P < 0.01) in these three lines. Among the identified proteins, prostate stem cell antigen (PSCA) was upregulated and eukaryotic elongation factor 1 alpha (EEF1α) was downregulated in the HPV+ cell lines. Immunofluorescence and western blotting analyses confirmed these results. Moreover, PSCA and EEF1α were differentially expressed in two clinical series of 50 HPV+ and 50 HPV− oral cavity carcinomas. Thus, our study reveals for the first time that PSCA and EEF1α are associated with the HPV-status, suggesting that these proteins could be involved in HPV-associated carcinogenesis.Human papillomavirus (HPV) was recently recognized as a new risk factor for head and neck squamous cell carcinoma. For oropharyngeal cancers, an HPV+ status is associated with better prognosis in a subgroup of nonsmokers and nondrinkers. However, HPV infection is also involved in the biology of head and neck carcinoma (HNC) in patients with a history of tobacco use and/or alcohol consumption. Thus, the involvement of HPV infection in HN carcinogenesis remains unclear, and further studies are needed to identify and analyze HPV-specific pathways that are involved in this process. Using a quantitative proteomics-based approach, we compared the protein expression profiles of two HPV+ HNC cell lines and one HPV− HNC cell line. We identified 155 proteins that are differentially expressed ( Head and neck cancers (HNCs) constitute a heterogeneous group of tumors that often arise in the oral cavity, oropharynx, hypopharynx, and larynx. HNC is the sixth most common cancer, with as many as 466,831 new cases diagnosed in men in 2008 [Although the relationship between HPV infection and patient prognosis seems clear in oropharyngeal carcinoma, this relationship is less evident in the other anatomical sites affected by HNC, such as the oral cavity, larynx, and hypopharynx. The meta-analysis performed by Ragin and Taioli, which examined the relationship between HPV and overall survival, did not show any differences between HPV+ and HPV− patients with cancers at nonoropharyngeal sites . RecentlProteomic analysis represents a promising approach for identifying HPV-related signaling pathways. However, a paucity of literature exists regarding the biology of HPV-mediated head and neck tumors. A small number of proteomic studies have been conducted, and these investigations have identified HPV-specific protein candidates in HNC. Additional proteins with altered expression levels were previously identified using 2D electrophoresis followed by mass spectrometry. S100A8, a calcium-binding protein, is a powerful biomarker of HPV18 infection in oral SCC patients  and is iHere, we used a quantitative proteomic-based approach to visualize major changes in protein expression between HPV+ and HPV− HNSCC cell lines. Among these proteins, we selected two candidates to validate our proteomic approach and studied their involvement in the carcinogenesis of HPV+ head and neck cancers. To this end, we performed immunohistochemistry on two clinical series  to support our results. In summary, this study aimed to establish a proteomic signature of HPV infection in head and neck cancer in order to better understand the mechanisms by which HPV drives head and neck carcinogenesis.2 atmosphere. The FaDU and 93VU-147T cell lines were grown in Dulbecco's Modified Eagle Medium  supplemented with 10% FBS, 2% L-glutamine, and 1% penicillin/streptomycin at 37°C in a humidified 95% air-5% CO2 atmosphere. The culture medium was changed three times each week, and the cells were passaged when they reached 90% confluence. The cell lines used in this study, which were derived from head and neck squamous cell carcinomas, are described in For total protein extraction, cells were washed twice in cold PBS and centrifuged, and the cell pellets were stored at −80°C. Protein extraction was performed using 6 M guanidinium chloride . The solution was then ultrasonicated for 3 × 10 sec  and incubated for 20 min at room temperature. The supernatant was recovered by centrifugation , and the protein concentration was determined according to the Bradford method, using bovine gamma-globulin as a standard.The proteins were reduced, and their cysteines were alkylated using an ICPL kit (SERVA). The proteins were recovered via acetone precipitation and digested into peptides using trypsin at an enzyme/substrate ratio of 1 : 50 overnight at 37°C. The next day, trypsin digestion was stopped by adding 0.1% formic acid.μg) were separated on a 25 cm C18 column  using a linear gradient (5–35% over 120 min) of acetonitrile (ACN) in water containing 0.1% formic acid at a flow rate of 300 nL min−1. To obtain the highest possible retention time stability, which is required for label-free quantification, the column was equilibrated with a 10× volume of 5% ACN before each injection. Mass spectra (MS) were acquired across 400–1500 m/z in high-resolution mode with a 500 msec accumulation time. The precursor selection parameters were as follows: intensity threshold 200 cps, 50 precursors maximum per cycle, 50 msec accumulation time, and 15 sec exclusion after one spectrum. These parameters led to a duty cycle of 3 sec per cycle, ensuring that high-quality extracted ion chromatograms (XICs) were obtained for peptide quantification.Protein identification and quantification were performed using a label-free strategy on an UHPLC-HRMS platform (Eksigent 2D Ultra and AB SCIEX TripleTOF 5600). The peptides  was used to conduct a database search against the UniProt Trembl database (09/30/2011 version), which was restricted to P value lower than 0.05 across the 3 biological replicates analyzed were taken into account for metabolic characterization. Fold changes were assessed using Student's t-test. Finally, proteins identified with 1 peptide were validated manually.For peptide quantification, PeakView was used to construct XICs for the top 5 peptides of each protein identified with an FDR lower than 1%. Only unmodified and unshared peptides were used for quantification. Peptides were also excluded if their identification confidence was below 0.99, as determined by ProteinPilot. A retention time window of 2 min and a mass tolerance of 0.015 m/z were used. The calculated XICs were exported into MarkerView, and they were normalized based on the summed area of the entire run. Only proteins presenting a fold change higher/lower than 1.5/0.6 with a 5 cells/well in 12-well plates containing sterile round glass coverslips and grown at 37°C and 5% CO2 for 5 days. The cells were washed with PBS and fixed with 4% paraformaldehyde for 15 min. The fixed cells were rinsed with PBS, permeabilized with 0.1% Triton X-100 in PBS for 15 min and blocked with 0.05% casein for 20 min. Then, the cells were treated overnight with primary antibodies against PSCA  and EEF1α (anti-EEF1A1 rabbit antibody (N-term), Abgent, Huissen, The Netherlands), which were diluted 1 : 50 in blocking solution. The next day, the cells were washed with PBS containing 0.1% Triton X-100 and incubated with Alexa Fluor 488-conjugated anti-rabbit IgG  for 1 h. The cells were washed with PBS containing 0.1% Triton X-100 for 15 min, rinsed with distilled water for 10 min and mounted with Vectashield Mounting Medium containing DAPI (Vector Laboratories). The cells were observed by confocal microscopy using an Olympus FV1000D laser scanning inverted microscope . The exposure time of each photo was 27.59 s/frame, pictures were captured at 1600 pix/1600 pix, and the pixel time was 10.0 μs/pix. The background noise was adjusted in the same manner and to the same level for each picture. Each picture was analyzed semiquantitatively.Cells were seeded at a density of 5 × 10μL 20× reducing agent (Fermentas) were added to each protein extract, and the sample volume was brought to 20 μL with deionized water. The samples were heated at 95°C for 5 min, and 30 μg of proteins was separated on 4–20% Mini Protean Gels . After electrophoresis, the proteins were electrotransferred onto nitrocellulose membranes . Nonspecific binding sites were blocked by incubation with PBS containing 5% nonfat milk at room temperature for 1 h. Immunodetection was performed overnight at 4°C using anti-EEF1α (anti-EEF1A1 rabbit antibody (N-term), Abgent, Huissen, The Netherlands) and anti-PSCA  antibodies, which were diluted 1 : 100 in PBS containing 2% nonfat milk. The membrane was washed three times with PBS and incubated for 1 h at room temperature with HRP-conjugated goat anti-rabbit IgG , which was diluted in PBS containing 2% nonfat milk. The bound peroxidase was detected using the SuperSignal West Femto kit (Roche), and the bands were visualized by exposing the membranes to photosensitive film .Proteins were extracted from cells using BugBuster Protein extraction reagent , and the protein concentrations of the extracts were determined using a Bio-Rad protein assay . Four microliters 4× LDS sample buffer  and 1 We examined 100 formalin-fixed, paraffin-embedded oral SCC specimens obtained from patients who underwent radical curative surgery between January 2004 and December 2008 at Saint Pieter's Hospital (Brussels) or the EpiCURA Center (Baudour). The tumors were classified according to the TNM classification of the International Union Against Cancer. β-globin levels were assessed using real-time quantitative PCR to verify the quality of the DNA in the samples and measure the amount of input DNA.HPV detection and typing of paraffin-embedded tissues were performed as described in our previous work . DNA extμm thick sections mounted on silane-coated glass sides. The paraffin-embedded tissue specimens were deparaffinized in toluene, soaked in ethanol, and then soaked in PBS. They were pretreated in a pressure cooker (11 min for PSCA and 6 min for EEF1α) in a 10% citrate buffer solution (for EEF1α) or a 10% EDTA solution (for PSCA) to unmask the antigens. Then, the sections were incubated in 0.06% hydrogen peroxide for 5 min to block endogenous peroxidase activity, rinsed in PBS, blocked with Protein Block , and incubated at 4°C overnight with rabbit anti-PSCA  or anti-EEF1α . The next day, the tissues were incubated with Post Blocking Antibody for 15 min, followed by PowerVision  for 30 min. The slides were washed with PBS between incubation steps. Finally, the localization of the antibody/antigen complex was visualized by staining with DAB , and the sections were counterstained with Luxol Fast Blue and mounted with a synthetic medium. To exclude antigen-independent staining, controls, for which the incubation step with the primary antibody was omitted, were examined. In all cases, these controls were negative.All tumors samples were fixed for 24 h in 10% buffered formaldehyde, dehydrated, and embedded in paraffin. Immunohistochemistry was performed on 5 α immunoreactivities in all tumor areas using an optical microscope . The mean intensity (MI) was defined as follows: 0 (negative), 1 (weak), 2 (moderate), and 3 (strong). The percentage of immunopositive cells  was categorized as follows: 0 (0% positive cells), 1 (1–25%), 2 (26–75%), and 3 (76–100%). Statistical analysis was performed using the Mann-Whitney test to compare the MI and LI values between the HPV+ and HPV− samples.Two independent investigators, who were blinded to the clinical details of the patients, assessed PSCA and EEF1Protein profiling using label-free quantification was conducted to identify proteins whose expression was altered by HPV infection. To elucidate the specific effects of HPV in head and neck carcinogenesis and identify potential candidates, we compared the differential patterns of protein expression between one HPV− cell line (FaDU) and two HPV+ cell lines (93VU-147T and UPCI-SCC90). Proteins extracts were analyzed in triplicate for each cell line using tandem mass spectrometry.For this analysis, we were interested in proteins that had increased or decreased expression levels and are clinically relevant.P values of <0.01; 56 of these were downregulated, and 99 were upregulated (α (EEF1α) (Accession number: Q6IPS9) expression was four fold higher in the HPV− cells than the HPV+ cell lines. Its upregulation was recently reported to be associated with increased cell proliferation and oncogenic transformation.Analysis of the three cancer cell lines identified 2221 proteins, among which 155 were differentially expressed between the HPV− and HPV+ cells with significant egulated . Two intα by immunocytochemistry in six head and neck cancer cell lines: 3 HPV+ cell lines  and 3 HPV− cell lines . The results of the immunofluorescence analysis of PSCA in all cell lines are presented in To confirm our mass spectrometry results, we studied the expression of PSCA and EEF1α between HPV+ and HPV− cell lines. EEF1α was primarily nuclear, but it was also diffuse throughout the cytoplasm. We also noted a marked difference in the expression of this protein in both cell populations (HPV+ and HPV−). In fact, as expected, confocal microscopy examination of EEF1α revealed an increase in the intensity of the immunofluorescence signal in the HPV− cells  in terms of the labeling index (LI), which corresponds to the percentage of immunopositive cells.Among the 50 HPV+ cases, qRT-PCR targeting 18 HPV subtypes revealed that 100% of the cases were infected by HPV-16, with two coinfections, HPV-53 and HPV-39. After confirming our results α expression in the same clinical series (50 HPV+ OSCCs versus 50 HPV− OSCCs). EEF1α was localized in both the nucleus and cytoplasm, but significantly stronger staining intensity was observed in the nucleus  values between the HPV+ and HPV− tumors was calculated using a nonparametric Mann-Whitney test (P = 0.03)  .Recent advances have been made in our understanding of the relationship between head and neck carcinogenesis and HPV. Strong evidence indicates that HPV+ HNSCC comprise a subclass of tumors with a different biology and different clinical properties and that affects specific demographic populations. HPV+ tumors occur in a younger age group, originate more frequently in the oropharynx, and have a lower T stage compared to HPV− tumors . At the Despite the progress made in the field of HPV-related HNSCC, a paucity of literature exists with respect to studies investigating the biology of HPV infection in head and neck carcinogenesis. Disease predictors are important from both the clinical and molecular perspectives. Current HNSCC treatments are frequently associated with adverse side effects, and 50% of HNSCC patients die within two years of their initial diagnosis because two-thirds of patients have advanced cancer (stage III or IV) at diagnosis , 20. TheOver the past decade, technological advances have been made in the field of proteomics, leading to the identification of specific proteins that are differentially expressed in tumor and control specimens. Mass spectrometry is undoubtedly the most powerful technology for proteomics. The most current mass spectrometers present high resolving power and mass accuracy, allowing for the detection and quantification of thousands of proteins. Thus, clinical proteomics is a powerful diagnostic and prognostic technology. However, advances in the proteomics field have resulted in publications describing numerous potential cancer markers that must be clinically validated prior to the development of a diagnostic test.P values of <0.01. The strength of our study lies in the clinical validation of our potential candidates. Indeed, there is a limitation in using cultured cells rather than clinical specimens, as the proteomes of cells grown in vitro may not accurately reflect those of in vivo cancer cells. However, if the selected protein candidates are further investigated by immunohistochemistry (IHC) using patient tissue samples, the proteomic analysis of cultured cells is entirely valid for the identification of putative candidates. Ye et al. identified 40 differentially expressed proteins between three paired oral SCC cell lines with different metastatic potentials. They were able to confirm their results by IHC and, consequently, identified superoxide dismutase 2 (SOD2) as a predictive marker for the diagnosis of metastasis [In our study, we used liquid chromatography coupled to electrospray ionization tandem mass spectrometry to analyze tryptic peptides from three cell lines (2 HPV+ and 1 HPV−). This technology allowed us to identify and quantify 2221 proteins, among which 155 were differentially expressed between the HPV− and HPV+ cells with significant Similarly, we validated several of the differentially expressed proteins between the HPV− and HPV+ populations in our study using three different methods. Immunocytochemistry and western blotting confirmed our mass spectrometry results, and IHC also demonstrated those statistically significant differences in 50 HPV+ and 50 HPV− oral SCC specimens. In fact, HPV+ oral carcinomas overexpressed prostate stem cell antigen (PSCA) compared to HPV− oral carcinomas. PSCA was discovered fifteen years ago. It is a glycosylphosphatidylinositol (GPI)-anchored cell surface protein belonging to the Thy-1/Ly-6 family . PSCA waPSCA seems to be involved in cell growth regulation and to play some roles in signal transduction. Other members of the Ly-6 superfamily are involved in cell adhesion, cell migration, and the regulation of T lymphocyte regulation –30. PSCAα, was overexpressed in the HPV− cell line. EEF1α is a GTP-binding protein that interacts with aminoacyl-tRNA to recruit and deliver it to the A site of the ribosome during the elongation phase of protein translation. In addition to its role in protein translation, EEF1α is involved in cell migration, cell morphology, protein synthesis, actin cytoskeleton organization, and the modulation of apoptosis sensitivity [α has been defined as a putative oncogene [α expression is associated with increased cell proliferation, oncogenic transformation, and delayed cell senescence [Our second candidate protein, EEF1sitivity , 34. Dueoncogene . This proncogene . In contnescence –39. EEF1nescence . Severalnescence , 41.α in head and neck carcinogenesis caused by viral infection, and their functions remain to be elucidated. This study will aid in our understanding of the mechanisms used by HPV to promote the development of head and neck cancers. In conclusion, PSCA and EEF1 meet several criteria, suggesting that they are involved in the biology of HPV-related HNSCC; however, further studies should be conducted to confirm our observations in a larger clinical series. Moreover, it will be interesting to perform functional experiments to understand the signaling pathways disrupted by HPV infection. By silencing several proteins, we plan to study the impact of gene extinction on cell proliferation, migration, invasion, and apoptosis to better understand the mechanisms used by HPV to drive carcinogenesis.To date, no clinical studies have demonstrated the involvement of PSCA or EEF1


Previous studies indicated that oral squamous cell carcinomas (OSCC) might be related to human papilloma virus (HPV) infection. However, up to now, there still lacks a large sample study to analyze the relationship between OSCC in a Chinese population and oral HPV infection. In the present study, we used a meta-analysis to evaluate the relationship of OSCC with HPV infection in a Chinese population.The reports on HPV and OSCC in a Chinese population published between January, 1994, and September, 2011 were retrieved via CNKI/WANFANG/OVID/MEDLINE databases. According to the inclusion criteria, we selected 18 eligible case-control studies. After testing the heterogeneity of the studies by the Cochran Q test, the meta-analyses for HPV and HPV16 were performed using the fixed effects model.P>0.05).The overall positive rates of HPV and HPV16 in OSCC were 58.0%  and 47.47% , respectively; which were significantly higher than those in normal controls 10.44%  and 7.1% . Quantitative meta-analysis revealed that, compared with normal controls, the combined odds ratios of OSCC with HPV or HPV16 infection were 12.7 (95% CI: 8.0–20.0) and 9.0 (95% CI: 5.1–15.6), respectively. Both Begg's test and funnel plots revealed that no publication bias was found in this present study (High incidences of HPV infection (mainly involving HPV16) were found in the samples of Chinese OSCC. For the Chinese population, HPV infection elevates the risk of OSCC tumorigenesis. Prophylactic HPV-vaccination may reduce the burden of HPV-related OSCC in China. As a member of the papillomavirus family of viruses, human papillomavirus (HPV) can infect humans by attacking the squamous cell of skin and mucous membranes. Based on the different nucleotide sequences, HPV can be divided into more than 200 genotypes by DNA sequencing, and 85 HPV genotypes have been well characterized The keywords HPV, human papillomavirus, oral, oral cancer, head and neck cancer, tongue cancer, squamous cell carcinoma, oral carcinoma, buccal cancer, oral lesions, and Chinese population and China in address in Chinese language or in English language were used alone and in combination to search. The retrieved databases included China National Knowledge Infrastructure (CNKI)/Wanfang Database/OVID/MEDLINE. Finally, a total of 361 citations published between Jun, 1994 and Oct, 2011 were identified.The literatures included in the present study meets the following criteria: case-control studies; the participants belong to a Chinese population; a clear description of diagnostic methods was needed and the detection methods were reliable. The literatures excluded in this study were mainly due to the following reasons: lacks normal control group; reviews; the literatures include the repeated data; the research design isn't scientific and reasonable. Study identification and selection is elaborated in the flow diagram . Of 361 The data related to this study were extracted by two independent reviewers (ZCT and LY) based on a standardized data extraction program designed in advance. Any discrepancies were resolved by consensus or in consultation with a third reviewer (DCL). The data related to this study were shown in P value less than 0.05 was considered significant for the heterogeneity. If no heterogeneity, a fixed-effect model was applied using the Mantel-Haenszel method The dichotomous data of HPV or HPV16 positive results in OSCC group and normal control group was summarized. OR and 95% confidence interval [CI] of OR were calculated for assessing the association between HPV or HPV16 infection and OSCC risk. The analysis of the heterogeneity of between-study was performed using the Chi-square-based Q test P<0.05). Therefore, a fixed-effect model was applied. Quantitative meta-analyses showed that, compared with normal oral mucosa the combined odds ratio of OSCC with HPV and HPV16 infection were 12.7 (95% CI: 8.0–20.0) and 8.95 (95% CI: 5.1–15.6), respectively. The test for overall effect showed that the P value was less than 0.00001 (Z = 10.9). Forest plot analyses were seen in P>0.05).The overall positive rates of HPV and HPV16 in OSCC were 58.0%  and 47.5% , respectively; which were significantly higher than those in normal controls 10.4%  and 7.1% .Tests for the heterogeneity showed that, the Chi-square values for HPV and HPV16 were 9.98 and 14.82, respectively  and undifferentiated carcinoma. Oral or mouth cancer most commonly involves the tongue. However, the tumorigenesis of OSCC is complicated. Smoking and other tobacco use are associated with about 75 percent of oral cancer cases There have some elucidated mechanisms of tumorigenesis of OSCC related to HPV infection. HPV viral sequences can integrate into the cellular DNA and the HPV “early" genes such as E6 and E7 can be expressed by host cells and then promote malignant transformation. In addition, E6 has a close relationship with the cellular E6-associated protein, which is involved in the degradation of p53 protein (a tumor suppressor factor can prevent cell growth and stimulates apoptosis in the presence of DNA damage), thereby cause tumors. In vitro studies suggested that the E6 protein of the HPV types implicated may inhibit apoptosis induced by ultraviolet light P<0.01).In 1983, OSCC was firstly reported to be associated with HPV infection The quantitative meta-analysis showed that, compared with normal oral mucosa, the pooled odds ratios of OSCC with HPV and HPV16 infection were 12.7 (95% CI: 8.0–20.0) and 9.0 (95% CI: 5.1–15.6), respectively. While another previous meta-analysis of the included literatures published in English-language journal between 1980 and 1998 revealed that The funnel plots of included studies showed that, the graphics is basically symmetric and all points is concentrated in the central funnel, indicating that no publication bias was found in this study. As a result, we believed that higher HPV infection incidences were found in the samples of OSCC of a Chinese population; HPV infection, mainly related to HPV16, elevates the risk of OSCC tumorigenesis. Therefore, HPV vaccines might also be a potential value in the prevention of OSCC. It is reported that, the HPV vaccines, which prevent infection with the HPV16/18 that cause 70% of cervical cancer, may lead to further decrease However, the previous reports showed that the patients with HPV-positive oropharyngeal cancers had a lower risk of dying or recurrence than do those with HPV-negative cancers However, this meta-analysis may have two potential limitations. Firstly, most of the literatures included the present study didn't consider the potentially interfering factors, such as gender, age, socio-economic conditions, and lifestyle . Especially for lifestyle, some studies have shown that a close relationship was confirmed to be between OSCC and HPV infection. Besides, although OSCC group were diagnosed by pathological check, but lacked the subgroup analysis such as tumor location, clinical stage as well as degree of differentiation and so on. Considering that HPV infection plays an important role in tumorigenesis of OSCC, more scientific studies are worthwhile to do in the future.


Cervical cancer is the greater cause of cancer death in women in many developing countries. Persistent infection with human papilloma virus (HPV), primarily high risk types 16 and 18, is recognized as a causal and essential factor for the development of cervical cancer. We aimed to determine the distribution of high-risk HPV genotypes in archival biopsies with cervical carcinoma in patients from Mazandaran Province, Northern Iran.(R) HPV HCR DNA genotyping kit. The prevalence of HPV infections was assessed in low and high grade cervical lesions by age.A total of 98 paraffin-embedded cervical samples consisted of 63 Squamous Cell Carcinomas (SCC), 4 Adenocarcinomas, 19 Cervical Interaepithelial Neoplasia grade I (CIN-I), 4 CIN-II and 8 CIN-III diagnosed during 2009–2011, were selected to perform high risk HPV genotyping using AmpliSensOf the 98 cervical samples analysed by DNA PCR, 78 (79.59%) were positive for HPV DNA. HPV was detected in the 52 of SCC, 4 of Adenocarcinomas, 14 of CIN-I, 4 of CIN-II, and 4 of CIN-III for HPV. From the 78 HPV positive samples, 23 (29.5%) samples were positive for HPV type 16, 32 (41%) were positive for HPV 18, 19 (24.4%) were positive for HPV 45, and 4 (5.1%) of cervical specimens were positive for HPV 39.This study provides valuable baseline data for future assessment of the impact of current prophylactic vaccination programs that is protective against the two most common oncogenic types of HPV found in cervical cancer, HPV-16 and HPV-18, but not against other high-risk mucosal HPVs, 39 and 45, reported in this population. Cervical cancer is one of the most common types of cancer affecting women worldwide with over 500,000 new cases estimated and over 250,000 deaths each year. The greatest burden of disease is in developing countries, but in spite of better screening and surveillance cervical cancer is still a significant burden also in developed world -3. The rHigh-risk HPV 16 and 18 are found with the highest frequencies in cervical cancer and account for approximately two thirds of all cervical carcinomas worldwide ,13, withThis cross-sectional study, took place during 2009–2011 by approval of the Scientific Ethics Committee of the Mazandaran University of Medical Sciences, Sari. A total of 98 formalin-fixed and paraffin-embedded cervical tissue fragments were retrieved from patients in attendance at Imam khomini Hospital Infections Disease Center, Mazandaran province, Northern Iran. Serial sections (4-7 μm thick) were cut from each specimen. Separate disposable items such as gloves, feather blades and tubes were used to minimise any cross-contamination between samples. The first and last sections were used for histopathological evaluation and intermediate sections were transferred into sterile micro-tube for HPV DNA detection. Additional information was obtained from each patient and collected by means of a questionnaire .(R), Russia). The kit is based on simultaneous amplifying in a one tube (multiplex-PCR) of four types of HPV DNA and allows the user to detect infections and co-infections of high risk HPV genotypes; HPV-16, 31, 33, 35, HPV-18, 39, 45, 59 and HPV-52, 56, 58, 66.PCR amplification for detection of HPV twelve types DNA were run in three tubes. HPV-positive and negative samples were included for every sample run.Sections of paraffin embedded samples in each tube went through deparaffinization with xylene, and rehydration in graded ethanol. Genomic DNAs from tissue section were isolated using the AccuPrep® Genomic DNA Extraction Kit  according to the manufacturer’s guidelines. HPV genotyping was performed using the HPV- HCR Genotype-Eph kit  of abnormal cervical specimens were SCC, 4 (4.1%) were adenocarcinoma, 19 (19.4%) were CIN-I, 4 (4.1%) were CIN-II and 8 (8.2%) were CIN-III. The results of high-risk HPV typing on these samples showed that HPV-DNA was found in 56 (83.5%) of cervical carcinoma specimens. This was reported at a much higher prevalence in Italy (99.3%) , Haifa (The main HPV types detected in our study are HPV 18 in 32 (41%), HPV 16 in 23 (29.5%), HPV 45 in 19 24.4%) and HPV 39 in 4 (5.1%) of the HPV positive cases. Worldwide, approximately 70% of women diagnosed with cervical cancer are found to carry HPV type16 and HPV type18 [.4% and HThe geographical variations in HPV distributions may be due to the difference in the number of samples in each study and also the cultural attributes in some countries. Establishment of distinctive religious and cultural limitations on having multiple sexual partners in countries like Iran can prevent people from being involved in risky sexual contacts, which consequently can lower the risk of HPV infection. On the other hand, limited awareness of Iranian women with respect to HPV and its implication in cervical cancer aetiology, and also lack of educational programmes on HPV can be the reasons for 79.59% of HPV genital infection reported in this study.Previous investigations in different parts of Iran -27 demonThis is in contrast with our findings that highlight HPV type 18 (29.85%) and HPV types 16 (28.35%) as the most prevalent type detected from HPV positive cases and HPV types 45 (19.4%) and 39 (6%) being found to a lesser extent in our cervical carcinoma group. This may be attributable to geographical variation in different parts of Iran that needs to be investigated further.In comparison to previous studies in Iran, this study has provided more detailed information on the pattern of the relative prevalence of high risk HPV infections in low and high grade cervical lesions by age. HPV types 16 and 18 were found in all age groups. This can be a characteristic feature of the population where HPV transmission continues into middle age and may contribute to cervical cancer incidence. Women in this population may also have decreased ability to clear HPV infections, possibly due to age-related reduced immune responses.Our results also showed that the prevalence of HPVs were peaked among women with SCC and fell with decreasing grade lesions to CINs. This observation is consistent with other reports, which highlighted the highest HPV positivity rate in SCC samples, but lower rates of HPV prevalence in other histological classifications ,29.Even though a significant HPV positivity was expected in all cervical samples, our results show there was a contrast in the percentage of HPV detected CIN3 (50%) and SCC (82.5%) compared to other cervical sample types, where the HPV detection rate was 100%. There may be a number of reasons for this, such as sample size and the time length individual samples may have been stored in paraffin before testing occurred. To account for any differences in the sample storage, extremely sensitive HPV detection methods were administered and therefore the results are reflective of what was found using these sensitive methods.In summary, an important strength of this study is that in addition to detection of the most common types of cervical cancer related HPV (type 16 and 18), we have detected other high risk types of HPV DNA, 39 and 45, in samples collected from women with cervical lesions. This, in line with other studies on the prevalence of HPV genotypes in cervical cancer, provides a baseline to assess the impact of current HPV vaccination.The presence of HPV genotypes 16 and 18 in 58.2% cervical cancer in this study will strongly support the implementation of current vaccination against HPV infection (aimed to prevent HPV 16 and 18) in north part of Iran. However, since the protection induced by the current vaccines is predominately type specific, the identification of HPV45 and HPV 39 genotypes in this study can be good evidence for generating a new broad spectrum prophylactic vaccine that can prevent greater than 56% of cervical cancers.There may still be other genotypes of HPV that can be detected in different geographical areas. Therefore, further investigation on HPV genotyping in the wider population would be required.The authors report no competing of interests.MH and GHA were responsible for the overall planning of the study. TGM was responsible for sample collections from the patients. AR participated in study design and helped revise the manuscript. OE carried out the histopathology evaluation of the samples. AH carried out DNA extraction and PCR analysis. MH drafted the manuscript; GHA interpreted results, revised and finalised the writing of the manuscript. All authors read and approved the final manuscript.


High risk human papillomaviruses (HR-HPV) are known to be extremely common, sexually transmitted infections, but more information is needed regarding the absolute risks of type-specific HR-HPV infections in the years following sexual debut.We conducted a survival analysis of 3,737 women aged 18–25 from the control group of the Costa Rican Vaccine trial to determine the absolute risks of HR-HPV infections at 12 months, 24 months, and end of follow-up (average of 50.7 months). To corroborate determinants of infection, we used Cox proportional hazards methods to assess associations between demographics and sexual risk behaviors and incident HR-HPV.Cumulative incidence for HR-HPV infections was 51.3% at the end of the study period. The most common incident types were HPV52 (15.4%), HPV51 (13.6%), and HPV16 (12.4%). Type-specific cumulative incidence corresponded closely with type-specific prevalences, except that HPV16 was more prevalent than predicted by incidence, suggesting greater persistence. The strongest predictors of incident HR-HPV infections as a group in a multivariate analysis were the expected correlates of sexual behavior of the woman and her partner, such as being single  or divorced/widowed , having multiple HPV infections at enrollment , and current smoking . In women who reported being having only one lifetime sexual partner (being in a monogamous relationship), the strongest predictors of HR-HPV included not living with sex partner  and age of sex partner .We confirm the extremely high incidence of HR-HPV in young women, emphasizing the importance of vaccinating young girls before sexual debut. Human papillomavirus (HPV) infections are easily transmitted by sexual contact and are very common among sexually active young women ,2. ApproThe determinants of HPV infection acquisition, all types combined, are better defined. Risk for HPV acquisition is strongly associated with sexual behavior ,3,5. TheCharacterizing more fully the incidence of new HR-HPV infections in young sexually active women would be informative for models of HR-HPV transmission and vaccination impact. The Costa Rican Vaccine Trial (CVT) control arm provides an opportunity to study a large number of recently acquired HR-HPV infections within a group of young women aged 18–25 years old. Such a young and narrow age range enables us to look at the acquisition of new HR-HPV infections, presumably around the onset of sexual activity. The primary purpose of this study was to define the absolute risk of HR-HPV and type-specific HR-HPV infection in our cohort of young women. Secondarily, we wished to add to the already substantial literature on determinants of transmission. We were also interested in confirming or refuting our prior belief that different HPV types  have similar predictors.The study population consisted of all 3,739 women participating in the control group of the Costa Rican Vaccine Trial ). Study design and procedures have been described elsewhere [All participants were administered an enrollment questionnaire by a trained interviewer. The questionnaire elicited information on level of education, marital status, household facilities, menstrual history, sexual, reproductive, and contraceptive history, smoking, and family history of cancer. Among women who reported being in a monogamous relationship, additional questions were asked about their sexual partner including their age, education, circumcision status, sexual history, and smoking history. All study protocols were reviewed and approved by the National Cancer Institute and Costa Rican Institutional Review Boards.10 primers followed by DNA enzyme immunoassay detection of amplimers. HPV typing on positive amplimers was performed using line probe assay (LiPA25). All HPV positive samples that were HPV16/18 negative by LiPA25 were tested by type-specific PCR. Carcinogenic HPV types, termed high-risk HPV (HR-HPV) in this analysis, included 16,18,31,33,35,39,45,51,52,56,58, and 59 . Other HPV types, characterized as low-risk HPV (LR-HPV) included 6, 11, 34, 40, 42, 43, 44, 53, 54, 66, 68, 70, 73, 74, and unknown types. The typing assay does not distinguish HPV68 and HPV73: they were included conservatively as low-risk.Exfoliated cervical cells were collected using a Cervex brush and rinsed into a vial of 20 mL of PreservCyt solution during the pelvic exam. Samples were tested for HPV DNA by polymerase chain reaction (PCR) amplification with SPFFor each individual HPV type, we estimated the 12 and 24 month and overall cumulative incidence. Incidence implied new detection of that HPV type for women who were or became sexually active who were completely HPV negative at enrollment or who were positive only for other types. Months at risk were calculated on the basis of number of months from the enrollment visit to detection of an incident HPV type or at the last follow-up visit, if a woman remained negative for that type throughout the study. Event times were calculated as the midpoint of the intervals between the last HPV type negative and the first HPV type positive visit.Socio-demographic, sexual behavior, contraceptives, smoking and reproductive history characteristics measured at enrollment, as well as prevalent HPV infection and number of prevalent HPV types were evaluated as possible determinants of incident HR-HPV and LR-HPV infection. Continuous questionnaire variables were categorized based on trends in the data when appropriate. The five questions regarding household facilities were characterized as an overall measure of socioeconomic status (SES). Women who had all five household amenities  were considered to have high SES while women who had less than all five were categorized as low SES. The variable, years of sexual activity was calculated by subtracting the women’s age at sexual debut from her age at enrollment. Questionnaire variables that only pertained to women having only one lifetime sexual partner were evaluated separately. From now on these women will be referred as being in “monogamous” relationships.Cox proportional hazards regression models were used to evaluate the relationship between enrollment questionnaire variables and time to incident HPV infection. Variables that were significant at the p ≤ 0.10 level or below in the univariate analysis as well as those that were of particular interest for this study, (i.e. circumcision status and condom use) were considered in multivariate regression models. The final models included variables that were significant at the p ≤ 0.05 threshold and collinearity between covariates was assessed by calculating variance inflation factors for each variable after fitting the mulitvariable regression models. Two women were excluded for having a questionnaire that was deemed unreliable by the interviewer, leaving a total of 3,737 women in the final analysis. All analyses were conducted using STATA .On average, women attended 4.476 screening follow-up visits throughout the study. Total person-months at risk was estimated to be 114,062.8 months for all HR-HPV types (data not shown). Table Table Among women who were in monogamous relationships Table , youngerIn the multivariate model shown in Table To determine if infection with the three most prevalent HR-HPV types influenced the subsequent incidence of other HR-HPV types, we conducted a Cox proportional hazard analysis in which prevalent infection with HPV16, HPV52, or HPV51 was used as a predictor of incident infection with other HR-HPV types. As predicted, we found that risk of incident HR-HPV infection of any type significantly increased if a woman was positive for HPV16, HPV52, or HPV51 at enrollment (data not shown).In the univariate analysis of risk factors for HR-HPV incidence, we observed similar associations for HPV16, HPV51, and HPV52 to all HR-HPV types.Our study confirms the high incidence of HR-HPV among sexually active young women. HPV16 was the most common prevalent type at study enrollment and HPV52 was the most frequently detected incident type, followed by HPV51 and HPV16. A graphical representation of prevalent and incident HPV types confirms a correlation between frequency of prevalent types and incident types, with the exception of HPV16. The high prevalence of HPV16 relative to its incidence might suggest longer persistence. As expected, risk of incident HR-HPV infection was strongly influenced by variables related to sexual behavior. Both HR-HPV and LR-HPV had similar risk predictors for incident infection.The high incidence rates we observed in our study are expected from a population of young sexually active women and are similar to other prospective studies of HPV incidence among young women in the United States, Denmark, Colombia, Brazil, and Canada ,9-12. MaWe did not find evidence suggesting that condom use was protective against HR-HPV acquisition, if anything our data shows an increased risk of HR-HPV infection with condom use, although this association was rendered insignificant when controlled for other measures of sexual behavior. There is conflicting data in the literature, several cross-sectional studies have shown that condom use by male partners does not reduce the risk of HPV infection in women , yet othWe found no association between circumcision of male partners and reduced HR-HPV risk among women in monogamous relationships. The lack of association in our study could be due to reliance on the woman’s report of her male partner’s circumcision status, which may be unreliable. Furthermore, only one-third of the women included in this study responded to this this question, therefore the estimation of male partner’s circumcision stats may have been inflated. Previous studies have shown that male circumcision is protective against HPV transmission from male to female partners ,24. HoweLimitations in our study included the fact that we were only able to use baseline questionnaire data and therefore were not able to evaluate associations between time-dependent variables and risk of incident HR-HPV infection. Specifically, we did not have any follow-up information on a woman’s sexual activity. Therefore, we do not know if women who reported being a virgin at enrollment, later became sexually active during the course of the study. This may bias our estimates of incidence, because we cannot determine whether these women are truly at risk. However, HPV is also transmissible through modes other than nonpenetrative sexual contact, and HPV has been detected among virgins in other studies ,27. The Our study provides good estimates of HR-HPV, LR-HPV, and type specific HR-HPV infections among a large cohort of sexually active young women. We have confirmed the sexually transmissible nature of HR-HPV infections and demonstrated a lack of association between condom use and female-reported, male partner circumcision status and HR-and LR-HPV acquisition. Other risks for HR-HPV infection included high socioeconomic status and smoking history, while lifetime use of birth control pills was protective for incident HR-HPV. Our findings demonstrate that HR-HPV is extremely common and underscore the importance of vaccinating young women before they become sexually active.The authors declare that they have no competing interests. Vaccine was provided for our trial by GLAXOSMITHKLINE Biologicals, under a clinical trials agreement with NCI. GLAXOSMITHKLINE also provided support for aspects of the trial associated with the regulatory submission needs of the company. NCI and Costa Rican investigators make final editorial decisions on this publication; GLAXOSMITHKLINE has the right to review/comment. The funding agency did not have any involvement in the design of the study; the collection, analysis, and interpretation of the data; the writing of the article; or the decision to submit the article for publication.MC: Analyzed data, drafted manuscript. MS: Involved in the conception and design of the study and provided critical revisions of manuscript. ACR: Made substantial contributions to conception and design of study and acquisition of data and provided critical review of the manuscript. SW: Biostatistician, involved in the design of the study and provided critical revisions of manuscript. AH: Made substantial contributions to conception and design of study and acquisition of data and provided critical review of the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/13/308/prepub


Objectives. Consensus guidelines establish that HPV testing should not be used to manage adolescents with atypical squamous cells of undetermined significance (ASC-US). This study aimed to estimate the impact on follow-up of HPV testing after the first-time ASC-US diagnosis. Methods. From January 2009 to December 2010, all ASC-US diagnoses in adolescents were retrieved. Results. 1950 cervical cytologies were received from this population and 335 cases (17.1%) were reported as ASC-US. A total of 287 cases were included in the study. Cases were divided into control group  and case group . On follow-up, in the control group, 43.4% patients had cytology, and 56.6% patients had no follow-up. The case group was divided into negative HPV (60 cases) and positive HPV (181 cases). In the negative-HPV group, 41.7% had cytology and 58.3% had no follow-up. In the positive-HPV group, 41% had cytology, 22% underwent colposcopy, and 37% had no follow-up. Patients with positive-HPV results were more likely to have follow-up than patients in the control and negative-HPV groups . Conclusions. HPV infections are common in adolescents. A positive HPV test cannot predict which women will develop carcinoma. Adherence to current guidelines is recommended in this population. Human papillomavirus is one of the most common sexually transmitted infections in young women with prevalence rates as high as 82% in some populations . The majCervical cancer is extremely rare in adolescents with only approximately 0.2% of cases of cervical cancer diagnosed under the age of 20 ; in our The study was approved by the University of Florida (UF) Institutional Review Board (IRB). Our laboratory receives cervical cytology from community based primary care clinics run by family practice specialized physicians as well as UF-based gynecologists. All gynecologic cytology is processed using ThinPrep  liquid based preparation and HPV test is performed on site using the Digene Hybrid Capture II (HCII) method on residual cytologic material.From January 2009 to December 2010, all cases diagnosed as ASC-US in adolescent patients were retrieved. The inclusion criterion was patients under 20 years of age and first time ASC-US diagnosis. Follow-up data of these cases were collected until 1 year after the initial diagnosis of ASC-US. The one-year period of follow-up was chosen because, according to 2006 screening guidelines (in effect in 2009-2010), adolescents with ASC-US should have repeat cytology at 1 year, and additional tests, including HPV testing, were not expected before that time . The purEnrolled patients were divided into two groups: (1) control group (HPV test was not performed/requested); (2) HPV group (HPV test was performed). This latter group was further divided into positive-HPV group and negative-HPV group. Follow-up within 1 year was classified into three possibilities as follows: (1) repeat cytology, (2) colposcopy with biopsy, and (3) no follow-up.During the study period, 1950 Papanicolaou tests (Pap test) were received from this patient population. Three hundred thirty-five cases (17%) were reported as ASC-US. Two hundred eighty-seven cases (287) met the inclusion criteria, meaning that the patient did not have history of previous abnormal cytology in our electronic medical record system and no history of dysplasia was provided on the pathology requisition. One hundred fifty-nine (55%) of the patients had at least one follow-up within one year. One hundred fourteen (71%) of these patients belong to the positive-HPV group. Seventy-five percent (75%) of patients with ASC-US in which the high-risk HPV test was performed had a positive result (181/241 cases).The control group included 46 cases with ASC-US. As a follow-up, in the control group, 20 patients had repeated cytology (43.4%), 26 patients had no follow-up (56.6%), and no patient underwent colposcopy. One patient was diagnosed with high grade squamous intraepithelial lesion (HSIL) on follow-up cervical cytology. This patient did not undergo colposcopy during the study period. A subsequent Pap test performed one year later revealed ASC-US. HPV test was negative.The positive-HPV and negative-HPV groups included 181 and 60 patients, respectively.In the positive-HPV group, a total of 114 (63%) patients had follow-up. Seventy-four patients had repeated cytology (41%), 40 patients underwent colposcopy (22%), and 67 had no follow-up (37%). Four patients were diagnosed with cervical intraepithelial neoplasia 2 (CIN-2) on follow-up biopsy during colposcopy. Of these patients, one underwent cone biopsy (conization) with the diagnosis of cervical intraepithelial neoplasia 3 (CIN-3), one patient underwent loop electrosurgical excision procedure (LEEP) with the diagnosis of CIN-2, one patient did not received surgical treatment and had a subsequent negative Pap test, and one patient was lost to follow-up.In the negative-HPV group, 25 patients had cytology follow-up (41.7%), 35 patients had no follow-up (58.3%), and no patient underwent colposcopy. No cases of HSIL were found in this group.Invasive carcinoma was not seen in our study during the follow-up period.Data are summarized on Tables Risky behavior is a major cause of morbidity and mortality among adolescents , 3. AccoScreening guidelines published in 2006 established that adolescents with ASC-US should be followed up with annual cytology. In addition, HPV testing should not be used under the age of 21 years and if it is inadvertently performed, a positive result should not influence management , 5. FurtIn 2012, updated guidelines for screening and management of cervical dysplasia were published. These recent guidelines recommend no screening in adolescents. Additionally, since cervical cancer risk remains low through age 25 years, HPV infections are common, and lesions often regress; a less intensive management for young women with abnormal cytology was advised .Some of the reasons behind these recommendations included the current knowledge that HPV infection, ASC-US, and even LSIL are common in adolescents. There is currently good evidence that in adolescents more than 90% of HPV infections, LSIL cytology, and CIN 1 lesions regress within 3 years . TherefoAfter 2006 guidelines publication and dissemination, we expected the use of HPV test in adolescents with ASC-US to be totally discontinued or at least gradually decreased. However, in our study, the majority of ASC-US girls (83.9%) had HPV test performed after first known ASC-US diagnosis. Patients with positive-HPV results were more likely to have follow-up in comparison with the control group and the negative-HPV group . Only patients with a positive-HPV result underwent colposcopy.Based on the results from the case group (HPV tested patients) on follow-up, the positive predictive value (PPV) of a positive-HPV test for significant dysplasia (moderate or severe dysplasia) in our population was only 3.5% and 0% for invasive carcinoma. For statistical analysis, the three groups were compared using 95% confidence intervals calculated using SPSS 17.0 . Results showed that there was a significant difference in follow-up between the HPV-positive group and the other two groups. No difference was detected between the control and the HPV-negative groups . This maAlthough five patients were detected with HSIL, CIN 2, and CIN 3 during the study period , there iIn spite of the discussion, it is valid to mention here that although HPV testing and cervical screening are currently not recommended in adolescents, guidelines are developed to assist clinicians in patient management; however they may not apply to all clinical scenarios , meaningDue to the results of the study and after the 2012 guidelines were published, our department joined efforts with the Department of Obstetrics and Gynecology (OB-GYN) and started an educational campaign targeted to educate community physicians about the adequate use of cytology and HPV and promote the use of current guidelines while treating adolescents. Additionally, our cytology requisition form was changed to modify the HPV options and accommodate current accepted guidelines. A prospective study is currently undergoing to evaluate the impact of 2012 guidelines and our efforts to disseminate them (data not available).Since low- and high-risk HPV infections are highly prevalent in adolescents and are commonly transitory, HPV testing is of little predictive value and should not be used to decide the management of adolescent with ASC-US cytology. An abnormal result cannot determine which patients currently may have or will develop cancer in the future and can result in more harm than benefit for the patient. Overuse of diagnostic and therapeutic procedures at this young age may increase morbidity and compromise reproduction in the future due to cervical incontinence. Our experience highlights a significant health care problem that is probably not limited to our setting. Education and adherence to guidelines will help reduce health care costs and emotional and physical patient harm without compromising early cervical cancer detection and treatment.


Extreme environmental conditions can give rise to extreme adaptations. We document growth, sexual maturation and fecundity in two species of African annual fish inhabiting temporary savanna pools.Nothobranchius kadleci started to reproduce at the age of 17 days and size of 31 mm and Nothobranchius furzeri at 18 days and 32 mm. All four study populations demonstrated rapid growth rates of up to 2.72 mm/day . Both species may produce diapausing embryos or embryos that are able to hatch in as few as 15 days, resulting in a minimum generation time as short as only one month. Incubation on the surface of damp peat moss results in high embryo survival (73%) and a high proportion of rapidly developing embryos (58%) that skip diapauses and hatch in less than 30 days. We further demonstrated that rapid growth and maturation do not compromise subsequent fecundity.Our data suggest that both species have the most rapid sexual maturation and minimum generation time of any vertebrate species, and that rapid maturity does not involve paedogenesis. Furcifer labordi) with a post-hatching lifespan of only four to five months . he genus ,25. For N. furzeri , with a known maximum age of 11 weeks  was reported in Neotropical annual fish, Austrolebias nigripinnis[A. nigripinnis is markedly lower than in Nothobranchius[High adult mortality risk experienced by oduction ,2. A notgripinnis. In genebranchius,12,24, wbranchius.N. kadleci female (Pungwe population) . Nothobranchius normally lay 5 to 50 eggs daily and each egg is laid singly [N. furzeri and N. kadleci to elevate their energetic allocation to reproduction if conditions are favorable. Environmental conditions in Nothobranchius habitats vary temporally and spatially [Fertilization success gradually increased from 21 to 63 days  and C. elegans (4 days), but considerably shorter than lab mice (70 to 80 days) [sensu Caswell (2001) [N. kadleci and N. furzeri to produce a large number of generations in rapid succession.In this study we found a proportion of embryos to develop within 15 days at 25.0 ± 0.2°C and Valenzano 80 days) . The cal80 days) . Likewisl (2001)  in the wA further important outcome of our tests of laboratory protocols is that over 50% of embryos skipped all diapauses and developed within a short period (30 days), along with a good survival rate (73% for embryos) for embryos developing on a damp peat moss. This extraordinary short generation time and high embryo survival in the lab makes them ideal model species for aging research .Nothobranchius is strictly determined by filling and desiccation of their habitats and thus strongly linked to the alternation of rainy and dry seasons [Nothobranchius distribution [The natural generation time of wild  seasons ,26. Howeribution ,30 whereN. furzeri and N. kadleci are species with the earliest known maturation schedule and shortest generation time among vertebrates. Their ontogeny is associated with extremely rapid growth rates and is not compromised by pedogenesis as sexually mature Nothobranchius fishes possess all the characteristics of typical adult teleosts. Nothobranchius appear to display high phenotypic plasticity with broad reaction norms for life history traits, representing an adaptation to the unpredictable conditions of seasonal pools to which they are adapted.We demonstrated that Nothobranchius furzeri from Gona Re Zhou Reserve; NF91: Nothobranchius kadleci population code MZCS91; NF108: Nothobranchius kadleci population code MZCS108; NF121: Nothobranchius furzeri population code MZCS121; NF220: Nothobranchius furzeri population code MZCS220; NF222: Nothobranchius furzeri population code MZCS222; NF430: Nothobranchius kadleci population code MZCS430; TL: Fish total length.GLM: Generalized linear model; GRZ: Inbred captive strain of The authors declare that they have no competing interests.RB, MP and MR conceived and designed the study. RB and MP performed the experiments, MR performed the statistical analysis, RB, MP and MR wrote the manuscript. All authors read and approved the final manuscript.Nothobranchius fishes.MP and RB are postdoctoral researchers in MR’s lab working on evolutionary ecology using fish models. They are working, in particular, on the life history evolution in annual


Hand hygiene was introduced in the 850-bed Hong Kong Baptist Hospital (HKBH), using the promotional techniques recommended by the WHO including a formal kick-off signing ceremony, hospital-wide posters, talks and use of role models. Significant improvement in compliance was observed in 2008 from 41% to 58% (p<0.01). Subsequently from 2009 to 2011, it remained below the 55% level in spite of various promotional activities. This might be related to campaign fatigue.To improve hand hygiene compliance with the involvement of ICLNs.HKBH has 99 infection control link nurses (ICLNs) in 26 clinical areas and from these focus groups were formed. These identified four key deficiencies and for each a program was implemented to resolve with the help of the ICLNs:Help your doctors for excellence in Hand Hygiene: ICLNs reverse the low compliance amongst doctors, the accompanying nurses would squirt alcohol hand rub for them during ward rounds.1. Competition for "Speaking Walls" poster: ICLNs helped their own ward to produce self-made posters, as the present reminders were deemed ineffective. It was believed that self-designed posters would have a better effect.2. Identification locations for Point-of-care hand rubs: ICLNs reported that there were strategic locations without hand rub facilities. 22 such locations were identified.3. Hand Hygiene education program and daily checklist for health care assistants (HCAs): This was implemented by the ICLNs for the reduction in compliance for HCAs is the highest.4. After implementing the four programs, the hand hygiene compliance rate increased to 83% in 2012 , which is significant (p<0.01) compared to 2009-2011. The alcohol hand rub consumption showed a similar trend in 2012, increasing from 8.1L per 1000 patient-days in 2011 to 9.1L in 2012.The strategies described could optimize the end users' participation as not only were ideas extracted from the ICLNs but they also helped in implementing their own ideas. New innovations are vital as Hand Hygiene has been introduced now for a long time and campaign fatigue is likely to occur.None declared.


High-risk human papillomavirus (H-HPV) infection is linked to cervical neoplasia but its role in detecting cervical glandular lesions (GLs) is unclear. Carbonic anhydrase IX (CA-IX) is a hypoxic biomarker that is highly expressed in neoplastic cervical GLs. The diagnostic utility of these biomarkers was evaluated by the Gynecologic Oncology Group in Japanese women with a cytological diagnosis of atypical glandular cells.Immunostaining was used to detect CA-IX in a conventional Pap smear. Immunoreactivity of CA-IX was interpreted by a panel of pathologists blinded to the histological diagnosis. Polymerase chain reaction was used to detect H-HPV in a liquid-based cytology specimen.in situ or invasive carcinoma, were observed in 37/88 (42%) of women. CA-IX testing alone (n=88) had a sensitivity of 89, 100 or 73% for SCLs, GLs or significant squamous lesions (SLs), respectively, with a false negative rate (FNR) of 14%. Testing for H-HPV (n=84) had a sensitivity of 65, 53 or 80% for SCLs, GLs or SLs, respectively, with a FNR of 22%. The combination of CA-IX and H-HPV testing had a sensitivity of 97, 100 or 93% for SCLs, GLs or SLs, respectively, with a FNR of 5%. Among eight H-HPV-negative GLs, six (75%) had a diagnosis of lobular endocervical glandular hyperplasia (LEGH).Significant cervical lesions (SCLs), defined as cervical intraepithelial neoplasia , adenocarcinoma The combination of CA-IX and HPV testing improved the diagnostic accuracy. The low rate of H-HPV positivity in the GLs was associated with coexisting LEGH independent of H-HPV. Cervical cancer is the second most common cause of cancer death in women worldwide, with an estimated 510 000 newly diagnosed cervical cancer cases and 288 000 deaths annually . Althougin situ (AIS) and invasive carcinoma. The rate of invasive carcinoma has been reported to be up to 10% ’, replacing the term ‘AGCs of undetermined significance’, thereby classifying glandular cell abnormalities exceeding those typical reactive changes, but lacking features diagnostic of adenocarcinoma, into the following three categories: AGC of unclear cell origin, atypical endocervical cells and atypical endometrial cells  strain(s) is widely accepted to be an important aetiologic factor for cervical cancer , it was observed that virtually all AGC associated with AIS and adenocarcinoma expressed high levels of CA-IX antigen, but this biomarker was rarely detected in the benign cervical cells/cervical tissues. This suggested the possibility that the expression of CA-IX may serve as a useful biomarker for diagnosing AIS and adenocarcinoma , 2000.In 1998, the Gynecologic Oncology Group (GOG), a national multi-institutional clinical trials group, supported by the US National Cancer Institute, conducted a study of women with a cytological diagnosis of AGC. In total, 25 institutions in the United States and 11 institutions in Japan participated in the trial. Here, we report the results from the Japanese cohort. The objective of this study, included in GOG protocol #171, was to determine whether CA-IX expression in a conventional PS is a diagnostic biomarker for a SCL in Japanese women with a cytological diagnosis of AGC and to explore the diagnostic value of H-HPV testing alone or in combination with CA-IX.GOG protocol #171 initiated accrual in the United States in 1998 and in Japan in 2003. The criteria of AGC diagnosis for patient enrollment were based on the 1991 Bethesda System classification and conventional PSs were used. PCR was used to detect H-HPV DNA in corresponding liquid-based cytology specimens. The protocol closed for accrual in both countries in 2005.Women over the age of 18 years, with a referring diagnosis of AGC, who were expected, on a clinical basis, to undergo complete histological evaluation of the cervical transformation zone within 6 months of the initial cytological diagnosis were enrolled. Patients with a history of endometrial hyperplasia and/or carcinoma of the uterine corpus, cervix and vagina; previous or concurrent chemotherapy and/or radiation to the uterine corpus, cervix and vagina; or HIV infection were excluded. All patients received colposcopic examination, cervical biopsy, endocervical curettage and/or an endometrial biopsy as clinically indicated, as well as either loop electrosurgical excision cone biopsy of the cervix with an endocervical curettage, a cold knife cone biopsy of the cervix with/without an endocervical curettage or a hysterectomy within 6 months of the initial cytological diagnosis of AGC. An endometrial biopsy or curettage was obtained in all premenopausal and postmenopausal women, as well as in all patients with a negative cone biopsy of the cervix. Patients with a negative diagnosis after the cervical cone biopsy, but not undergoing a hysterectomy, were to be followed with routine PS screening every 6 months for 2 years. Informed consent consistent with federal, state and local requirements was obtained before enrollment. Before activation, the protocol was approved by the National Cancer Institute, Division of Cancer Prevention and the GOG Human Research Committee, and annually by the Institutional Review Board at each of the participating institutions.After enrollment, two cytology specimens were collected before surgical procedures were performed. At first, a spray-fixed conventional study PS was taken with a spatula and cytobrush and stained according to the Pap method. A second sample was obtained with a sampling device and suspended in 20 ml of the PreservCyt Solution  for H-HPV DNA testing.Histological samples were taken by punch biopsy, endocervical and/or endometrial curettage or loop electrosurgical excision or cold knife cone excision at colposcopy and/or hysterectomy, as clinically indicated. Specimens were fixed in 10% formalin, paraffin embedded, sectioned and stained with hematoxylin and eosin.Hematoxylin and eosin-stained slides of the most abnormal lesions from each diagnostic procedure were reviewed centrally by teams of two pathologists from the GOG Pathology Committee who reached a consensus diagnosis. Disparities were arbitrated by a third GOG pathologist. A positive diagnosis, coded as a SCL, reflects the presence of CIN2, CIN3, AIS or invasive carcinoma in the cervix. A negative diagnosis represents the absence of SCLs, including CIN1 and atypia. Atypia was defined as glandular and SLs in which cellular atypia falls short of AIS and CIN1. Evaluation of the entire cervical transformation zone was required to make a negative diagnosis, but not to make a positive diagnosis. The significant lesions were restricted to the cervix and there was no case of vaginal dysplasia/neoplasia without the coexisting cervical lesions or lesions outside the uterus identified in the study. Retrospective evaluation of the histology in all cases with the knowledge of the H-HPV and CA-IX status was conducted by one pathologist (SYL). The hematoxylin and eosin-stained sections of all GLs, and those cases that were negative for H-HPV DNA, but expressed CA-IX, were also independently reviewed by a second pathologist (WHR) who was blinded to the clinical data.vs weak/negative) and immunoreactive patterns . Strong positivity represented dark brown immunoreactivity that was easily identified at a low-power magnification ( × 4 or × 10). The diffuse staining pattern was defined as more than 50% of the cytologically atypical or normal endocervical cells in the smear showed immunoreactivity to CA-IX. The patterns A, B, C and D were defined as follows: (A) when individual atypical cells and/or cell clusters showed specific immunoreactivity that was either diffuse or focal; (B) when normal-looking endocervical cells exhibited focal or diffuse strong specific positive staining; (C) when the normal endocervical cells showed focal, but weak staining and; (D) when there was non-specific faint cytoplasmic positivity or lack of staining observed at × 40 magnification . The PCR method used was a minor modification of published procedures . Sensitivity, specificity, positive predictive value and negative predictive value, interpreted as the risk of a SCL among women who test negative for H-HPV and/or CA-IX, and overall accuracy were calculated using the definition of FNR as 1 minus negative predictive value for women diagnosed by CA-IX or H-HPV status, individually or jointly, relative to histological diagnosis. When used in combination, the following decision rule was employed: if either CA-IX or HPV was positive, then the case was said to be test positive.n=3) and incomplete histological evaluation of the cervix (n=1). The age distribution of the patients ranged from 29 to 80, with a median age of 46. Details are given in n=37), 15 (41%) harboured a significant SL, including CIN2 (n=3), CIN3 (n=11) and squamous cell carcinoma (n=1), and 22 (59%) had a significant GL, comprising AIS (n=14), invasive adenocarcinoma (n=7) and adenosquamous carcinoma (n=1). Details are presented in A total of 92 Japanese women with a cytological diagnosis of AGC were enrolled in the study. Four women were excluded because of the unsatisfactory study PS  cases showed positive staining for the CA-IX in the conventional study PS. Among these positive cases, 33 (55%) had a SCL, including 11 SLs and 22 GLs. Thus, CA-IX immunoreactivity identified 33 of 37 (89%) SCLs with sensitivities of 73% for SLs and 100% for GLs. The overall specificity was 47%, with a FNR, defined as one minus the negative predictive value, equal to 14%, which indicates that 14% of the negative diagnoses were incorrect. Details are presented in The PCR-based H-HPV genotyping could only be performed in 84 of the eligible Japanese women with a liquid-cervical cell sample. H-HPV DNA was detected in 29 35%) cases. Patient ages ranged from 29 to 71, with a median age of 43 for the H-HPV positive group and 49 for the H-HPV negative group . Among t5% cases.Combined CA-IX and H-HPV testing detected the additional 11 H-HPV-negative cases (two CIN3 and nine GLs). Thus, the overall sensitivity for SCLs, GLs or SLs was 97, 100 or 93%, respectively, with a specificity of 42%, and a FNR of 5% .et al for lobular endocervical glandular hyperplasia (LEGH) (n=3), glandular hyperplasia (n=2), atypia (n=1), CIN2 (n=1) and GLs (n=6) by the panel of pathologists at the GOG central review. Six of 13 cases (43%) with LEGHs had coexistent in-situ (n=3) and/or extremely well-differentiated invasive adenocarcinoma, also termed ‘minimal deviation adenocarcinoma’ (n=3). All 13 LEGHs were HPV negative, but showed positive CA-IX expression in the PS. None of H-HPV-positive GLs had a coexistent LEGH histology. The results of retrospective histological evaluation and the corresponding H-HPV and CA-IX data are shown in Retrospective histological evaluation was conducted on all cases by one of the authors (SYL). The diagnosis was confirmed in all cases but two. Those two cases were entered in the GOG database as CIN3 and adenosquamous carcinoma, respectively, but no SCLs could be identified retrospectively. Surprisingly, when the criteria proposed by Nucci a (LEGH) . All of In agreement with previous studies, a wide spectrum of benign and clinically significant lesions was identified in follow-up evaluation of patients with a diagnosis of AGC enrolled in the Japanese cohort of GOG protocol #171. The rate of significant uterine lesions in published studies of AGC has ranged from 17 to 80% , with ranges of 0–34%  for women with GLs, 5–43%  for those with SLs and 0–23%  for those with invasive carcinomas, with most of the latter being of endocervical or endometrial origin . In thisn=8) identified six cases that were associated with LEGH. The diagnosis of LEGH in each specimen was confirmed by positive immunostaining of the lesion with an anti-MUC-6 antibody (H-HPV DNA detection has been strongly linked with SCLs; however, published data on the utility of H-HPV testing in AGC diagnosis is limited. Our recent data suggested that, in the United States, H-HPV testing is a useful biomarker for identifying SCLs in women with a cytological diagnosis of AGC, and has an overall sensitivity of 97, 100 or 96% for SCLs, GLs or SLs, respectively, and a FNR of 1% (None of the reported cases of LEGH contained H-HPV (Our current study confirms the diagnostic utility of CA-IX in detecting glandular neoplasia of the cervix , 2000. PDespite the relatively low detection rate of CA-IX for SLs, and H-HPV for GLs, the combined CA-IX/H-HPV testing had a sensitivity of 97, 93 and 100% for SCLs, SLs and GLs, with a FNR of 0.05%. If the results of retrospective review were used for data analysis then the combined CA-IX/H-HPV testing had a sensitivity of 100% for SCLs with 0% FNR. Thus, the study concludes that the combination of CA-IX with H-HPV testing significantly improved the diagnostic accuracy in Japanese women with AGC diagnosis; the low rate of H-HPV positivity in the GLs was probably attributed to coexisting LEGH independent of H-HPV; CA-IX testing identified all cases of GLs associated with LEGH; and lastly, most H-HPV-positive specimens contained a single genotype. The replacement protocol, GOG #237, opened to accrual in February 2009 to prospectively test the conclusions reported herein.


Rates of acquisition and clearance of cervical human papillomavirus (HPV) during a 3-year period in women 51 years of age were compared with rates in younger women to provide data on cervical screening for women >50 years of age. Paired, cytologically negative, archived cervical smears taken 3 years apart from 710 women in Nottingham, United Kingdom, were retrieved and tested for HPV infection with polymerase chain reaction (PCR) with GP5+/6+ primers. Seventy-one (21.3%) of 333 women 51 years of age who were HPV negative at baseline were positive 3 years later. This percentage was higher than the corresponding acquisition rates among women 21 (15.2%), 31 (14.1%), and 41 (13.3%) years of age, although these differences were not significant. This retrospective study shows that HPV-negative women >50 years of age can acquire HPV and, therefore, require cervical screening. This approach also allowed us to estimate rates of HPV persistence and clearance during the same 3-year interval for women whose baseline smears were HPV positive.Anonymous data were provided by the Nottingham Cervical Screening Laboratory, which holds computerized details of all cytology smears of women who have lived in Nottingham since 1987. This laboratory also stores all specimens from women for whom routine cervical screenings were conducted by general practitioners in this area. All specimens used for this study were cytologically normal and had been stored for >10 years  but were scheduled for routine destruction.Women eligible for this study were 21, 31, 41, or 51 years of age when a normal baseline smear was taken in 1988. We intended to select a sample of 400 women 51 years of age and 100 women from each of the other age groups to participate in the study. The main entry criterion was a normal cervical smear taken in 1988, followed by a technically adequate subsequent smear (of any cytologic grade) taken from October 1990 to December 1991. The sample for women 51 years of age was supplemented by including women whose baseline smear was obtained in 1989 and who had a subsequent smear taken before July 1992; all eligible women of this age were selected. Women 21, 31, or 41 years of age were selected randomly from the list of participants who met the main entry criteria. For all participants, results from subsequent cytology examinations conducted through December 2002 were provided by the Nottingham Cervical Screening Laboratory, including any cervical biopsy results from which histologic confirmation of cervical intraepithelial neoplasia  or cervical cancer was obtained.When cytology slides were retrieved for HPV testing, all identifiers  were replaced with a unique study number. Only investigators based at the cytology laboratory  had access to clinical data. These investigators did not have access to HPV testing results for specific patients. Another investigator (M.G.) was responsible for linking HPV test results from the same patient with subsequent cytology results and had access to data on screening histories but not information through which patients could be identified. The study was approved by the Nottingham City Hospital ethics committee.Cytology slides were immersed in xylene (40 mL) for 2–3 days to remove coverslips. Cells were then scraped with a sterile scalpel blade into an Eppendorf tube containing ethanol (1 mL) and centrifuged to remove any trace of xylene. DNA was extracted by using Qiagen  DNA extraction kits  , acquisition, and clearance across the 4 ages were compared by using the χ2 test for trend.HPV acquisition was calculated as the percentage of women who were HPV negative at baseline but HPV positive on follow-up (and the converse applied for HPV clearance). Clearance of baseline infections was assumed if the equivalent test at follow-up was negative. Nonsymmetric confidence intervals were calculated to measure acquisition and clearance, as these are more appropriate when observed percentages are low . Of the study sample (N = 710), 11 were subsequently excluded because of a cervical abnormality before their baseline smear. Follow-up smears could not be retrieved for 27 women; the smear was cytologically abnormal in 20 cases, and the follow-up smear could not be retrieved in the 7 remaining cases. Sixteen women were excluded because an HPV result could not be ascertained at either time because of insufficient DNA. The final sample was 656 women. The mean length of time between the baseline and follow-up smear was 3.08 years (range 1.98–3.84 years). For 76% of the sample (n = 499), the time gap was 2.75–3.25 years. The average time gap between the smears did not vary substantially between age groups.Rates of HPV infection in this sample are shown in 2 trend = 3.18, p = 0.07). Clearance of HPV infection was lowest for women 51 years of age, although again, no substantial differences were observed between this rate and those for the other 3 ages, and the trend in clearance rates across the 4 ages was not significant . When analyses for overall HPV acquisition and clearance were conducted again and included the 20 women with abnormal cytology at follow-up, assuming that these were HPV positive, the results for both HPV acquisition and clearance remained similar.Age-specific rates of HPV acquisition and clearance  are shown in 2 trend 0.08, p = 0.77) or HPV18 . Three women  acquired both HPV16 and HPV18 infections at follow-up, after testing negative for both at baseline. Of the 27 HPV16 or HPV18 infections that were found at baseline over all 4 ages, only 3 were still present at the time of follow-up. This finding indicated overall clearance rates were close to 90% for both HPV16 and HPV18  to be misclassified as incident infections, therefore overestimating rates of HPV acquisition. This study relied on archived cervical smears taken a decade earlier to detect HPV infection. While studies have validated the use of this type of specimen by showing agreement between results from archived and fresh specimens from the same women (A less understood but still plausible reason for these results also relates to the use of archived cervical smears; the possibility of cross-contamination of samples at the time of collection could have accounted for false-positive results at the second phase of testing. This contamination could have occurred either where cervical smears were taken or at the screening laboratory during fixation and staining. In a study from Sweden, this risk of cross-contamination was largely excluded when smears with registration numbers adjacent to the HPV-positive study smears were also tested for HPV (This report also contains details of HPV infection 3-year clearance rates for women who tested positive in the first phase of the study. As baseline rates of HPV infection in this sample were 10%–25%, depending on age , few sme–In the United Kingdom, screening intervals were recently lengthened to 5 years for women 50–64 years of age, after an analysis of UK screening data showed that the protective effect after a negative cervical smear persisted longer for older women (


We briefly highlight the growing body of recent evidence linking unprotected oral sex with the development of some types of head and neck cancer in younger patients. These tumours appear to be increasing in incidence although the development of more sensitive methods of HPV detection may be a confounding factor. Usually these cancers present with advanced metastatic disease causing high morbidity and mortality [The incidence of head and neck squamous cell carcinoma (HNSCC) has been increasing over the last 30 years. It is the 6ortality ,2. The pInterestingly however, in the US the percentage of smokers reduced from 42.5% 1965) to 20.9% (2005) with an associated fall in the traditional subset of HNSCC, however the rates of oropharyngeal cancer especially of the tongue and tonsil have increased again over the last 30 years and are also reflected in European studies . This re65 to 20.Viral genes interfere with cell replication control mechanism. Many other viruses are known to predispose to tumours Table . VirusesRecent data have now attributed a human papillomavirus (HPV) aetiology to a subset of head and neck cancers particularly tongue base  and palatine tonsils  ,10. HPV The HPV family of DNA-viruses preferentially infects squamous epithelial cells with over a 100 separate genotypes and more than 40 of which cause genital infections. They represent the most common sexually transmitted disease worldwide with nearly 80% of infection rates in sexually active adults in the US of at least one HPV type by the age of 50 years. The peak prevalence occurs during early sexual activity (adolescence and young adulthood) which declines with age with HPV 16 being the most common . Most peHuman papillomavirus (HPV) as a risk factor was first suggested in 1983 by Syrjänen et al., who noted that 40% of the studied cancers contained histological and morphological similarities with HPV-associated lesions . SubsequWork by D’Souza and colleagues recently showed HPV infection is likely to be sexually acquired  with incEstablishing the evidence for a causal role for HPV in a subset of HNSCC has been compounded by the heterogeneity of the tumours and multiple primary anatomical sites. These studies employed different detection techniques; different sampling methods and different storage procedures. The non-standardization of these multiple variables has confused the issue .Serological and molecular markers of HPV infection are also associated with increased risks of HNSCC. Hansson et al. found a strong association between the detection of high-risk HPV DNA in the oral cavity and oropharyngeal carcinoma  after adjusting for alcohol and tobacco usage .The most common types of infection associated with cancer are HPV-16 . It is eThe pathogenesis of viral induced oncogenesis is presumably similar to cervical cancer development but heterogeneity and the existence of multiple pathways to carcinogenesis is highly likely. This is also likely to reflect a difference in life cycles of the different HPV subtypes in different mucosal locations, with an associated difference in mucosal immune responses . It mustThe high risk human papilloma viruses (types 16 and 18) contain the transforming genes E6 and E7. Although these gene products have a multitude of effects, in essence E6 binds to and inactivates the p53 suppressor gene resulting in tolerance of DNA errors and tumour entrenchment. Whilst E7 binds the RB suppressor gene, causing a loss of control over the cell cycle resulting in continuous proliferation. This interaction between viral derived products and intrinsic cellular proteins inhibits apoptosis in infected cells, enabling them to both survive and undergo continuous growth which inhibits differentiation. This has been found quite readily in oral keratinocytes and epithelial cells .Accumulating evidence suggests that HPV + status is an important prognostic factor associated with a favourable outcome after treatment in head and neck cancers -23. ProsFinally, it may be that the subgroup of head & neck tumours often in younger patients are induced by sexually transmitted viruses and we would advice caution to oral-sex practitioners and promote the use of barrier methods . We would also advocate the early specialist referral of any patient presenting with i.e. persistent unilateral oropharyngeal symptoms or signs (>3weeks) even in the absence of the ‘traditional predisposing factors’  to head & neck squamous cell carcinoma. One would welcome vaccination schedules which include the HPV subtypes associated with head and neck cancer. Unfortunately not all the commercially available vaccines include the subtypes in question although there is some evidence of limited cross-over protection.The authors declare that they have no competing interests.TU, WJ, MA and HR designed the study, carried out the literature research and manuscript preparation. TU, WJ, MA, HR and HS were responsible for critical revision of scientific content and manuscript preparation and review. All authors contributed to conception and design and approved the final version of the manuscript.