that negative results were not caused by excessive DNA fragmentation due to prolonged preservation in paraffin. 48 samples tested positive for the presence of HPV DNA. DNA was present in sufficient amounts to be submitted for sequencing in 24 samples, for which a BLAST search was performed to assign sequences to known HPV types. HPV 16 was identified as the single genotype present in all 24 samples.β-globin was not detected by the test, suggesting that the samples did not contain DNA suitable for analysis. Of these 27 samples, 16 also tested negative for HPV DNA using the GP5+/GP6+ detection method; these samples were tested for the presence of β-globin using PCO3/PCO4 probes (110 bp). All 16 tested positive for β-globin, confirming that negative results were unlikely to result from excessive DNA fragmentation. Among the 128 samples that tested negative for HPV DNA using the LA-HPV detection method, 27 were judged invalid as P = 0.0017; cf. P < 0.0001) and N (log-rank P = 0.0112) separately.Overall survival was statistically significantly different based on TNM .DFS and LRC were also significantly different according to TNM . This is probably related to the fact that many cancer subsites were rare in our population. We therefore decided not to conduct a statistical analysis of interaction using the terms “primary site ∗ HPV status.” However, within the largest subgroup of patients suffering from oropharyngeal HNSCC, survival was significantly different according to HPV positivity (P = 0.002). Patients with HPV− oropharyngeal SCC had a median overall survival of 2.46 years, while median survival was not reached at a minimum of 4.63 years of followup for HPV-positive patients  (cf. P = 0.0005). Since there were statistically significant differences between the HPV+ and HPV− populations, a COX analysis adjusting for age, T, N, and treatment period  and regimen was conducted and showed that the difference in overall survival remained significant .For HPV+ and HPV− cases, respectively, median overall survival was 8.89 and 3.09 years (002) cf. . This trP = 0.0014). For HPV 16+ cases specifically and HPV 16− cases, respectively, median DFS was 8.89 and 3.53 years (log-rank P = 0.0010).Disease-free survival (DFS) for HPV+ and HPV− cases was 8.89 years and 2.10 years, respectively (log-rank P = 0.0048).As for OS, the difference in DFS remained significant after adjustment for T, N, age, and treatment period and regimen . For HPV 16-negative cases, median LRC was 3.09 years, while it was not reached for HPV 16-positive cases (P = 0.0001). This persisted on multivariate analysis .LRC for HPV-negative and HPV-positive cases was 2.17 years and 8.89 years, respectively . Toxic effects that were evaluated included cutaneous toxicities, mucitis, nausea and vomiting, need for gavage, grade 3-4 neutropenia, per-treatment hospitalisation, and per-treatment deaths. P = 0.0013, P = 0.0061, and P = 0.0008, resp.; cf. Overall survival, DFS, and LRC were statistically significantly different based on HPV genotype  median overall survival was 8.89 years; while median OS was 2.04 years for patient receiving low-dose (Cisplatin 6 mg/m2 daily or Carboplatin 25 mg/m2 daily), chemotherapy. For patients receiving high-dose chemotherapy, that is, concurrent chemotherapy (Cisplatin 100 mg/mP = 0.0001).DFS was also 8.89 years for patients receiving high-dose chemotherapy and 1.63 years for patients receiving low-dose chemotherapy (P < 0.0001).Median LRC was 8.89 years and 1.92 years for patients receiving high- and low-dose chemotherapy, respectively (P = 0.0006). This effect was present although not statistically significant in the low-dose chemotherapy, probably due to the small number of patients in that subgroup (n = 43).HPV subgroup remains a statistically significant predictor of survival even in the high-dose chemotherapy group (which includes most of the cohort) with a median OS of 6.5 years in the high-dose chemotherapy HPV-negative group compared to a median OS not reached in the high-dose chemotherapy HPV-positive subgroup .Previous studies have reported improved outcomes in HPV-associated oropharyngeal cancer , 30, 31,HPV prevalence may have been underestimated in our trial because of the reduced sensitivity of PCR in FFPE samples . HoweverOther studies have suggested that the biological behaviour of HPV-positive tumours may be altered by tobacco use , 4, possAn increasing proportion of oropharyngeal cancer is linked to HPV, which, along with tobacco use, is the strongest independent determinant of OS in OSCC patients treated with chemoradiotherapy , 39. Tho2 regimen (for a cumulative dose of 300 mg/m2) [In this cohort, treatment with 3-week high-dose chemotherapy proved to be more advantageous. This regimen is the most widely used chemotherapy regimen in combination with radiotherapy , 41. Pre0 mg/m2) . TreatmeThis large study, with a cohort from one centre, confirms that HPV status is strongly associated with improved prognosis among H&N cancer patients receiving CRT and should be a stratification factor for all clinical trials including HNSCC cases. Separate trials in HPV-positive and HPV-negative oropharyngeal cancers will be needed to device the optimal treatment for each of these distinct entities, with the focus in HPV-positive cancers being to determine whether a decrease in treatment intensity and consequential toxicity can be achieved without compromising currently achieved outcomes. The comparison for a new therapy could consist of a concomitant boost-accelerated-fractionation regimen of radiotherapy or a standard-fractionation regimen, combined with concurrent, high-dose cisplatin, as both methods lead to similar results in terms of overall survival .


Human papillomaviruses (HPV) are classified into high-risk HPV and low-risk HPV. The most common high-risk HPV types in cervical cancer are HPV 16 and 18, and the most common low-risk types causing genital warts are HPV 6 and HPV 11. In this study, applying novel AllGlo fluorescent probes, we established a quadruplex quantitative PCR method to simultaneously detect and differentiate HPV 6, 11, 16 and 18 in a single tube.The specificity, the sensitivity, the detection limit, the reproducibility and the standard curve of this method were examined. Finally, clinical samples that had been tested previously by TaqMan PCR and HPV GenoArray (GA) test were used to verify the accuracy and sensitivity of the method.1 to 102 copies/test and a linear detection range from 101 to 108 copies/test. The mean amplification efficiencies for HPV 6, 11, 16, and 18 were 0.97, 1.10, 0.93 and 1.20, respectively, and the mean correlation coefficient (r2) of each standard curve was above 0.99 for plasmid templates ranging from 103 to 107 copies/test. There was 100% agreement between the AllGlo quadruplex quantitative PCR, HPV GA test and TaqMan uniplex qPCR methods.The assay has a sensitivity of 10AllGlo quadruplex quantitative PCR in a single tube has the advantages of relatively high throughput, good reproducibility, high sensitivity, high specificity, and a wide linear range of detection. The convenient single tube format makes this assay a powerful tool for the studies of mixed infections by multiple pathogens, viral typing and viral load quantification. More than 120 Human papillomaviruses (HPV) genotypes have been found throughout the world. The most common high-risk HPV types in cervical cancer are HPV 16 and 18, and the most common low-risk types causing genital warts are HPV 6 and HPV 11 HPV genotyping is mainly determined by molecular methods of detecting viral DNA The AllGlo probe is the latest generation of fluorescent quantitative probes developed by AlleLogic Biosciences Corporation . It replaces the quencher with a fluorophore that is identical the first fluorophore. Both of the fluorophores are in a quenched state when the probe is intact, but become dequenched when the probe is cleaved. An AllGlo probe generates two light-emitting fluorophores per cleavage event, leading to greater fluorescence gain in fluorescence PCR and lower Ct value . In addiIn this study, we used AllGlo probe and developed a method to simultaneously detect four types of HPV  in a single-tube format. This quadruplex assay was validated with an existing uniplex TaqMan assay and a HPV GA test. We carried out a series of control experiments and demonstrated the high specificity, sensitivity, and repeatability of this approach.Clinical specimens which included vaginal secretions (220 specimens), leucorrhoea (30 specimens), cauliflower excrescence warts (200 specimens), and other tissues (10 specimens)were collected from Hangzhou First People’s Hospital, China. The patients aged 20 to 50 years.Informed written consent to utilize their specimens for this study was obtained from the patients . After pre-treatment, all of the specimens were subject to DNA extraction, to detection of HPV genotype by HPV GA test , and then to quantitative detection of viral loads  by fluorescence quantitative PCR . The positive samples with known concentrations were conserved at –80 °C for future use.10 copies/µl in TE (pH 8.0) buffer for storage at –20°C.We used PCR to gain anticipated size of DNA fragments for each HPV target gene  and thenSecretion swab specimens were dissolved in normal saline. Tissues and cauliflower excrescence warts were infiltrated in 4% sodium hydroxide solution and comminuted with metal bar. The dissolved samples were oscillated and resuspended for 1 min, followed by centrifuging for 5 min . The supernatant was discarded, and the sediment was resuspended in 50 µl of DNA extraction solution . After heating at 100 °C for 10 min, the sample was centrifuged for 5 min . The supernatant was saved for qPCR analyses.Conserved regions sequences of the four HPV type-specific genes were obtained from Gene Bank. After sequence comparison by CLUSTRALW software, the most conservative regions were used to design the primers and probes with Primer Premiers 5.0. By BLAST sequence comparison, the primers and probes with the best specificity were selected. The primers and AllGlo probes used in the multiplex qPCR were synthesized by Chaoshi Bio-company . To ensure the specificity and sensitivity of the multiplex qPCR, all primers and probes were designed to have similar Tm . To enha2 copies/µl to105 copies/µl), 2 µl primer and 1 µl probe . Each PCR reaction was performed in triplicate. A blank plasmid without an HPV DNA insert and other HPV types DNA  was used as a negative control, and deionized water was used as the no-DNA blank control. The cycling conditions were a 5-min incubation at 95°C followed by 45 temperature cycles moving between 95°C for 10 s and 52°C for 20 s . The specificity and sensitivity of primers and probes were determined according to the lowest Ct value, the highest gain in fluorescence and detection limit of the uniplex fluorescence qPCR, and the quadruplex qPCR described in the following sections referred to the parameters of the uniplex fluorescence qPCR.To determine the specificity and the sensitivity of the primer and the probe, the uniplex fluorescence quantitative PCR experiments were first carried out on a Qiagen Rotor Gene 6000 fluorescence quantitative PCR instrument, which can simultaneously detect five wavelengths. Parameter variables such as cycle temperatures, concentration of primers and probe, the detection limit and replicating steps were all optimized. For every reactions, each 25-µl PCR reaction contained 11 µl of deionized water,10 µl of 2 × qPCR Mix , 1 µl individual HPV plasmid DNA template and determined the compromise annealing temperature used for multiplex detection was 58?.2 copies/µl to105 copies/µl), 8 µl mixture of all four sets of primers and 4 µl mixture of all four sets of probes , 2 µl of deionized water. Other parameter such as cycle temperatures, annealing temperatures and replicating steps were chosen from the uniplex qPCR. Each PCR reaction was performed in triplicate. The negative and blank control test of quadruple qPCR were performed in the same manner as the uniplex qPCR.Quadruplex quantitative PCR was performed in the same manner as the uniplex qPCR assay described above, PCR mixtures contained 10 µl of 2 × qPCR Mix, 1 µl individual HPV plasmid DNA temple  was used as a negative control, and deionized water was used as the no-DNA blank control. Fluorescence qPCR test was carried out in triplicate to (1) determine the minimum detection limit of this method, (2) determine the detection sensitivity and specificity of fluorescence quantitative PCR for the four types of HPV and (3) construct standard curves.Four individual HPV plasmid DNA stocks and one mixed stock that comprised all four types HPV plasmids DNA were made with concentrations ranging from 1.0 × 103, 104, 105, 106, and 107, as well as primers and probe at optimized concentrations and combinations. The qPCR assay were performed in duplicate, and a standard curve was constructed by the instrument’s software.Each quantitative PCR reaction, which was used to construct a standard curve, contained mixture of the four types of HPV plasmids  at final concentrations (copies/µl) of 102, 104, 106,and 108(copies/test). Five side-by-side quadruplex quantitative PCR assay were carried out using the aliquots plasmids DNA in triplicate to determine the mean Ct value. The CV% between each aliquot was calculated to determine the replicability of our method.Five aliquots plasmids DNA were made from the mixed stock of four types HPV plasmids and stored at –20 °C, The selected concentrations of each HPV type were 10270 clinical samples conserved at –80 °C whose HPV genotype and viral loads  had been previously determined were divided into nine groups , each grTitrations of primer/probe combinations gave a positive result in uniplex qPCR and Quadruplex qPCR. The 400/200 combination had the highest amplification efficiency and the lowest Ct value . The opt1, 102, 101, and 102 respectively  were used to construct a standard curve with the AllGlo quadruplex fluorescence quantitative PCR system. The mean amplification efficiencies for HPV 6, 11, 16, and 18 were 0.97, 1.10, 0.93 and 1.20, respectively, and the mean correlation coefficient (r2) of each standard curve was above 0.99.Five concentrations of plasmids of each type  were PCR-amplified and sequence-confirmed in order to ensure the reliability of the fluorescence qPCR. Plasmids containing the target gene were cloned by T-A cloning for the preparation of standard DNA stock. By optimizing the concentrations and combinations of primers and probes in AllGlo quadruplex quantitative PCR, we finally resolved the issue of mutual interference among primers in multiplex PCR and established a single-tube quadruplex fluorescence quantitative PCR technique based on the AllGlo probe. This new approach allows simultaneous detection and typing of four HPV types in a single tube. The rAllGlo quadruplex quantitative PCR has the advantages of relatively high throughput, good reproducibility, high sensitivity, high specificity, and a wide linear range of detection. The convenient single-tube format makes this assay a powerful tool for the study of mixed infections caused by multiple pathogens, viral typing and viral load quantification,meanwhile, this method could be used in single nucleotide polymorphism (SNP) and mutation analysis, prenatal diagnosis and genetic disease screening, drug efficacy analysis, tumor gene expression detection, and so on. The assay will facilitate molecular epidemiological studies and drug development.Supporting Information S1The DNA target sequences of HPV  inserted in plasmid was confirmed by Sanger sequencing.(DOC)Click here for additional data file.


Wolfsburg HPV Epidemiological Study (WOLVES) is a population-based cohort study on HPV infections and associated diseases in the pre-vaccination era in young women in Wolfsburg, Germany.Women born 1983/84 or 1988/89 were invited to participate. Participants were recruited in gynecology practices, and completed a questionnaire with socioeconomic, sexual and medical data including vaccination status. Pelvic examination with Pap smear and HPV testing (HC2 = Hybrid Capture 2) was obligatory. HC2-positive and 10% of HC2-negative samples were tested for specific HPV types with SPF-10-PCR, and in inconclusive cases with DNA sequencing. Women with genital warts (GW) and those with atypical Pap smears were transferred for colposcopy. GWs were classified as typical condylomata acuminata (TCA), flat condyloma (FC) and seborrheic wart-like (SWL).In total, 1258 subjects were recruited from the target population of 2850 (44.1%). Overall the prevalence of HC2 low-risk (LR) types was 8.5%. HPV6 was the most frequent LR type (2.1%), followed by HPV42 (1.1%), HPV11 and HPV44 (each 0.4%). LiPA showed a low sensitivity for HPV types 42, 90 and 91, which were detected only by HC2 and HPV sequencing. Nine women (0.7%) were transferred with incident GW: five TCA, two FC and two SWL. All TCA were associated with HPV6 in corresponding cervical swabs and warts. Tissues of SWL contained HPV6 (n = 1) and HPV16 (n = 1). The cumulative life-risk for GW was 1.4% in the 1988/89 and 4.8% in the 1983/84 cohort. Eight of 107 HC2-LR + and five of nine cases of GW had concomitant abnormal Pap smears. All CIN lesions could be linked to high-risk HPV types but borderline and low-grade abnormal smears were explained by vaginal and cervical TCA in four cases.HC2 was a specific test for the detection of established and potential LR types. In this first WOLVES analysis, HPV6 was the most frequent HPV type and the single LR type linked to disease. The observed GW incidence of 715 per 100,000 fits well with estimates of healthcare providers. Although life risks for GW were lower than in Scandinavian analyses, the societal burden within the WOLVES populations was considerable. Most clinical data on the frequency of genital warts (GW) are published from clinics specialized in sexually transmitted infections (STI) or other health institutions with a focus on the treatment of diseases of the lower genital tract, while studies estimating the impact of GW on health budgets are based on data from healthcare providers ,2. NeithThere is also an ongoing controversy about the best approach to measure the burden of HPV infections in the general population. In epidemiological and screening studies, Hybrid Capture 2 (HC2) showed a lower analytical sensitivity for high-risk HPV (HR-HPV) compared with different PCR-based methods. However, the clinical sensitivity of HC2 for HR-HPV-associated lesions like cervical intraepithelial neoplasia grade 3 (CIN3) and cervical cancer ranged from 93% to 100% in several controlled and randomized controlled trials. Therefore, HC2 is considered to be the best validated HPV test for cervical cancer screening . FurtherWe have used data from a population-based cohort study in Wolfsburg, Germany and HC2 testing to determine the prevalence of HPV infection and associated disease. Here we report the first results on the prevalence of LR-HPV types and associated diseases. Detailed analysis of HR-HPV types will be published separately.Wolfsburg HPV Epidemiological Study (WOLVES) is a population-based cohort study on the prevalence and incidence of HPV infections and associated diseases in women of two predefined birth cohorts (born in 1984/4 or 1988/9) recruited between 19 October 2009 and 31 December 2010. The study was designed to measure the role of HPV infection in the pre-vaccination era and changes over a 5-year observation period. The residents’ registration office provided a list of all women born either 1983/84 or 1988/89 with a first residency in Wolfsburg city and 2850 women were invited by letter to attend cervical cancer screening. The invitation included information on the intention of the study and the voluntary participation. To be recruited, women had to attend one of 18 gynecologists in private practices in the city of Wolfsburg for routine Pap smear screening. Participants had to give written consent and complete a short standardized questionnaire in the private practice. Then they underwent a pelvic examination with visualization of the uterine cervix. Pap smears were taken using spatula and endocervical brush. A second sample was then obtained with a Qiagen Cervical Sampler , and suspended in 1 ml of specimen transport medium  for HPV DNA testing.The The questionnaire included questions on education, birth country, marital status, pregnancies, parity, contraception, smoking, number of sexual partners, age at sexual debut, history of abnormal Pap smears, STI and GW. Furthermore, the referring gynecologist collected information on HPV vaccination status by checking the certificate of vaccination.Patients were transferred for colposcopy if they suffered from GW and/or had abnormal Pap smears conspicuous of high-grade lesions or had Pap smears classified as borderline/low-grade and tested positive for high-risk (HR)-HPV.Colposcopists classified the type of transformation zones according to the Barcelona nomenclature of the International Federation for Cervical Pathology and Colposcopy (IFCPC) . In caseA variety of skin and mucosal lesions may be classified as GW. Lesions were included in the category “genital warts” only if they were classified on colposcopy as:1. “typical condylomata acuminata” (TCA) for lesions that showed the typical acuminate morphology Figure 2. “flat condylomata” (FC) for genital papillomas with a more hyperkeratotic and papillomatous surface and flat condylomata with a smooth surface and non-pigmented papules Figure 3. “seborrheic wart-like lesions” (SWL) of the dry skin of the external anogenital area if intraepithelial neoplasia was excluded on histology Figure .Naevi, vulvar intraepithelial neoplasia (VIN), hirsutoidpapillomas and molluscacontagiosa were excluded.Colposcopists and pathologists were blinded to HPV genotyping results at the time of classification of GW.All primary HPV testing was undertaken using the HC2 assay . All samples were analyzed for the presence of at least 13 HR-HPV types  following the manufacturer’s instructions. Therefore, a positive HR-HPV result in this study refers to a patient positive for one or more of the 13 HR-HPV types included in the probe mix. Infection with LR-HPV types was also investigated by HC2. The LR probe mix included HPV types 6, 11, 42, 43 and 44. Samples were considered positive if they attained or exceeded the FDA-approved threshold of 1.0 pg HPV DNA/ml, which corresponds to 1.0 relative light unit (RLU).All samples that tested positive for LR-HPV and/or HR-HPV with HC2 and 10% (every tenth) of all HC2-negative samples underwent HPV genotyping. HPV genotyping was performed as described previously using SPF-10-PCR, followed by Reverse Line Probe Assay LiPA Extra (SPF-10-PCR) . BrieflyAny lesion suspicious of high-grade neoplasia had to undergo histological assessment and patients with GW were tested for histological confirmation of the diagnosis with biopsies. Paraffin embedded tissue blocks of GW and/or cervical intraepithelial neoplasia (CIN), vulvar intraepithelial neoplasia (VIN), and vaginal intraepithelial neoplasia (VAIN) underwent HPV-genotyping with SPF-10 and LiPA. Data for the prevalence of HR-HPV, cofactors associated with the risk of HR-HPV infection, and the association between specific HR-HPV subtypes and abnormal Pap smear test results will be published separately.The study was designed to provide a one-time cross sectional study of the 1983/84 cohort, whereas the 1988/89 cohort will be followed by annual examination for 5 years. In the year 2014/15, women born 1993/94 will be invited for a one-time examination. Overall WOLVES will allow investigation of individual changes in HPV infection during follow-up and a comparison of prevalence of HPV infections in different age cohorts over time.WOLVES was approved by the ethics committee of the physicians’ association of Lower Saxony in Hannover, Germany (Bo/07/2009).All statistical analyses were undertaken by an independent statistician who did not participate in the collection of data.U Test) and multivariate (logistic regression) analyses. Statistical analyses were performed with the validated program Testimate Version 6.5 from IDV Gauting . Logistic regression was calculated using SAS.The association between HPV infection and explanatory variables was analyzed in univariate  of 1504 registered women born 1983/84 and 599 (44.5%) of 1346 women born 1988/89 were recruited. The socioeconomic status of the recruited population reflected the general population in Wolfsburg.In the 1983/84 cohort, 52/659 (7.9%) cervical samples tested positive for LR-HPV types with HC2, while in the 1988/89 cohort the corresponding prevalence was 55/599 (9.2%) .Two of 107 HC2-LR positive samples failed in HPV genotyping with SPF-10-PCR. LR-HPV genotypes 6, 11, 40, 42, 43, 44, 54, 61, 70, 72 and 81, as defined by Munoz , were deBased on SPF-10-PCR genotyping of HC2-LR positive cases, HPV6 was the most frequent LR type (n = 26), followed by HPV11 (n = 5), HPV44 (n = 5), HPV43 (n = 2), HPV54 (n = 1) and HPV70 (n = 1).In 43/105 samples, genotyping showed either no HPV types at all or only HR types  and two cases associated with HPV74.Thirty women had a history of GW, 26/659 (3.9%) in the 1983/84 cohort and 4/599 (0.7%) in the 1988/89 cohort. Among cases with GW prior to the start of WOLVES, 22 underwent pharmaceutical treatment and eight were treated surgically. Another five women born 1983/84 and four born 1988/89 were transferred to colposcopy because of first-time GW.The colposcopy classification was TCA in five cases, FC in two cases and SWL in two cases. Both cases of SWL and one FC tested negative for HC2-LR, while all five cases of TCA and one FC were associated with cervical HC2 samples that tested positive for LR-HPV. Genotyping detected HPV6 in all five cervical samples of patients with TCA either as single HPV or as one of multiple HPV types. In two TCA cases, biopsies were taken and PCR genotyping confirmed HPV6 in the tissues. The remaining patients with TCA and FC refused biopsies. Both SWL underwent histological assessment. One case of warts close to the pubic mount was associated with HPV6, while the second case of SWL of the greater lips of pudendum contained HPV16.All patients with TCA and SWL were smokers and not HPV vaccinated. One patient with FC of the perineum was a smoker and received a full course of quadrivalent HPV vaccination 3 years prior to diagnosis. The cervical sample tested negative for HC2-LR types. The remaining patient with FC never smoked and was not vaccinated. The cervical sample tested positive for HR- and LR-HPV types. Genotyping showed HPV types 51, 52 and 66.Overall 8/107 LR-HPV-positive women showed abnormal Pap smears, including five cases of low-grade squamous intraepithelial lesion (LSIL) and three cases with borderline cytology. All occurred exclusively in women who tested positive for HC2 LR and HR, while no case was observed in HC2-LR-positive/HC2-HR-negative participants.Five of nine patients with GW had abnormal Pap smears classified as borderline (n = 1), low-grade squamous intraepithelial lesion (n = 3) and high-grade squamous intraepithelial lesion in one patient with SWL whose cervical sample tested positive for HR-HPV, but negative for LR-HPV types.The colposcopic evaluation of all five TCA cases excluded any cervical abnormality in three women with normal or borderline Pap smears. The remaining two cases had Pap smears classified as LSIL. One case of TCA was diagnosed with a CIN1 lesion. PCR genotyping of the CIN1 tissue detected HPV51 as single HPV type. The remaining TCA cases had cervical condylomata acuminata, the tissue contained HPV6 as single HPV type.WOLVES has provided the first real-life data on the epidemiology of LR-HPV and associated diseases in Germany. Of particular interest is the observation that the prevalence of LR-HPV is declining in the 1983/84 cohort, suggesting that spontaneous regression is common. Furthermore, the results from this analysis also suggest that the number of sexual partners is the most important risk factor for LR-HPV infection. The reported HC2-LR prevalence of 7.9–9.2% is concordant with the 8.5% prevalence in 25–34 year-old Italian women observed by Rossi and coworkers . The ItaHPV6 was the most frequent of all identified LR-HPV types (26 cases) and, to date, the single LR type associated with clinical disease in our study population. One focus of WOLVES is a careful colposcopic distinction between HPV-related lesions in groups with different specific morphologies. We hypothesize that TCA are linked exclusively to HPV6 and HPV11, whereas the approximately 10–17% of GW associated with other HPV types observed in a number of different studies may be explained by FC and SWL .In the WOLVES study population, the majority, but not all, external GWs were confirmed as TCA. All five cases with typical acuminate warts were associated with HC2-LR-positive cervical samples and detection of HPV6 by SPF-10-PCR genotyping, while cervical samples of both cases with SWL lesions tested negative for HC2-LR, although HPV6 and HPV16 were detected in the wart tissue. The number of GW classified colposcopically is still too small for an assessment of our hypothesis of HPV type-specific clinical pictures, but we hope that follow-up data over 5 years from WOLVES and other studies will provide definitive evidence.The observed incidence of 715 GW per 100,000 women (9/1258) during the observation period corresponds well with calculations based on healthcare providers’ studies and cross-sectional studies for urban regions in Western Germany and shows that these approaches may provide reliable data . In the Although we found a significant burden of disease, neither incidence nor life-time risk for GW reached the high levels observed in four Nordic countries . HoweverThe results of the multivariate logistic regression showed that only a higher number of sexual partners increased the risk for LR-HPV type infection. Other risk factors such as age at sexual debut or smoking were not significant. Nevertheless, it is notable that eight of nine patients with GW were current smokers. Although the numbers are too small for statistical analysis, our observation is consistent with a concept that risk factors for the acquisition of HPV infection are different from those (co-factors) associated with the development of clinically relevant disease. The number of sexual partners and smoking have been identified as risk factors associated with HPV infection in other recent studies, although these data related to overall prevalence of HPV rather than LR-HPV specifically ,27.LR-HPV types are purported to cause a substantial proportion of low-grade and borderline findings on cytology. We also found an increased rate of abnormal Pap smears (8/107), but most cases were related to HR-HPV types in women with multiple HPV-type infections. Three out of five patients with TCA had abnormal Pap smears and two of these abnormalities were linked to vaginal and/or cervical condylomata.WOLVES is the first population-based trial that provides real-life data on the epidemiology of LR-HPV in young women in Germany. Our standardized approach to investigate the epidemiology of LR-HPV types in young German women with the use of HC2 seems feasible. Genotyping of an HC2-negative control group shows that a small number of probably latent infections will be missed by this concept, but HC2-LR accurately detected all LR-HPV types, defined by Munoz et al., and related types (e.g. HPV74) that were described in recent studies as LR-HPV. We speculate that HC2-LR may be useful in discriminating future LR from HR-HPV types. The observed LR-HPV prevalence of 8.5% is in accordance with results from similar studies in Italy and Spain ,28.HPV6 was the most common LR type found in the WOLVES population, followed by HPV types 42, 11 and 44. In contrast, the prevalence of HPV6 was only 2.1% and less than one-quarter of HC2-LR cases were related to HPV6, while all cases of LR-HPV-associated diseases were exclusively related to this type. Of 26 cases with cervical HPV6 infections, five presented with TCA, one with FC and three showed abnormal Pap smears. We expect to diagnose lesions related to other LR-HPV types during future follow-up, but in accordance with other investigations our findings underline the peculiar role of HPV6 and question the clinical relevance of LR-HPV infections other than HPV types 6 and 11.Most established co-factors in the genesis of HR-HPV infections and associated neoplasia and GW showed no association with LR-HPV infections in the WOLVES study population. The single co-factor associated with a significantly increased risk for LR-HPV was a high number of sexual partners. This may be different for the development of GW, which were observed almost exclusively in smokers, and further research is mandatory. Similarly, our hypothesis of an HPV type-specific clinical morphology of lesions needs to be confirmed in larger patient numbers. To date, all TCA were related to HPV6.KU Petry received speaker’s honorarium from Beckton Dickinson, Qiagen and Roche Diagn. R Schulze-Rath is employee of Sanofi Pasteur MSD. T Iftner has received institutional grants from GSK, Gen-Probe, Sanofi-Pasteur MSD and Hologic. The remaining authors declare no conflict of interest.KUP was responsible for the overall study design, data analysis, interpretation, writing of the manuscript and data collection. AL was responsible for data collection and interpretation. AJ was responsible for statistical analyses and interpretation. AI was responsible for HC2-testing, HPV genotyping and sequencing, data collection and interpretation. SS was responsible for trial coordination, quality control and data collection. RSR was responsible for trial coordination and interpretation. TI was responsible for the study design, HPV testing, data collection and interpretation. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/12/367/prepub


A higher incidence of cervical cancer and human papillomavirus (HPV) infection has been reported in northern Canada and in First Nation, Métis and Inuit women, with some evidence to suggest that the HPV type distribution in these populations may be different from the rest of Canada.The objective of this study was to measure the HPV type prevalence in Labrador women to determine if significant differences in HPV types could reduce the effectiveness of HPV vaccination.The prevalence of HPV types was determined in 1,370 women presenting for routine pap screening in Labrador between February and November 2010. Cervical cytology and HPV genotyping were performed on the same liquid-based cytology specimens.The overall prevalence of HPV was 21.4%; cytological abnormalities were found in 8.8% of the participants. HPV 16 and 18 were the most common high-risk HPV types. These two types were found in 52.4% of high-grade lesions. The prevalence in HPV infections was comparable across the Labrador regions.The present results support the potential effectiveness of the HPV immunization program in Labrador. Every year in Canada, approximately 1,300 women are diagnosed with cervical cancer and 450 die from it –5. Not sOver 50 different human papillomavirus (HPV) types infect the anogenital tract, some of which are referred to as high-risk (HR) types for cancer . These H2 inhabited by approximately 26,000 people with a predominantly Aboriginal population. The Aboriginal population living in Labrador is composed of Métis, Inuit and First Nations. We were interested in determining the distribution of HPV types in women living in Labrador and carried out a surveillance study using a convenience sample of women attending routine cervical screening.Labrador is a vast region of 294,330 km®, BD Diagnostics) samples were collected as per standard practice and shipped to the Newfoundland Public Health Laboratory in St John's. Specimens were aliquoted for HPV genotyping and shipped to the National Microbiology Laboratory (NML) in Winnipeg, Manitoba. Cytology was carried out according to provincial guidelines and practices at the Eastern Health Cytology Laboratory in St John's. Cytology and HPV testing were carried out independently in a blinded fashion.Women in Labrador who presented for routine Papanicolaou (Pap) test screening in 17 Labrador communities between February 2010 and November 2010 were introduced to the study. Liquid-based cytology , the South coast  and the Central region . According to the 2006 Canadian census, approximately 40% of the population in the Central region self-identified as Aboriginal, while at least 87% self-identified as Aboriginal in the North coast and South coast. Univariate comparisons were made using χEthics approval was obtained from the Public Health Agency of Canada ethics boards. Labrador-Grenfell Health facilitated the project and community stakeholders reviewed the manuscript before being submitted for publication.Of the 1,370 women that were enrolled in the study, 293 (21.4%) were infected with at least one HPV type and 218 (15.9%) tested positive for at least one HR-HPV type. The infected women had a total of 398 HPV infections; 77 of HPV-positive women had multiple infections. The age of the participants ranged from 13 to 86 years . Overall, 90.4% of participants had a normal cytology, 3.5% had an ASCUS, 3.7% a LSIL, 1.5% a high-grade lesion  and 0.8% had an unsatisfactory Pap test.At least one HR-HPV type in the IARC Group 1 (carcinogenic) was detected in 12.5% of participants; 4.6% were positive for at least one IARC Group 2 type (probably or possibly carcinogenic); and 5.6% tested positive for multiple HPV infections . Study pInfections with species A9, which includes types closely related to HPV 16, were the most frequent (32.2% of infections), including HPV 16 (12.3%), HPV 33 (4.5%), HPV 58 (3.5%) and HPV 67 (3.8%) . SpeciesHPV 16 and/or 18 were found in 2.7% of women with normal cytology, 27.3% of women with low-grade cervical lesions  and in 52.4% of those with high-grade lesion . HPV 16 The prevalence of HPV infection in the study participants 21.4%) is in the upper range of results that have been reported worldwide in women with normal cervical cytology . A study.4% is inHPV 16 was the most prevalent HPV type observed in the present study, which is similar to that previously reported worldwide ; HPV 18 The prevalence of multiple HPV infections in this study was 5.6%, or 26.2% of HPV-positive women. This proportion of multiple infections is in the middle of the worldwide range of 11.5–42.4% for women with a normal cervix . It is, Labrador has a predominantly Aboriginal population , especially outside Happy Valley-Goose Bay. In the 2006 Canadian Census, 38% of the residents of Happy Valley-Goose Bay  self-identified as Aboriginal, while at least 87% of the people living outside Happy Valley-Goose Bay self-identified as Aboriginal. Higher prevalence of HPV in Aboriginal groups living in Canada has previously been reported (Our study shows that the distribution of HPV types in women undergoing routine cervical cancer screening in Labrador is in accordance with observations reported in Canada and worldwide. These results indicate that the current HPV vaccine against HPV 16 and 18 is likely to have the expected effectiveness in preventing cervical cancer in Labrador. A careful investigation of the underlying risk factors would help guide strategies for the prevention of cervical cancer in the population of Labrador.


Head and neck cancer (HNC) incidence, mortality and survival rates vary by sex and race, with men and African Americans disproportionately affected. Risk factors for HNC include tobacco and alcohol exposure, with a recent implication of human papillomavirus (HPV) in the pathogenesis of HNC. This study describes the epidemiology of HNC in the United States, examining variation of rates by age, sex, race/ethnicity and potential HPV-association.We used the North American Association of Central Cancer Registries (NAACCR) Cancer in North America (CINA) Deluxe Analytic Data to analyze HNC incidence for 1995–2005 from forty population-based cancer registries. We calculated age-adjusted incidence rates and incidence trends using annual percent change by age, sex, race/ethnicity and HPV-association.Males and Non-Hispanic Blacks experienced greater HNC incidence compared to women and other race/ethnicity groupings. A significant overall increase in HNC incidence was observed among HPV-associated sites during 1995–2005, while non HPV-associated sites experienced a significant decline in HNC incidence. Overall, younger age groups, Non-Hispanic Whites and Hispanics experienced greater increases in incidence for HPV-associated sites, while HNC incidence declined for Non-Hispanic Blacks independent of HPV-association. In particular, for HPV-associated sites, HNC incidence for Non-Hispanic White males aged 45–54 increased at the greatest rate, with an APC of 6.28% (p<0.05). Among non HPV-associated sites, Non-Hispanic Black males aged 0–44 years experienced the greatest reduction in incidence , while a greater decline among the older, 55–64 year age group  occurred in females.This study provides evidence that HPV-associated tumors are disproportionately affecting certain age, sex and race/ethnicity groups, representing a different disease process for HPV-associated tumors compared to non HPV-associated tumors. Our study suggests that HPV tumor status should be incorporated into treatment decisions for HNC patients to improve prognosis and survival. In the United States, it is expected that 36,540 new cases of oral, pharyngeal, and laryngeal cancer, also known as head and neck cancer (HNC), will be diagnosed in 2010, and 7,880 deaths will occur from HNC HNC incidence, mortality, and relative survival rates vary with respect to sex and race. Incidence and mortality rates are more than twice as high in men as in women, and are greatest in men who are older than 50 years of age It is well known that intensity and duration of alcohol and tobacco consumption are established risk factors for HNC. Over 80% of HNC cases are directly attributable to alcohol and tobacco exposure, however, the other 15–20% of cases generally consist of non-smokers and non-drinkers With the implication of HPV infection as an emergent risk factor for HNC, it is crucial to describe the distribution and incidence of HNC by sub-sites potentially associated with HPV and those not associated with HPV. The disparity of HNC among sex and race/ethnicity groups also needs better understanding to determine the potential causes for such large differences in incidence. This study uses 11 years of HNC incidence data (1995–2005) from the North American Association of Central Cancer Registries (NAACCR) Cancer in North America (CINA) Deluxe Analytic Data to investigate HNC trends in incidence by potential HPV-association with further stratification on age, sex and race/ethnicity. In comparison to SEER data, a greater geographic coverage and a larger sample size can be obtained with utilization of the CINA data, allowing for more in-depth analyses. This may yield patterns in HNC incidence that could not be seen in smaller sample sizes. The objectives of our study were to assess the incidence of HNC in the United States between 1995 and 2005 and to investigate incidence trends of HNCs putatively associated with HPV infection.Cases of HNC were identified through the NAACCR CINA Deluxe Analytic Data for the diagnosis years 1995 to 2005. The source of this data is from NAACCR data submissions as of December 2007. Support for cancer registries is provided by the state, province, or territory in which the registry is located. In the U.S., registries also participate in the National Cancer Institute's Surveillance, Epidemiology, and End The CINA Deluxe dataset uses a joint race/ethnicity variable generated from race and ethnicity information found in medical records, which were then enhanced by the NAACCR Hispanic Identification Algorithm (NHIA) Another important data issue occurs with the use of multiple staging systems during the eleven year time period of data collection. The stages of HNC diagnosed from 1995–2000 were classified according to the 1977 SEER summary staging system criteria, HNC diagnosed from 2001–2003 were classified according to the 2000 SEER summary staging system, and HNC diagnosed from 2004–2005 were classified according to the derived 2000 SEER summary staging system. The use of multiple staging systems can lead to potential misclassification and affect the stage distribution of the population.To identify cases in the CINA deluxe analytic data file between 1995 and 2005 with HNC  we extracted the following HNC sites according to the topography codes in the International Classification of Diseases for Oncology, Third Edition (ICD-O-3) We also classified all HNC cases into two groups based on proclivity for HPV infection: HPV-associated sites and non HPV-associated sites. Since we were unable to determine the true HPV status of the tumors we relied on professional expertise and available literature to determine which anatomic sub-sites are potentially associated with HPV Counts and percent distribution were computed by sex, age, race/ethnicity, and cancer stage for all HNC sites, as well as those sites associated with HPV infection and those sites not associated with HPV infection. HNC incidence counts, age-adjusted incidence rates  and corresponding 95% confidence intervals (95% CIs) were calculated by sex, race/ethnicity, cancer stage and proclivity for HPV infection. In addition, race/ethnicity specific rate ratios and corresponding 95% CIs were calculated using Non-Hispanic Whites as the referent group. Lastly, we calculated the annual percent change (APC) in incidence rates from 1995–2005 to assess whether incidence trends differ across time for HPV-associated and non HPV-associated sites as well as age, sex and race/ethnicity. All data analyses were conducted using the SEER*Stat software, Version 6.2.4 .As shown in For HNC diagnosed in HPV-associated and non HPV-associated sub-sites, specific patterns emerged with respect to age . IndepenAge-adjusted incidence rates of HNCs were highest among Non-Hispanic Blacks, followed by Non-Hispanic Whites and Hispanics . The samFurther investigation of the trends in the APC of the AAIR by HPV-association, age, sex and race/ethnicity can be found in Our analysis of HNC incidence in the United States reinforces previous observations that HNC incidence rates disproportionately affect men and Non-Hispanic Blacks We found that a greater proportion of the subjects in this study with HNC in HPV-associated sites were diagnosed at younger ages compared to non HPV-associated sites. Gillison, et al. also reported a similar trend where HPV-positive patients were younger by about 5 years on average when compared to HPV-negative patients Subjects diagnosed with HNC in HPV-associated sites were more commonly diagnosed at a regional tumor stage compared to non HPV-associated sites, which were more commonly diagnosed at a localized stage. The same trend was seen among the stage-specific AAIRs. Other studies have found similar results, with HPV-related HNC cases diagnosed at more advanced stages than non HPV-related sites Since the 1980s, HNC incidence rates have been declining Several studies have found that patients with HPV-positive tumors are less likely to smoke and consume alcohol compared to patients with HPV-negative tumors Stratifying incidence rates over time by race/ethnicity revealed interesting patterns among certain race/ethnicity groupings. Among HPV-associated sites, the increasing trend in HNC incidence was mainly due to Non-Hispanic Whites. Interestingly, HNC incidence rates for Non-Hispanic Blacks are decreasing across HPV-associated sites as well as non HPV-associated sites. It was shown that in a study of HIV positive individuals, Caucasians had a greater risk of oral HPV infection than African Americans There were several limitations to the current study. Due to the nature of the CINA data, we were unable to obtain individual risk factor and HPV level data on each HNC case. Inferences cannot be made as to individual level characteristics, but this study provides important information from a population perspective. The second limitation, and the most notable, is the classification of anatomic sub-sites as HPV-associated and non HPV-associated. We were unable to determine the HPV status of each tumor, and relied on previous literature to determine which anatomic sub-sites were potentially associated with HPV. The classification of HPV-association could result in misclassification; however without HPV typing results on each individual, the classification of HPV tumor status can only be estimated by previous etiologic knowledge presented in the literature. Another limitation results from unavailable data on tobacco or alcohol use within the cancer registries. Without this knowledge, we cannot establish the effect of tobacco and alcohol use on HNC incidence and infection with HPV.This study has shown that HNC cases associated with HPV infection represent a different disease process and mechanism compared to HNC cases caused by alcohol and tobacco use. The findings of this study suggest that determination of HPV tumor status could be important in the decision of treatment therapies for patients. With the identification of HPV-tumor status, treatment could be tailored to the individual patient to improve prognosis and survival. In addition, the current treatment options could be improved by development of vaccine-mediated therapy or the use of biological modifiers for preservation therapy of organs within the head and neck region.


The COMP in urine was also determined by both QCM and EIS for comparison. A highly sensitive, user friendly and cost effective analytical method for the early stage OA diagnosis has thus been successfully developed.Current methods for diagnosing early stage osteoarthritis (OA) based on the magnetic resonance imaging and enzyme-linked immunosorbent assay methods are specific, but require specialized laboratory facilities and highly trained personal to obtain a definitive result. In this work, a user friendly and non-invasive quartz crystal microbalance (QCM) immunosensor method has been developed to detect Cartilage Oligomeric Matrix Protein (COMP) for early stage OA diagnosis. This QCM immunosensor was fabricated to immobilize COMP antibodies utilizing the self-assembled monolayer technique. The surface properties of the immunosensor were characterized by its FTIR and electrochemical impedance spectra (EIS). The feasibility study was based on urine samples obtained from 41 volunteers. Experiments were carried out in a flow system and the reproducibility of the electrodes was evaluated by the impedance measured by EIS. Its potential dynamically monitored the immunoreaction processes and could increase the efficiency and sensitivity of COMP detection in laboratory-cultured preparations and clinical samples. The frequency responses of the QCM immunosensor changed from 6 kHz when testing 50 ng/mL COMP concentration. The linear regression equation of frequency shift and COMP concentration was determined as: y = 0.0872 x + 1.2138 (R OA is a prevalent cause of pain and disability in a considerable proportion of the aging population. No method or drug has been proven to stop disease progression or make cartilage rejuvenate. There is no proper detection method to diagnose the initial cartilage degradation of OA and to determine exact therapies. Planar radiographs were used in detecting joint space width, but the cartilage destruction could only be determined from radiographs when significant cartilage degradation has occurred. Therefore, early diagnostics of OA symptoms by biochemical methods or sensor systems is an urgent necessity. A delayed gadolinium-enhanced magnetic resonance imaging of cartilage (dGEMRIC) method was designed to examine glycosaminoglycan changes in articular cartilage during the development of OA. However, dGEMRIC is not available in most clinic facilities, it tests are lenghty and patients are also exposed to high radiation doses when cartilage tissue is measured by this method. On the other hand, biological markers might provide sufficient information to reveal dynamic changes of the cartilage. Several studies have shown that serum levels of cartilage oligomeric matrix protein (COMP), which is abundant in OA cartilage, are a sensitive marker for cartilage degradation detection and thus a potential prognostic marker providing important information on metabolic changes occurring in the cartilage matrix in joint diseases –4. The Ci.e., COOH, OH and NH2) of self-assembled monolayers (SAM) and immunocapture antigens such as COMP or other targets. The QCM can consequently detect mass changes due to these molecular interactions on the surface of the QCM.Considerable efforts have been directed towards the development of simple biosensors for the detection of viruses –11. BiosSauerbrey first described the relationship between frequency shift and mass change on the crystal surface in air . The freMost biochemical diagnoses of cartilage degradation use synovial fluid from invasive operations at diseased sites or in serum. There is very little literature in which the COMP concentration in urine of OA patients has been defined. In order to develop an easy to perform and homecare system for monitoring the cartilage degradation, a non-invasive simple QCM-based sensor was developed in this research. The efficiency and sensitivity of the sensor were also evaluated to further enhance its practicability in early OA diagnosis2.2.1.2HPO4, 2 mM KH2PO4, pH 7.4) and bovine serum albumin were purchased from Sigma . Thioctic Acid  was obtained from ACROS (USA). The electrolyte potassium ferricyanide (K3[Fe(CN)6], SHOWA), potassium ferrocyanid (K4[Fe(CN)6], SHOWA) and potassium chloride  were analytical grade. Doubly distilled water was used throughout the experiments. The feasibility study was carried out using urine samples from 41 persons including 14 males and 27 females, collected from healthy personnel and hospital OA patients. The samples were provided by E-Da hospital, Kaohsiung, Taiwan, and analyzed without further treatment.COMP Human, Mouse Monoclonal Antibody, Clone:16F12 was purchased from BioVendor  and Recombinant Human COMP (>90%) was purchased from R&D Systems . N’-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride , medium for preparing phosphate buffer saline  alcohol solution at room temperature for 24 h in the darkroom. Afterwards, it was rinsed thoroughly with ethanol and dried with nitrogen gas and stock at room temperature for further used.The QCM sensor , coupled inside a flow injection system, was a 10 MHz quartz crystal with a 3.8 mm diameter gold electrode. Each of the gold electrodes was pretreated by electrochemical cleaning in 0.5 M H2.3.−1) in PBS solution was placed on the electrode to conjugate at 4 °C for 12 h and then rinsed by PBS. Afterwards, the electrode was blocking by 5% bovine serum albumin (BSA) for 1.5 h. Finally, the electrode was rinsed with PBS and then dried by nitrogen gas. The coupling agent, 0.2 M 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) was used to activated the prepared TA monolayer for 3 hr at room temperature and then rinsed with ethanol and dried as aforementioned. Then 20 ìL of COMP monoclonal antibody  and the impedances of the electrodes were analyzed by electrochemical impedance spectrometry . The EIS analysis was accomplished in a three-electrode mode system wherein the modified gold electrode, a screen-printed carbon electrode and an external Ag/AgCl electrode were working, counter and reference electrode, respectively.2.4.63−/4− and KCl.3.3.1.In order to determine the chemical structure of the modified gold electrode, each electrode sample was characterized by FTIR. −1 and 2,900 cm−1, respectively. It suggested that the thiotic acid was successfully modified onto the electrode surface. In −1 and the enhanced N-H vibration peaks at 3,400 cm−1 implied that the carboxylic acid group was activated by EDC. In For the thiotic acid modified electrode the C=O and C-H functional groups appeared at 1,700∼1,730 cmct) of Fe(CN)63−/4− redox pair at the BSA/anti-COMP/EDC/TA/gold electrode. The reproducibility of the modified gold electrodes was evaluated by examining the impedance for each electrode. The impedance of the COMP antibody immobilized electrode and the BSA treated electrode were evaluated by EIS at 30 °C. All electrochemical measurements were performed in a three-electrode electrochemical cell. A screen printed carbon and a screen printed Ag/AgCl electrodes were used as the counter and reference electrode, respectively. A QCM was introduced as the working electrode. A thioctic acid monolayer was formed by the SAMs technique, and then the functional group of TA monolayer was activated by EDC. Finally anti-COMP and BSA were immobilized on modified electrode area successfully. Impedance spectroscopy (EIS) studies demonstrated that the formation of antibody–antigen complexes increased the electron-transfer resistance  when detecting COMP, the calibration standard curve of human COMP concentration was established according to the kit protocol using the concentration range from 4–128 ng/mL. The QCM sensor device for COMP detection should also have high response and sensitivities to reflect the real concentrations of COMP in detecting sample. In this study, a linear relationship between COMP concentration and frequency shift at 26 °C was shown in 2 = 0.9957), where y is absolute value of frequency shift and x is COMP concentration in ng/mL. The frequency shifts were observed in the range of 1–200 ng/mL, which showed the QCM sensors have higher sensitivity and faster response than the ELISA method in the real time detection, thus allowing the possibility of estimating COMP concentrations in unknown samples.The linear regression equation is y = 0.0872 x + 1.2138 (R3.4.ct) of Fe(CN)63−/4− redox pair at the BSA/anti-COMP/EDC/TA/gold electrode. Because of the relatively large surface area for EIS analysis, the sensitivity of the electrode was higher at relatively low COMP concentrations. Nevertheless, the operating procedure of EIS is much simpler than the ELISA method and it could therefore be applied as a homecare system.Since the impedance of the electrode was significantly altered by the electrode surface condition, we exploited the EIS technique to detect the COMP concentration. The modified electrodes were immersed in 20 μL of solution with the desired COMP concentration for 5 min and then rinsed with PBS. Due to the different amounts of COMP adsorption, the impedance of the electrodes changed with the COMP concentrations as shown in 3.5.This QCM based sensor needs to be further evaluated for its rapid and sensitive detection of COMP in practical clinical specimens of OA. We collected 41 urine samples including patients with OA and normal persons to compare the measurement results of the QCM and electrochemical immunosensor. According to the ELISA kit obtained from BioVendor , the COPThe trends of the results obtained by QCM and EIS were similar. Comparisons of ELISA data, QCM results and the reference values of COMP concentrations , showed 4.Clinical diagnosis of OA is difficult, especially in the early stages of cartilage degradation. Most methods for OA diagnosis involve invasive collection of specimens or radiophotography that could expose patients on the radiation. Most patients that have been defined as OA have cartilage erosion with fast degradation of cartilage, so early OA detection is important for early cartilage protection or medical treatments. A detection system and device for easy operation, like the device for detecting blood glucose, should be helpful for homecare. This study used the well known COMP antibody biomarker immobilized on a QCM sensor and established its stable detection properties at room temperature. From the data of this study, such a QCM sensor showed the same sensitivity as EIS, and the values of COMP of volunteers also reflect the grades of cartilage degradation determined by clinical diagnosis. In addition, the analytical procedures of this QCM immunosensor are direct and simple in real time without multiple labeling and separation steps. The experimental results suggested that a highly sensitive and user friendly QCM sensor has been successfully developed for early stage OA diagnosis.


The prevalence of human papillomavirus (HPV)–associated head and neck cancers is increasing, but the prevalence of oral HPV infection in the wider community remains unknown. We sought to determine the prevalence of, and identify risk factors for, oral HPV infection in a sample of young, healthy Australians. For this study, we recruited 307 Australian university students (18–35 years). Participants reported anonymously about basic characteristics, sexual behaviour, and alcohol, tobacco and illicit drugs use. We collected oral rinse samples from all participants for HPV testing and typing. Seven of 307 (2.3%) students tested positive for oral HPV infection , and six of them were males (p = 0.008). Compared to HPV negative students, those with oral HPV infection were more likely to have received oral sex from more partners in their lifetime (p = 0.0004) and in the last year (p = 0.008). We found no statistically significant associations with alcohol consumption, smoking or numbers of partners for passionate kissing or sexual intercourse. In conclusion, oral HPV infection was associated with male gender and receiving oral sex in our sample of young Australians. The incidence of squamous cell carcinoma of the head and neck (HNSCC), more specifically of oropharyngeal squamous cell carcinoma (OPSCC), is increasing. Sites particularly affected are those of the tonsils and base of tongue We recruited 307 students (ages 18 to 35 yrs) from an Australian university. Students were informed about our study by emailing flyers, posting on social media through student organisations and providing information in lectures. Students were approached after lectures as well as at campus where our data collection room was set up. Participants were asked to self-complete a paper-based 4-page questionnaire. Potential participants were informed that the investigators would not record any identifying details, and that their anonymity would be preserved. Students who agreed to participate were given a questionnaire and a tube for collection of oral rinse sample, both labelled with the same unique identification number. The questionnaire covered basic demographics , sexual behaviour , and alcohol, tobacco and illicit drugs use as well as history of genital infections, abnormal pap smears and Gardasil vaccination . It also included two questions about knowledge of protection from contracting a sexually transmitted infection from engaging in oral sex. In the questionnaire we included the following descriptions: ‘one drink’  =  a glass of wine, middy of beer or nip of spirits; ‘Non-smoker’ - You have smoked 100 or fewer cigarettes in your life-time; An ‘illicit drug’ refers to illegal or controlled substances; ‘Recreational drug’ refers to drugs used for non-medical purposes, not including cigarettes or alcohol; ‘Passionate kissing’ refers to open-mouthed kissing; ‘Oral sex’ refers to a man's or woman's mouth on a partner's genital area; ‘Sexual intercourse’ refers to vaginal intercourse and anal intercourse only and does not refer to oral sex; and ‘Mouth jewellery’ refers to jewellery worn on the lips or tongue requiring piercing of the skin.Participants rinsed their mouth with 7 ml sterile saline (0.9%) for 30 seconds. All oral rinse specimens were frozen at −20°C within 3 hours of collection. Specimens were later thawed at ambient room temperature, vortexed and 3 ml was used for DNA extraction. DNA was extracted in batches of 24 samples at a time, using the QIAamp DNA Mini Kit following the QIAGEN supplementary protocol for Isolation of genomic DNA from saliva and mouthwash (QA19v Jul-10).All participants were given a copy of the Study Information Sheet; consent was obtained by completing the anonymous questionnaire. The study was approved by the human research ethics committees of the Queensland University of Technology (1200000292) and the QIMR Berghofer Medical Research Institute (P1508).2 (Applied Biosystems). Forty cycles of amplification was performed on an Eppendorf Mastercycler Gradient PCR machine after an initial step of 10 minutes denaturation at 94°C. Each cycle for the GP+ PCR consisted of 94°C for 1 min, 40°C for 2 min and 72°C for 1.5 min, plus a final elongation step at 72°C for 4 min. Each amplification cycle for the β-globin PCR consisted of 94°C for 1.5 min, 50°C for 1.5 min and 72°C for 1.5 min, plus a final elongation step at 72°C for 4 min. In each batch of tests, H2O was used as a negative control. HeLa cells  were used as a positive control in both PCR reactions. PCR amplicons were analysed by electrophoresis  and identified under UV light. To minimize the risk for contamination we used different pipettes and rooms for DNA extraction, preparing the PCR solution, adding DNA samples to PCR solution and electrophoresis analysis.The DNA samples extracted from the participants' oral rinse specimens were analysed by PCR for the presence of HPV with the general mucosal HPV primer GP5+/GP6+ http://blast.ncbi.nlm.nih.gov).The HPV positive PCR products were purified with the Agencourt® AMPure PCR purification kit (Agencourt Bioscience) in a magnetic 96-ring SPRIplate®. The sequencing reaction contained the purified PCR products together with 3.25 μM of primer and BigDye Terminator (Applied Biosystems). The sequencing reaction was performed in an Eppendorf Mastercycler Gradient PCR machine. After an initial step of 96°C for 2 min, 20 cycles followed, each with 96°C for 10 s, 50°C for 5 s and 60°C for 2 minutes. The sequence reactions were purified with the Agencourt® CleanSEQ dye-terminator removal kit (Agencourt Bioscience) in a magnetic 96-ring SPRIplate®, and purified sequence reactions were analysed with an automated DNA sequencer (ABI model 3100). The DNA sequences obtained were compared with available sequences in GenBank through the BLAST server .We compared HPV positive with HPV negative individuals to identify characteristics associated with HPV positivity. For comparisons, we used the t-test for normally distributed continuous variables and for categorical variables we used the χ2 test (or Fischer's exact test when the expected number in any cell was <5). Odds ratios (ORs) and 95% confidence intervals (CIs) were obtained using logistic regression models. For some exposures, categories were combined to permit effects to be estimated. All significance tests were two-sided tests at α = 0.05. We used SAS (version 9.2) for all analyses.The mean age of the 307 participants was 22.0 years (range 18–35) and 62% were female. The majority of students  had had some type of sexual contact  in their lifetime. Of the students who reported prior sexual activity, 82% (n = 225) had engaged in oral sex, and 78% (n = 214) had had sexual intercourse. Thirty-two percent of the participants  had received at least one dose of the Gardasil® vaccine. Seven of the 307  students tested positive for oral HPV infection. All the positive samples had single HPV types detected. The most prevalent HPV type was HPV-18 which was identified in three of the seven oral HPV positive individuals (43%). HPV-16, -67, -69 and -90 were identified in one sample each. All samples tested positive for β-globin.We asked if the participants thought it was possible to get HPV from engaging in oral sex and 58% answered yes, 3% no, 8% not impossible, but rare, and 31% were not sure. We also investigated the knowledge of protection from STIs when engaging in oral sex and the majority of students (91%) thought that use of male condom would be protective. et al on 5,579 individuals, reported an overall prevalence of 6.9% Here we investigated the prevalence and determinants of oral HPV infections in a sample of young Australians. We obtained DNA samples from oral rinse samples and used PCR primers (GP+) to detect a wide spectrum of α-papillomaviruses The most commonly detected HPV type in our study was HPV-18 detected in three of the seven positive samples. HPV-16, -67, -69 and -90 were isolated in one sample each. HPV-16 and -18 are both common high-risk HPV types, while HPV-67, -69 and -90 are less commonly reported types isolated from vaginal intraepithelial neoplasia and cervicovaginal cells We found that oral HPV infection was significantly associated with male gender. This has also been reported in the largest oral HPV infection population-based study to date, with 10.1% in men and 3.6% in women Receiving oral sex was strongly associated with oral HPV infection in our population, but we found no associations with passionate kissing, giving oral sex or sexual intercourse (number or partners or frequency). Several other studies have found oral HPV infection to be associated with oral sex behaviours, number of partners as well as frequency Self-reported HIV diagnosis was found to be associated with oral HPV infection. HIV infection has previously been linked with increased odds ratio of prevalent oral HPV infection Some U.S studies have reported strong associations between marijuana use and oral HPV infection, both in healthy populations and HNSCC patients About one third of the participants of our study had received one or more doses of the Gardasil® vaccine, and the great majority of these students were females (95/98). Only one study has evaluated the effect HPV vaccination has on oral HPV prevalence, using the bivalent HPV-16/-18 Cervarix® in a Costa Rican population and found that oral HPV prevalence was much lower in the vaccinated population after 4 years follow-up Strengths of the current study include the collection of detailed lifestyle and sexual behaviour information from all participants. Also, a sensitive technique was used for determining the presence of α-HPV DNA in the oral rinse samples. However, while the sample was relatively large, the prevalence of oral HPV was low, and our study was underpowered to fully explore associations with all possible explanatory factors. While few of the associations were statistically significant, the magnitude and direction of the associations are plausible with current knowledge, and should be examined in larger datasets or through meta-analysis. In addition, our sample was recruited from a single tertiary education institution, and thus while our findings cannot be generalised to all Australians in the same age group, we believe they are likely to reflect the experiences of those in similar settings.In conclusion, we found that oral HPV prevalence was low in this sample of young Australians. Oral HPV infection was found to be associated with male gender, receiving oral sex and self-reported diagnosis of HIV. Given the low HPV DNA prevalence in this study, larger studies are required both in Australia and internationally to determine oral HPV infection in the general population.