The causal interpretation of cervical immune response to Human Papillomavirus (HPV) infection is complex and poorly characterized mainly due to the delicate balance that exists between viral infection, increase of inflammatory cytokines and host risk factors. This study aims to explore the significance of cervical immune mediators associated to cell survival, angiogenesis and interaction with immune response, in predicting the risk to develop HPV-related intraepithelial lesions.A panel of 48 cytokines and growth factors were explored in a selected cohort of 168 immunocompetent women including 88 diagnosed with low (LSIL) or high (HSIL) squamous intraepithelial lesions of the cervix and 80 with normal cervical cytology (NIL). HPV genotyping was performed by Linear Array HPV test and the soluble concentration of 48 immune molecules was analyzed using the Bio-Plex platform.The prevalence of single HR-HPV infection was 30% in NIL and 100% in LSIL and HSIL women. The expression of 13 cytokines, including interleukins IL-6, IL-3, IL-12p40, IL-12p70, IL-16, IL-18, LIF, of chemokines CCL7 (MCP-3), CXCL9 (MIG), CXCL12 (SDF-1α) and of the tropic factors VEGF, G-CSF, M-CSF were significantly associated with the presence of infection, with levels being higher in women with precancerous lesions compared to NIL HPV negative women. Only the growth factor GM-CSF was positively associated with the cytological abnormalities.The ability of HR-HPV to escape from innate immune recognition and to orchestrate the production of specific inflammatory and growth factors, involved in early inflammatory response and in the cell-proliferating phase of intraepithelial damage, was documented in women before the development of cervical lesions. Cervical cancer (CC) is the third most common cancer in women worldwide accounting for 13% of all female cancers in developed countries. Persistent infection with High Risk Human Papillomaviruses (HR-HPVs) is considered the major cause of CC . Most seHowever, the”in vivo” causal interpretation of mucosal cytokines expression is difficult, mainly due to the delicate balance between pro-inflammatory and anti-inflammatory cytokines which is affected by HPV and by host risk co-factors that can influence the progression of carcinogenesis.The aim of this study was to analyze the”in vivo” cervical cytokines profile of HR-HPV infections in the absence of additional cervical cancer co-factors that may drive the cytokine network. Th1/Th2, T-reg, Th17 cells, Th9 cytokines and growth factors linked to cell survival and angiogenesis, were studied in cervical exfoliates positive for single genotype of HPV from a selected cohort of young women living in geographical regions at average incidence for cervical cancer, and negative for sexually transmitted diseases (STD) other than HPV.The study was approved by the Institutional Scientific Board of the Institute for Maternal and Child Health—IRCCS “Burlo Garofolo” of Trieste, Italy and written informed consent was obtained from each participant in accordance with the principles outlined in the Declaration of Helsinki.Cervical cells and cervical-vaginal fluid samples were collected from a selected group of women recruited as outpatients at the Gynaecology Department Service, to detect HPV and evaluate cytokines concentration. Baseline eligibility criteria of enrolled women included: 20–35 years of age, Caucasian origin, no current use of tobacco, alcohol or contraceptives, no hospitalization in the preceding 6 months, no chronic systemic medication, no hematological evidence of autoimmune disorders (negative for antibody (Ab) anti-nucleus and Ab anti-DNA), positive Th1/Th2 response in previous diagnosed infections, not HPV vaccinated, negative for bacterial vaginosis and STD including Chlamydia Trachomatis, Neisseria gonorrhea, Trichomonas vaginalis, Micoplasma Hominis/Genitalium infections, “healthy” on the day of enrollment.Cervical cells were obtained using a cervex brush device , reaching the endocervical canal and touching both the ectocervical area and the transformation zone (T-zone), and transferred in 500 μL of sterile water. The cervico-vaginal fluid was collected using 3 mL of sterile water in a sterile plastic transfer pipette, vigorously flushed several times over the cervix. The material was collected with the same pipette and placed in a test tube on ice. Aliquots (0.1 mL) were prepared immediately and stored at -80°C. Cytopathological analyses were performed according to the Bethesda System 2001 .™ . Briefly, 25 μL of diluted Master Mix were added to each 0,2 mL tube, then 25 μL (50 μL for Trichomonas and Neisseria) of corresponding isolated DNA solution were added to each tube. The temperature profile was: 2 min at 50°C, 2 min at 94°C, 10sec at 94°C, and 40 sec at 60°C, for 50 cycles.DNA was extracted from cervical cell samples using the QIAamp DNA Blood miniKit  as indicated by the supplier, and stored at -80°C. The presence of C. trachomatis, Mycoplasma , Neisseria gonorrhoeae and Trichomonas vaginalis DNA was detected by Real Time PCR (RT-PCR) using a commercial kit . The amplification and PCR product detection were performed by CFX96HPV DNA was extracted from 500 μL of each exfoliated cervical sample, eluted in a total volume of 100 μL using a commercial kit  as indicated by the supplier.HPV detection and genotyping was performed by Linear Array HPV genotyping test (LA) (Roche Diagnostics) using biotinylated primers to define a sequence of 450 nucleotides within the polymorphic L1 region of the HPV genome. The pool of HPV primers is designed to amplify HPV DNA from 37 genotypes, including 20 HR/pHR-HPV types , and to amplify the β-globin human gene as a genomic DNA control. The LA assay was used manually as described by the manufacturer. At least one replicate of the linear array HPV positive (HPV-16) and negative controls was processed within each run.The main outcome of this study was the quantification of cytokines and growth factors concentrations, based on magnetic bead multiplex immunoassays . Luminex multiplex panel technology was used for simultaneous measurement of a panel of 48 analytes including cytokines, chemokines and growth factors .In brief, 50 μL of cervico-vaginal fluid, this amount falling within the linear range of the assay, and standards were added in duplicate to a 96 multiwells plate containing analyte beads. After incubation for 30 minutes at room temperature and washing, the antibody-biotin reporter was added and incubated for 10 minutes with streptavidin-phycoerythrin. The concentrations of the cytokines were determined using the Bio-Plex array reader . The Bio-Plex Manager software optimized the standard curves automatically and returned the data as Median Fluorescence Intensity (MFI) and concentration (pg/mL). This assay has a reported limit of detection of 1–20 pg/ml, depending on the cytokine target .The softwares Stata (v. 13.1) and GraphPad Prism (v. 5) were used for statistical data analysis. For homogeneity of presentation, the values of the cytokines were expressed as medians and interquartile ranges (IQRs). Comparisons between two groups were made using the t-test and the Mann-Whitney test. The Kruskall-Wallis one-way analysis of variance was used to compare more than two groups. When a significant p-value was found (p<0.05), a multiple comparison test was used to determine which groups were different.A total of 168 women (mean age 30 (SD±4) years) who fulfilled the baseline demographic and microbiologic criteria were enrolled in the study. The correlations of HPV infection and genotype distribution with the type of cervical lesion are described in A panel of 48 soluble immune proteins was measured in the cervical fluids for the evaluation of their expression profile in response to HR-HPV infection. The cytokine profiles of HPV-positive samples were compared to HPV-negative samples. A pattern of 22 molecules was found to be statistically associated with HR-HPV infection (p<0.001) while the remaining 26 did not differ significantly. The concentration of the molecules, measured as pg/ml, and the degree of statistical significance are reported in vs 90 pg/mL) than those found in HPV-/CYTO- women. When the severity of the precancerous cervical lesions (LSIL vs HSIL) was factored into the analysis , with concentrations that were 2 times higher  promotes the production of cytokines, including interleukins IL-1β, IL-6, IL-8, IL12, Tumor necrosis factor (TNFα) and interferons (IFNs), and creates a powerful pro-inflammatory environment . HoweverAlthough several studies have described the pleiotropic role of soluble factors in driving the progression of cervical intraepithelial lesions , the in In the current study, a number of cytokines and growth factors were found to be significantly associated with HR-HPV infection, regardless of the presence of altered cytology . In keepvs HSIL). A possible explanation could be that the majority of the studies involved this mediator were focused on cervical cancer. Conversely, our study examined the pre-cancer phase including only intraepithelial lesions of low and high grade. During this stage, HPV infected cells have not crossed the basal barrier and appear to be orchestrated by a pattern of induced mediators which are different from those observed in CC [A significant negative modulation was observed for cytokines usually involved in the recruitment and activation of immune cells and key factors during the resolution of an active HPV infection: a down-regulation of IL-9, IL -15 and IFN-γ was observed in all groups included in the study, regardless of the severity of cytological lesions. Growth factors promoting angiogenesis and cell proliferation were found to be over-expressed in infected women and the highest levels of concentration of these factors were measured in women with a cervical lesion. In the present study, the growth factor VEGF, one of the most extensively investigated molecules for its potent pro-angiogenic and mitogenic effect on vascular endothelium in vivo, was found to be significantly over-expressed in women with HPV infection and in women with abnormal cytology but not statistically associated with the severity of the cervical lesions  to induce the migration of antigen presentation cells and cytotoxic T-lymphocytes into tumor lesions ,43,44, iThe novelty of this study consists in the “in vivo” demonstration of the ability of HR-HPV to drive specific molecules of the local inflammatory response towards cell transformation. This process seems to occur independently of the capacity of the transformed-cells to evade the host immune response through molecules that are thought to deregulate the immune response in several tumors, as recently suggested for CXCL12 (SDF-1α) ,45. In tRecent data have suggested that the progression of HPV pre-cancerous lesions depend on both the suppression of cellular immunity, driven by the Th1 response, and the development of the immunosuppressive T-reg profile for neoplastic progression ,47. In aIn conclusion, data from this “in vivo” study demonstrate that HR-HPV influenced the production of specific inflammatory and growth factors which are involved in early inflammatory response, suggesting that they might trigger transformation and immortalization of epithelial cells, thereby establishing the conditions for the onset of cancer.


SALL3) and the expression of SALL3 in cervical cancer to explore the function of this gene in cervical cancer carcinogenesis.This study aimed to investigate the methylation status of the promoter region of spalt-like transcription factor 3 , matched pericarcinomatous tissue samples (n = 23) and normal cervix tissue samples (n = 17). MTT was used to measure the cell viability and proliferation capacity of SiHa and HeLa cells.The methylation status of SALL3 promoter was completely methylated in SiHa cells, unmethylated in C33A cells and partially methylated in HeLa cells. After treatment of SiHa and HeLa cells with 5 μM and 10 μM of 5-Azacytidine (5-Aza), respectively, the methylation level of the SALL3 promoter decreased and observed increase in the degree of unmethylation in a dose-dependent manner. Moreover, the relative expression of SALL3 mRNA increased as the concentration of 5-Aza increased in SiHa (p<0.05) and HeLa (p<0.05) cells. This above-mentioned increase in SALL3 mRNA in SiHa cells was more remarkable than that observed in HeLa cells. Cell proliferation capacity also decreased after administration of 5-Aza to SiHa and HeLa cells (p<0.05). Methylation of the SALL3 promoter was observed in 15 of 23 (65.21%) cervical cancer tissue samples, 15 of 23 (65.21%) matched pericarcinomatous tissue samples and 5 of 17 (29.41%) normal cervical tissue samples (p<0.05). SALL3 mRNA expression was significantly lower in cervical cancer and pericarcinomatous tissues compared with normal cervical tissues (p<0.05). In all cervix tissue samples, HPV infection was positively associated with hypermethylation of the promoter region of SALL3 , and the expression of SALL3 mRNA in HPV-positive tissues was lower than that in HPV-negative tissues (p<0.05).The SALL3 together with HPV involvement inactivated its function as a tumor suppressor and contributed to carcinogenesis in cervical cancer.The aberrant hypermethylation of  Cervical cancer ranks as the second leading cause of genital tract cancer-related mortality among women worldwide, and results in approximately 266,000 deaths each year according to the recent National Comprehensive Cancer Network (NCCN) Guidelines 2015 version)5 version, 4. NeveSALL3) is a member of the SAL family and is located on 18q23. SALL3 encodes a sal-like C2H2-type zinc-finger protein. Mutations in some of these genes are associated with congenital disorders in humans, which indicates their importance in embryonic development[et al[SALL3 was silenced by DNA methylation and that the protein product of this gene (SALL3) directly interacts with the PWWP domain of DNMT3A in human hepatocellular carcinoma (HCC), which suggested that SALL3 acts as a tumor suppressor in HCC. Love et al[SALL3 was recurrently mutated in Burkitt lymphomas. Furthermore, in CD133 (+) colorectal cancer cells (CRC), SALL3 was significantly up-regulated compared with CD133 (-) CRC cells[SALL3 was a potential biomarker for colon cancer[SALL3 is associated with the occurrence of several cancers. However, whether SALL3 is involved in the carcinogenesis of cervical cancer remains unknown.Spalt-like transcription factor 3 . The medium containing 5-Aza was replaced every 24 hours during a 72-hour period.1.0×103/ well SiHa or HeLa cells were seeded into 96-well plates and were cultured with 0, 5, or 10 μM 5-Aza. Throughout a 5-day period, the medium was replaced every 24 h, and the same concentration of 5-Aza was added. A 3--2, 5-diphenyl tetrazolium bromide (MTT) assay was used to assess the proliferative capacity of SiHa and HeLa cells after treatment with 5-Azacytidine according to a standard protocol. Cell viability was measured by the absorbance at 490 nm.3×10Twenty-three cervical cancer tissue specimens, 23 matched pericarcinomatous tissue specimens and 17 normal cervical tissue specimens were obtained from the First Affiliated Hospital of Xi’an Jiaotong University between January 2014 and December 2014. All patients were diagnosed by pathologic examination and none had received chemotherapy or radiotherapy prior to surgery. Every patient was voluntary for this research study and provided written informed consent.The cervical cancer samples were collected from the center of the tumors, while the samples of pericarcinomatous tissues originated 1.0 cm away from the cancerous tissue. Seventeen samples of normal cervix were obtained from the uterine cervix of patients who underwent a total hysterectomy due to benign uterine diseases. The average size of each sample was 0.5 cm×0.5 cm×0.5 cm. After the tissues were dissected, each sample was washed with sterilized PBS and stored at -80°C. All procedures were performed on ice.SALL3: 5’-ATTTTAGAATGGAAGGGAGTTCGTC-3’ (forward), 5’-ATAACCTCCTAAAACTTCCCCGAA-3’ (reverse) for methylation and 5’-ATTTTAGAATGGAAGGGAGTTTGTTG-3’ (forward), 5’-AAATAACCTCCTAAAACTTCCCCAAA-3’ (reverse) for unmethylation. Both of the PCR products were 197 bp. The thermocycler programs were as follows: 95°C for 10 minutes and 40 cycles of 95°C for 30 seconds, the annealing temperature for the methylated primer pairs was 51°C while that for the unmethylated primer pairs was 50°C for 30 seconds, and 72°C for 30 seconds followed by an incubation at 72°C for 7 minutes.The PCR products were separated on a 2% agarose gel, stained with Gelview and visualized under ultraviolet illumination. Each reaction was performed in triplicate.Genomic DNA was isolated from cells and tissues using a TaKaRa Mini BEST Universal Genomic DNA Extraction Kit  according to the manufacturer’s instructions. A total of 500 ng of the extracted DNA was bisulfite-modified with the EZ DNA Methylation-Gold™ Kit . The primer pairs used in the MSP were as follows: HPV-DNA of twenty-three cervical cancer tissue samples and seventeen normal cervix tissue samples were tested by polymerase chain reaction(PCR) and flow-through hybridization. 21 HPV genotypes were qualitatively tested by HPV genotyping test kit according to alkaline phosphatase system,including high-risk types HPV-16,18,31,33,35,39,45,51,52,56,58,59 and 68,low-risk types HPV-6,11,42,43,44 and common types in China HPV-53,66 and CP8304.SALL3: 5’-CAAAGCGAGCTCAGAAACAG-3’ (forward), 5’-CCTGATGCTCCAACTTCAAA-3’ (reverse); GAPDH: 5’-GCACCGTCAAGGCTGAGAAC-3’ (forward), 5’-TGGTGAAGACGCCAGTGGA-3’ (reverse). The reaction conditions were as follows: 95°C for 30 seconds, 40 cycles of 95°C for 5 seconds and 60°C for 30 seconds. We used the cycle threshold (CT) as the representative point. The relative expression of SALL3 mRNA in each group (fold-change compared with control) was calculated using the formula: RQ = 2-△△Ct. Each reaction was performed in triplicate.Total RNA was extracted from cells and tissues using TRIzol Reagent  according to the manufacturer’s instructions. The primers used for real-time quantitative PCR are as follows: p<0.05 were considered statistically significant.All the data were analyzed with SPSS version 18.0.Consecutive data were analyzed by the Wilcoxon rank sum test, Kruskal-Wallis H test or one-way ANOVA analysis. Categorical data were compared by Pearson Chi-Square test. All statistical tests were 2-sided, and differences where SALL3 promoter  to determine the SALL3 promoter methylation status in SiHa, C33A and HeLa cells. The results showed that this region in SiHa cells was completely methylated, whereas this region was completely unmethylated in C33A and partially methylated in HeLa cells  in the promoter . One paiLa cells .SALL3 in cell lines in which the SALL3 promoter is strongly methylated (SiHa and HeLa) after treatment with 5-Azacytidine (5-Aza), which can cause DNA demethylation. We measured the methylation by MSP and real-time Q-PCR. The results showed that the intensity of methylation of the SALL3 promoter in both SiHa and HeLa cell lines decreased, while the unmethylation of SALL3 gradually increased as the concentration of 5-Aza increased (SALL3 mRNA in SiHa and HeLa cells increased significantly in a dose-dependent manner after the administration of 5-Aza (p<0.05); the increased expression that was observed in SiHa cells was more remarkable than that observed in HeLa cells , which dSALL3 in cervical cancer tissues, we performed MSP in 23 cervical cancer tissues, 23 matched pericarcinomatous tissues and 17 normal cervical tissues. As shown in p<0.05)   except tl cancer .SALL3 mRNA in the 23 cervical cancer tissues, 23 matched pericarcinomatous tissues and 17 normal cervical tissues by real-time Q-PCR. Compared with the normal cervix, the average relative expression of SALL3 was lower in cervical cancer tissues (p<0.05) and pericarcinomatous tissues (p<0.01), but no significant difference was observed between the tumor tissues and pericarcinomatous tissues (p>0.05) (We detected the expression of (p>0.05) .SALL3 was methylated, and the other consisted of the tissues where that region was unmethylated. The average relative expression of SALL3 mRNA in the methylated tissues was lower than that in the unmethylated tissues (p<0.05)  . These rSALL3 promoter region in cervical cancer. In cervical cancer tissues, 14 of 18 HPV-positive samples were methylated (77.78%), and 4 were unmethylated (22.22%); 1 of 5 HPV-negative samples was methylated (20.00%), and 4 were unmethylated (80.00%). In normal cervix tissues, 2 of 6 HPV-positive samples were methylated (33.33%), and 4 were unmethylated (66.67%); 3 of 11 HPV-negative samples was methylated (27.27%), and 8 were unmethylated (72.73%). The combined statistical results were shown in p<0.05). In addition, we found a positive relationship between HPV infection and hypermethylation of the SALL3 promoter region in cervix tissues , which dSALL3 mRNA in the HPV-positive tissues was lower than that in the HPV-negative tissues .Our result was consistent with Shikauchi et al[et al[In our study, we have explored the feasibility of detecting hypermethylated chi et al and Yang al[et al.SALL3 methylation-related carcinogenesis. High-risk HPV (hrHPV)-induced immortalization and malignant transformation are accompanied by DNA methylation of host genes. Schütze et al[SALL3 methylation status in cervix tissues, the results demonstrated that genomic hypermethylation and lower mRNA expression in transcription level of SALL3 in HPV-positive cervical cancer tissues when compared with in HPV-negative tissues(p<0.05) and hr-HPV infection positively associated with hypermethylation of SALL3 promoter region ,which was consistent with Schütze and our previous study on cervical cancer cell lines, bringing us new evidence that HPV infection did have participated in the carcinogenesis of cervical cancer. However in our present study, the order of two events—hr-HPV infection and SALL3 methylation occurred and whether they had direct interaction (with E6/E7 or other gene loci) still remains unknown. We’ll keep on searching for the mechanism of carcinogenesis induced by HPV infection and DNA methylation in the following researches.Taken these together, the infection of HPV might participate in the mechanism of tze et alfound thaSALL3 methylated in promoter region, with 5-Azacytidine,an agent could play a role on DNA demethylation on a genome-wide scale. The results suggested that when the concentration of 5-Aza elevated generally, the level of methylation of SALL3 dropped off as well as unmethylated expression increased significantly in SiHa and HeLa(p<0.05). At the same time, the mRNA relative expression of SALL3 in SiHa and HeLa increase also and the increased extent in SALL3 totally methylated cell line SiHa was more remarkable than in HeLa, the SALL3 partial-methylated cell line, which suggested that demethylation of SALL3 could effectively reverse its expression in transcription level. Then, MTT assay showed that after treated with 5-Aza, the cell viability and proliferation of SiHa and HeLa significantly weakened which illustrated a cancer-inhibiting function of SALL3 in cervical cancer cells. In tissue samples, the Q-PCR results suggested that mRNA expression of SALL3 was higher in cancer and pericarcinomatous tissues than in normal cervix tissues(p<0.05), which shared the similar idea with previous studies[SALL3 methylated tissues, the mRNA of SALL3 was higher than in unmethylated tissues(p<0.05).Taken together, the results demonstrated SALL3 hypermethylation contributed to the down-regulation of gene expression in transcription level in cervical cancer.In order to further verify the relationship between methylation and gene expression, we treated SiHa and HeLa, which were strongly s studies. In addiSALL3 was of hypermethylation, and due to this mechanism down-regulated the mRNA expression and accelerated cell growth in cervical cancer tissues and cell lines. High-risk HPV infection as an etiology of cervical cancer participated in SALL3 methylation. This aberrant methylation status on the whole genome scale inactivated its function as a tumor suppressor and contributed to the carcinogenesis of cervical cancer.In conclusion, our research first demonstrated that the promoter of S1 Table(DOCX)Click here for additional data file.


Oral cancer is one of the commonest causes for mortality and morbidity with squamous cell carcinoma being the sixth most frequent malignant tumour worldwide. In addition to tobacco and alcohol, human papilloma virus (HPV) is associated with a proportion of head and neck cancers. As in cervical cancers, HPV types 16 and 18 are the cause of malignant transformation. HPV-positive cancers of head and neck have unique characteristics such as occurrence in a younger age group, distinct clinical and molecular features, and better prognosis as compared to HPV-negative carcinomas. They also possess the potential for prevention by using vaccination. The present review describes in detail the salient features of HPV associated oral squamous cell carcinoma (OSCC), its differences from HPV-negative OSCC, diagnostic features, and recent strategies in prevention and management. Oral cancer has been defined as “a neoplasm involving the oral cavity which begins at the lip and ends at the anterior pillar of the fauces” . SquamouCommonly associated with benign skin and oral lesions, recent reports suggest the involvement of HPV in oral and oropharyngeal cancers . Structure of HPV. HPV belongs to the family Papillomaviridae and is a small, nonenveloped DNA virus having diameter of 52–55 nm. The genome consists of double-stranded DNA bound to cellular histones and surrounded by a protein capsid [n capsid . The eign capsid .zur Hausen, in the 1970s, proposed that cervical cancer and condylomata acuminate may share a common viral etiology, that is, HPV 6–8 [A systematic review of HPV associated head and neck squamous cell carcinoma (HNSCC) found a prevalence of 25.9%. The prevalence was higher in oropharyngeal squamous cell carcinoma (OPSCC) (35.6%) compared to oral (23.5%) and laryngeal (24.0%) SCC . The ethOropharyngeal cancers associated with HPV have risk factors such as more number of sexual partners, oral-genital sex, and oral-anal sex. Marijuana use is an independent risk factor for HPV-positive HNSCC, and the risk increases with the intensity, duration, and cumulative years of marijuana smoking.Marijuana use may have a role in oropharyngeal cancers (OPCs) due to its immuno modulatory effect. Cannabinoids bind to the CB2 receptor of immune-modulatory cells in tonsillar tissue. This causes decreased immune response, reduced resistance to viral infections, and decreased antitumour activity . ToombakHerrero et al.  found thDu et al.  examinedThe oncogenic potential of HPV is because of its ability to incorporate E6 and E7 into the host genome leading to inactivation of the tumour suppressor genes p53 and p16 . E6 protDai et al.  found anThe carcinogenic effect of alcohol, tobacco, and other related carcinogens may be increased by HPV infection. Defective apoptosis, neovascularisation, and cellular immortality are also predominantly associated with HPV infection. Thus, HPV mediated carcinogenesis may be an integrated process due to the interplay of various factors. This led to the hypothesis of “a condemned mucosa syndrome” occurring due to viral infection and the associated cellular as well as genetic changes .The International Agency for Research on Cancer (IARC) conducted a multicenter case-control study of oral and oropharyngeal cancer in nine countries. They found HPV DNA in biopsy tissue of 3.9% of oral cavity cancers as against 18.3% of oropharyngeal cancers. HPV 16 was the commonest type (89.3%) . The effHPV-positive oral cancer occurs in younger age group as compared to HPV-negative cancers with an average age difference of 4–10 years. HPV-positive patients also have higher income and more years of education . The malThe common methods being used in diagnosis of HPV are viral DNA detection with polymerase chain reaction (PCR) or In Situ Hybridization and p16 detection by immunohistochemistry. Amplification of target DNA sequences by PCR followed by hybridization with dedicated probes is the commonest method for HPV detection and genotyping .A recent study used three methods together to determine the HPV influence on occurrence of HNSCCs. They used a combination of p16 immunohistochemistry, Consensus PCR HPVDNA, and In Situ Hybridization to better identify HNSCCs caused by HPV. All the HNSCCs which were HPV-positive by PCR and/or ISH were also p16 positive by immunohistochemistry (IHC), with IHC showing a very high sensitivity as single test (100% in both OSCC and OPSCC) but lower specificity (74% in OSCC and 93% in OPSCC) .Studies have suggested that p16 positivity can be used as a biomarker for HPV associated tumours and also as a prognostic factor in HNSCC . ChaudhaAntibodies to HPV can be detected in the serum and is a measure of the cumulative exposure of an individual to HPV infection . AntibodHPV DNA detection is low in exfoliated cells and saliva as compared to tissue. Immune response decreases in HIV patients and HPV detection rates are said to be higher. Adamopoulou et al.  found thThey are not associated with surface dysplasia or keratinisation.They exhibit lobular growth.They have infiltrating lymphocytes.Basaloid variants are common.HPV-positive tumours are usually well differentiated and the basaloid tumours have good prognosis [The pathologic features of HPV-positive tumours are different from HPV-negative tumours in the following:rognosis .Oral squamous cell carcinoma has significant mortality and morbidity rates despite the enormous amount of research in the subject . Higher Among the molecular markers, aberrations in chromosomes 3, 9, 11, 13, and 17 and tumour suppressor genes like p53 and pRb are implicated . CytokerAscorbic acid, b-carotene, and a-tocopherol are believed to have a protective effect. Ho et al.  found thOne of the methods proposed for prevention of HPV related oropharyngeal cancer (OPC) is vaccination. At present, there are two vaccines available: the HPV 16/18 vaccine Cervarix and the HPV 6/11/16/18 vaccine Gardasil . The CDCThe improved survival outcomes have led to the investigation of reduced radiation dose in HPV-positive head and neck cancers . OngoingHPV-positive head and neck cancers thus differ from HPV-negative tumours in epidemiology, clinical features, molecular profile, and response to therapy. Due to this, identification of HPV positivity is essential in all cases for better management of the patient.


P = 0.0283), in which all the patients in this group were smokers. Therefore, HPV did not change the survival of patients with SCC of the oropharynx in this study, indicating that factors other than tobacco need to be studied in conjunction with it, and the level of tobacco consumption is significantly higher in the group of HPV-negative patients.The purpose of this study was to evaluate the survival of patients with SCC of the oropharynx, according to the presence of HPV and tobacco consumption. A total of 37 patients were followed up for at least 5 years after being diagnosed with SCC of the oropharynx. The biopsy tissue was submitted to the polymerase chain reaction (PCR) and in situ hybridization (ISH) methods for broad determination of HPV presence, to identify the presence of high-risk viruses (16 and 18). 12 of the 37 (32.4%) samples were HPV positive, whereas the two specific types of virus were identified in two samples for HPV-16 and in no samples for HPV-18. We observed no significant effect of the virus in survival analysis, irrespective of tobacco consumption. The level of tobacco consumption was significantly higher in the group of HPV-negative patients ( Considering that the etiological factors most correlated with the development of squamous cell carcinoma (SCC) of the head and neck are tobacco and alcohol consumption, this study evaluated the isolated and associated effect of these two carcinogens, showing that alcohol does not act in an isolated manner, as does tobacco, but the interaction of these habits presents a significant effect on the formation of the tumors . When paSmoking and infection by HPV are the main independent prognostic factors for patients with SCC of the oropharynx, probably because they determine the molecular profile of cancer and, therefore, the response to therapy , 5; howeThe smoking habit is very common in patients with SCC of the head and neck, ranging from 66.6% to 89.4 in the studied population , 7, 8. TP = 0.0003).The correlation between tobacco consumption and the presence of HPV in patients with SCC of the oropharynx indicated that the number of smokers is higher in the group of HPV-negative patients and that after the science of diagnosis 66% of the smoker patients continued with the habit, in contrast with HPV-positive patients. This last one presented a lower level of tobacco consumption, and only 30% of the smoker patients continued with the habit after the science of diagnosis , as in tAs regards the influence of these two above-mentioned etiological factors, tobacco and infection by HPV in the local recurrence of SCC of the oropharynx, the study indicated that the group of HPV-positive patients did not undergo changes in the recurrence of the lesion, according to the consumption of tobacco, or not . HoweverWith regard to the risk for developing SCC of the head and neck in individuals with HPV-16, in spite of Dayyani et al.  having eThe study group was composed of 37 patients with carcinoma of the oropharynx, treated with curative intention at the Oral Oncology Center, UNESP, between 2005 and 2007. The study was approved by the Ethics Committee on Research in Human Beings, of the Araçatuba School of Dentistry (Protocol 2005/00689). The data were obtained from the patients' record chards, and follow-up was performed for at least five years.All cases underwent identical personal interviews regarding gender, age, tobacco smoking, and alcohol drinking. Patients reporting having never consumed at least one cigarette or one drink at a regular monthly basis were considered nonusers. Cumulative doses of tobacco exposure were calculated in terms of pack-years  . Most paβ-globin . A cancer sample was considered HPV-positive after the nPCR method had been performed in triplicate, with the use of the following primers: GP5+ and GP6+ (Life Technologies).The paraffinized tissue samples were submitted to the DNA extraction technique , DNA purity, and quantification test (NanoDrop ND-1000 UV-Vis) and PCR for gene control of human μm were placed on silanized slides (SIGMA) and submitted to the in situ hybridization reaction (ISH), using specific biotinylated probes for HPV 16 and 18 (DAKO). The ISH reactions were accompanied by a positive control (Kit GenPoint-DAKO), negative control , and reaction control, using a normal oral mucosa biopsy.Histological cuts of 5 P < 0.05. The overall survival curves were calculated by the Kaplan-Meier method. Survival was calculated from data at the beginning of diagnosis until death or the last date at which the patient was known to be alive. Statistical significance of the differences between the survival time was determined by the log rank test at a level of significance of P < 0.05.Comparisons were made between the clinical characteristics of the HPV-positive and HPV-negative cancers, using the Exact Fisher or chi-square test, with statistical significance being established at In total, 12 of the 37 (32.4%) tumor tissue samples were shown to be HPV-positive. By means of investigating the two specific types of HPV (16 and 18), two samples (16.6%) were positive for HPV-16 and no sample was positive for HPV-18.P = 0.4870). The male sex was predominant in our study group (89.1%) and 75% of the samples positive for HPV were from patients of the male sex (9/12), with the P value = 0.0908 (Most patients were diagnosed with 45 years of age or older (70.3%), and there was no statistically significance difference between the age of the patients and the HPV status , but this habit was significantly higher in the HPV-negative than in HPV-positive patients (P = 0.0283). The advanced clinical stage was observed in 67.6% of patients at the time of diagnosis (25/37), and the proportion of HPV-positive patients was higher in advanced stage oropharyngeal cancer , when compared to early stage oropharyngeal cancer (4 : 1), however not statistically significant . The Log-Rank test in accordance with viral presence and absence was applied in two ways; in the first test, all the patients were included irrespective of tobacco consumption , when compared with HPV-negative patients 36–50%) , 16, 17;0% , 16, In addition to various studies having affirmed an improvement in the prognosis of SCC of the oropharynx due to the presence of HPV , 9, 16, P = 0.1773).In spite of the survival of smoker patients with SCC of the oropharynx not being benefited by the presence of HPV, as much as the nonsmoker patients were, the authors showed that the HPV-positive smoker patients presented better survival when compared with the HPV-negative smoker patients , 8, 19. P = 0.0283). Certainly, other factors, such as the expression of p16 of the epidermal growth factor receptor (EGFR) and the association of single nucleotide polymorphisms (SNPs), may contribute to the survival results [When evaluating tobacco consumption by HPV-negative patients, studies have shown that this habit is significantly more prevalent in the group of HPV-negative patients , 9 as wa results , 21.According to our study group, sex and alcohol consumption did not present correlation with the presence of the virus; presence of HPV does not indicate a significant improvement in the survival of patients with SCC of the oropharynx, and the level of tobacco consumption is significantly higher in the group of HPV-negative patients.


P=0.008). For the genes CDH13, DAPK1, and RARB, both HPV-positive and HPV-negative OPSCC showed promoter hypermethylation in at least 20% of the tumors. HPV status was found to be an independent predictor of promoter hypermethylation of CADM1 (P < 0.001), CHFR (P = 0.027), and TIMP3 (P < 0.001). CADM1 and CHFR showed similar methylation patterns in OPSCC and cervical SCC, but TIMP3 showed no methylation in cervical SCC in contrast to OPSCC. Methylation status of neither individual gene nor CMI was associated with survival. These results suggest that HPV-positive tumors are to a greater extent driven by promotor hypermethylation in these tumor suppressor genes. Especially CADM1 and TIMP3 are significantly more frequently hypermethylated in HPV-positive OPSCC and CHFR in HPV-negative tumors.Oropharyngeal squamous cell carcinoma (OPSCC) is associated with human papillomavirus (HPV) in a proportion of tumors. HPV-positive OPSCC is considered a distinct molecular entity with a prognostic advantage compared to HPV-negative cases. Silencing of cancer-related genes by DNA promoter hypermethylation may play an important role in the development of OPSCC. Hence, we examined promoter methylation status in 24 common tumor suppressor genes in a group of 200 OPSCCs to determine differentially methylated genes in HPV-positive versus HPV-negative primary OPSCC. Methylation status was correlated with HPV status, clinical features, and patient survival using multivariate methods. Additionally, methylation status of 16 cervical squamous cell carcinomas (SCC) was compared with HPV-positive OPSCC. Using methylation-specific probe amplification, HPV-positive OPSCC showed a significantly higher cumulative methylation index (CMI) compared to HPV-negative OPSCC ( Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide Besides known risk factors such as alcohol consumption and tobacco use, human papillomavirus  has been identified as an independent risk factor for a subset of HNSCC, in particular OPSCC Epigenetic changes are thought to be an early event in the carcinogenesis in various human cancers and could (at least) partly be responsible for the difference between HPV-positive and HPV-negative in molecular and clinical behavior We selected all patients who were diagnosed with a first primary OPSCC at the University Medical Center Utrecht between August 1997 and December 2011. Exclusion criteria were a previous history of HNSCC, histologic abnormalities including inflammation, and dysplastic lesions, leaving 210 patients. Pathological, demographical, clinical, and survival data were retrieved from electronic medical records. Since we used leftover material, no ethical approval is required according to Dutch national ethical guidelines. Anonymous or coded use of leftover tissue for scientific purposes is part of standard treatment agreement with patients in our center μm paraffin blank slides using a scalpel. Tumor cell percentage was at least 30%. DNA isolation was achieved by suspending in direct lysis buffer  and overnight incubation in proteinase K  at 56°C. Proteinase K was then inactivated by heating the lysate to 100°C for 10 min. For further analysis the supernatant was used. DNA content was measured with a spectrophotometer. The DNA concentration of the samples was varying between 43.2, and 1901.9 ng/μL. The resulting DNA was stored at −20°C until use.Tumor and normal areas were identified on HE slides by a dedicated head and neck pathologist (S. M. W.) and corresponding areas were dissected from 5 Human papillomavirus type 16 (HPV-16) tumors were determined according to the algorithm of Smeets et al. For methylation analysis in tumor and control tissue, MS-MLPA  was performed according to the manufacturer's instructions for a set of 24 tumor suppressor genes . The choice for probe mix ME001-C2 was based on a thorough literature search indicating that genes specific for this particular tumor suppressor kit showed frequent methylation in head and neck cancer in association with prognosis and that hypermethylation of some promoter regions were associated with HPV in cancer of the cervix μL were diluted 1:1 in Milli-Q water  and above 1500 ng/μL were diluted 1:2 in Milli-Q water (Merck Millipore).The principle of MS-MLPA has been described elsewhere in more detail MLH1 and RASSF1A, two probes for different CpG islands were available. Mean value of two probes for same gene were used for analysis. Promoter methylation analysis in normal oropharynx and cervix (control) tissue was performed using the same method and tumor suppressor kit.Reaction products were separated by electrophoresis on an ABI 3730 capillary sequencer (Applied Biosystems). Methylation status analysis was performed with Genemapper software v4.1 (Applied Biosystems) and Coffalyser.NET analysis (MRC Holland) software. First intrasample normalization was carried out by comparing the signal of each probe by the signal of 15 internal reference probes in the MS-MLPA kit. The methylation percentage was then calculated for each probe as the ratio between normalized probe peaks from the undigested sample and the corresponding HhaI digested sample. For the genes, According to previous cell line experiments and previous experiences, the cutoff level for promoter hypermethylation was set at 15% Development of cancer of the cervix is causally related to infection with high-risk human papillomavirus, mainly types 16 and 18 n = 10) was added as control. The tissue microarray is cut in 4-μm paraffin sections using standard techniques. Immunohistochemistry was performed using a monoclonal-rabbit CHFR antibody  in a 1:300 dilution and CADM1 antibody  in a 1:10,000 dilution. For all stainings, slides were deparaffinized in xylene and rehydrated in decreasing ethanol dilutions. Endogenous peroxidase was blocked with H2O2 in PBS followed by antigen retrieval by boiling in an EDTA buffer, pH = 9. After a cooling down period of 30 min in the buffers, the tissue slides were incubated with the primary antibodies for 60 min at room temperature. For detection of the primary antibodies the tissue slides were incubated with poly-HRP goat/rabbit/rat . Between these steps, slides were washed with PBS. Finally, peroxidase activity was developed with diaminobenzidine for 10 min, slides were counterstained with hematoxylin, and dehydrated in increasing alcohol dilutions. Normal lung tissue was taken along as positive control.Hematoxylin and eosin (H&E)-stained sections were cut from each donor block and examined by an experienced head and neck pathologist (S. M. W.) to mark representative tumor regions. A fully automated tissue microarray instrument (Beecher Instruments) was used to acquire tissue cores from the donor block from our patient's cohort. From each block, three cores with a diameter of 0.6 mm were subsequently placed in an empty paraffin block. Tissue microarrays included 193 of 210 patients from our cohort. In addition, normal oropharynx , blinded to the clinical characteristics. A core was considered lost or inadequate if it contained less than 5% tumor tissue or when more than 95% of the core contained no tissue anymore. χ2 test (or Fisher's exact when appropriate) for categorical variables and ANOVA for continuous variables were used to compare the frequency of methylation for individual genes between HPV-positive and HPV-negative tumors. The same tests were used to compare frequency of methylation between cervical cancer and HPV-positive OPSCC. Correction for multiple comparisons was applied by resetting the 0.05 threshold to 0.05/24 = 0.002 (Bonferroni correction). The following clinicopathological features were dichotomized: tumor size (T1/2 vs. T3/4), stage (I/II vs. III/IV), nodal stage (0 vs. 1–3). Backward logistic regression was performed to compare methylation in HPV-positive and HPV-negative tumors and to compare HPV-positive and cervical cancer, taking significant differences between the two groups into account. Odds ratios (OR) and 95% confidence intervals (CI) were calculated. Patient survival was examined first using Kaplan–Meier survival curves, and differences between strata were tested using log-rank test. To adjust for additional variables related to patient survival, Cox regression analysis was used. Next, we explored the association between methylation status of CADM1 and CHFR and protein expression using the nonparametric Spearman's correlation coefficient between the mean protein expression levels of CADM1 or CHFR and methylation percentage as continuous variable. Next, we calculated the association between promoter hypermethylation dichotomized  of CHFR and CADM1 and protein expression of these proteins dichotomized (high vs. low) using Pearson χ2 test (or Fisher's exact when appropriate). Finally, we evaluated the relation between HPV status and CHFR or CADM1 expression (low vs. high) using Pearson χ2 test (or Fisher's exact when appropriate). All statistical analyses were performed using IBM SPSS 20.0 statistical software .The Mann–Whitney test was used to compare CMI between HPV-positive and HPV-negative cases and to calculate differences in CMI between HPV-positive OPSCC and cervical cases. Pearson The original group of OPSCCs consisted of 210 patients, however, in 10 cases the amount of DNA was insufficient, leaving 200 cases for further analyses. Of 200 cases stained for p16, 69 were scored as positive. Of 69 p16-positive tumors 43 were confirmed to be HPV-positive by PCR, of which 42 were high-risk HPV. All of the 42 high-risk HPV-positive were HPV16 and two were coinfected with HPV33 or HPV52, resulting in a HPV prevalence of 21%. HPV16 was detected most frequently in base of tongue, tonsillar fossa, and tonsil. Distribution of relevant clinical features between HPV-positive and HPV-negative OPSCCs is shown in Table P < 0.001) and HPV-negative tumors . HPV-positive OPSCC showed a significantly higher (P = 0.008) CMI compared to HPV-negative OPSCC . CMI was dichotomized using the median value (148) of the complete cohort of tumors. After multivariate logistic regression correcting for alcohol use, tumor size, lymph node status, and smoking, HPV presence was significantly associated with a high CMI (>148) (P = 0.004).The results of overall methylation (CMI) are shown in FigureCDH13 (40%), but the latter was still lower in control tissue  compared to tumor tissue . No hypermethylation was observed for BRCA1, BRCA2, HIC1, VHL, ATM, CD44, CDKN1B, and GST1P in both HPV-positive and HPV-negative tumors. Hypermethylation frequencies for CDH13, RARB, and DAPK1 were similar for HPV-positive and HPV-negative patients. Using logistic regression, thereby allowing for the controlling of confounders , patients positive for HPV16 had increased hypermethylated CADM1  or TIMP3 . In contrast, an inverse correlation was found between HPV status and methylation of CHFR . , no history of alcohol abuse (P = 0.007), or smoking (P < 0.001). Hypermethylation of CDH13 was inversely related to distant metastasis (P = 0.022). Hypermethylation of TIMP3 was more often present in patients with no history of smoking (P = 0.036). After correction for multiple comparisons, there was only significant correlation between promoter hypermethylation of CADM1 and smoking.The methylation status of each gene was tested for an association with the clinical characteristics age, sex, tumor size , cervical lymph node status (N), distant metastases (M), clinical stage, alcohol, and nicotine consumption. Promoter hypermethylation status was not associated with lymph node status, sex, or tumor size for any of the genes. Hypermethylation of n = 42) in comparison with cervical SCCs (n = 16). Compared to cervical SCC, we observed a significantly higher CMI in HPV-positive OPSCCs (P = 0.037). The high frequencies of hypermethylation in RARB (40% vs. 38%), CADM1 (50% vs. 44%), and DAPK1 (33% vs. 19%) were shared between HPV-positive OPSCC and cervical cancer. Hypermethylation was however less frequent in cervical cancer in most genes, particularly in CDH13 (P < 0.001) and TIMP3 (P = 0.025). After correction for multiple comparisons, there was no significant correlation between promoter hypermethylation of TIMP3 and tumor location (cervix vs. oropharynx). Univariate logistic regression analysis showed significant differences for alcohol, no other clinical feature was significantly different between the groups. After multivariate logistic regression analysis only methylation status of CDH13  was a strong independent predictor of location of HPV-related SCC.Methylation results could be obtained for 16/18 cervical SCC cases with clinical features as described in Table S2. Normal cervix control samples showed no promoter hypermethylation in the studied genes (Table S2). FigureP < 0.001) and small tumor size (T1–2) (P < 0.001) were correlated with increased 2 year overall survival as expected. Positive cervical lymph node status (N) (P < 0.001) and active smoking (P = 0.035) were associated with decreased overall survival. CMI dichotomized using a threshold of 148 was not significantly associated with patients' outcome. Patients with hypermethylation of CADM1 (P = 0.004) had however increased survival compared to patients who did not have CADM1 hypermethylation. Methylation status of no other individual gene was associated with patient survival. Combination score of HPV-related genes  with a cutoff value of 2 or more showed a significant better overall survival (P = 0.018). In Cox regression, CADM1 methylation, active smoking, HPV status, T status dichotomized, N status dichitomized, and the combined methylation pattern of HPV-related genes were included. CADM1 methylation, active smoking, and the combined methylation pattern of HPV-related genes were not independent prognostic factors. In the subset of HPV-positive and HPV-negative tumors, methylation status of none of the individual genes was associated with patients' outcome.HPV positivity . CADM1 expression was reduced in 89% (158/178) and CHFR expression in 47% of OPSCC. Using the cutoff value of 15% for promoter hypermethylation, the frequencies of CADM1 promoter hypermethylation in this cohort was 16% (28/178) and of CHFR promoter hypermethylation was 18% (33/181). No correlation was found between percentages of methylation and protein expression for CADM1 and CHFR as continuous variables , as well as categorical . Next, we investigated if CADM1 and CHFR protein expression by IHC was related to HPV status. CADM1 expression was not related to HPV status. Reduced protein expression of CHFR was significantly (P = 0.012) related to HPV-positive OPSCC in univariate analyses. To account for potential confounders of these results, we conducted a logistic regression analyses to adjust for baseline characteristics . After multivariate adjustment, reduced CHFR expression was an even more strongly related to HPV-positive OPSCC.For HPV-positive and HPV-negative OPSCC are driven by distinct carcinogenic pathways which are reflected in their diverse clinical behavior DAPK1, RARB, and CDH13 showed promoter hypermethylation in more than 20% of cases, both HPV-positive and HPV-negative tumors, indicating that these genes are often involved in HPV-positive and HPV-negative OPSCC carcinogenesis. These results are in accordance with previous studies evaluating promoter hypermethylation in HNSCC, although methylation rates vary widely in literature MS-MLPA of 24 common tumor suppressor genes resulted in the identification of three genes significantly differentially methylated in HPV-positive and HPV-negative OPSCC and three genes frequently methylated in both tumors. The genes CDH13 was lower in cervical SCC compared to HPV-positive OPSCC, indicating that methylation of the promoter region of this gene is more strongly associated with oropharyngeal region. It has to be noted however that using a cutoff value of 15%, CDH13 methylation was also a common event in control tissue, although at a lower frequency compared to OPSCC cases. In the literature there is no consensus on the threshold value for hypermethylation. Used cutoff values vary between studies ranging from 10% to 20% CDH13, the cutoff value to provide best discrimination between tumor and normal tissue  should be higher. In addition, using a higher cutoff value of 22%  instead of 15%, hypermethylation of CDH13 occurs still often in both HPV-positive and HPV-negative OPSCC (38 vs. 23%) and is not significantly different (P = 0.045) after Bonferroni correction.In addition, the frequency of promoter hypermethylation of TIMP3 and CADM1 showed a significantly higher methylation frequency in HPV-positive tumors compared to HPV-negative tumors. In addition, an inverse correlation was found between HPV16 infection and methylation of CHFR.Hypermethylation of genes in HNSCC in association with HPV status has been studied before. However, most of these studies used a small sample size, investigated a limited number of genes and in only four studies a cohort of tumors from exclusively oropharynx was evaluated in association with HPV status TIMP3 promoter methylation has been described in a wide range of tumors, including kidney, esophagus, colon, and breast cancer TIMP3 is a matrix metalloproteinase inhibitor and therefore able to inhibit growth, angiogenesis, invasion, and metastasis of tumors TIMP3 in a cohort of HNSCC, suggesting TIMP3 is important for HPV-positive OPSCC carcinogenesis. However, in cervical cancer no promoter hypermethylation of this gene was observed in our study, suggesting that methylation of this gene might be location specific.CADM1 methylation in OPSCC. CADM1 plays a role in cell–cell adhesion and reduced expression of this gene is correlated with lymph node metastasis CADM1 was also often methylated in cervical cancer  nor any combination of HPV-related genes that could independently predict overall survival, in the entire cohort or in the subset of only HPV-positive tumors. This may be explained by the fact that in multivariate analysis, HPV status is the strongest independent predictor for survival and promoter methylation of CADM1 is significantly associated with HPV status.This study has several limitations. MS-MLPA cannot distinguish between partially and fully methylated promoter regions, since this method measures methylation for only one CpG site in promoter region. However, for gene silencing not all CpG islands in promoter region have to be methylated CADM1, TIMP3, and CHFR was significantly different between HPV-positive and HPV-negative tumors. However, this is not reflected on protein level suggesting that other  mechanisms regulate the expression of these proteins.In conclusion, our findings suggest that promoter methylation is important in the development of OPSCC, in both HPV-positive and HPV-negative tumors. Promoter methylation was more common in HPV-positive compared to HPV-negative OPSCC. Hypermethylation of the genes


Background. The cytological interpretation of ASC-US represents a category of morphologic uncertainty. For patients with this result, other tests are necessary in order to determine the risk for cervical lesions.Materials and methods. 198 patients with ASC-US cytology have been analyzed between 2008 and 2013. All the patients included in the study have subsequently had a high oncogenic HPV testing and colposcopy risk. 103 (52%) patients tested positive for high risk HPV and 21 (10%) had associated colposcopy changes and precancerous and cancerous lesions identified through biopsy. 95 (48%) patients tested negative for HPV and none of these women had lesions at colposcopy. Results and discussion. High oncogenic risk HPV testing was proven useful in identifying the patients with ASC-US cytology who are at high risk for cervical lesions (100% sensibility). In this study, the HPV testing had a negative predictive value of 100%, which uselessly renders a further colposcopy evaluation. HPV testing for women with ASC-US is not specific in identifying women with cervical lesions (Specificity 53%) and this results from a high prevalence of limited HPV infections in an age group which is less than 30 years old. Conclusions: High risk HPV testing for women with ASC-US cervical cytology is useful in determining the risk for precancerous and cancerous cervical lesions. A positive result is associated with a high risk for cervical lesions (20%) and for these patients colposcopy is necessary. For women with a negative result, the risk for cervical lesions is practically null so colposcopy is not required.Abbreviations: HPV = Human Papilloma Virus, ASC-US = Atypical Squamous Cells of Undetermined Significance, CIN = Cervical Intraepithelial Neoplasia, DNA = Deoxyribonucleic Acid, Pap = Papanicolaou test, LSIL = Low Grade Squamous Intraepithelial Lesion, HSIL = High Grade Intraepithelial Lesion.  Based on solid evidence, regular screening of appropriate women for cervical cancer with the Pap test reduces mortality from cervical cancer. The benefits of screening women younger than 21 years are small because of the low prevalence of lesions that will progress to invasive cancer. Screening is not beneficial in women older than 65 years if they have had a history of recent negative tests. Regular Pap screening decreases cervix cancer incidence and mortality with at least 80% [2-4]. In spite of these good results, cervical cytology has limitations: low reproducibility for ASC-US and LSIL, lack of specificity for precancerous lesions in ASC-US, highly variable results among laboratories, poor performance in detecting adenocarcinoma, frequent screening requirement (every 1 to 3 years) [5-7]. It is now known that cervical cancer is caused by sexually acquired infection with certain high-risk types of HPV, HPV16 and 18 being responsible for 70% of the cervical cancers and precancerous cervical lesions [8]. Recent studies have focused on the importance of high risk HPV testing in screening for cervical precancerous and cancerous cervical lesions. The need for other screening methods rose from the limitations associated with cervical cytology.Cervical cancer is the fourth most common cancer in women, and the seventh overall, with an estimated 528,000 new cases in 2012. In Romania the incidence is estimated at 4343 per 100,00 and the 5 year prevalence at 14834 per 100,000  [9]. For patients with this result, other tests are necessary in order to determine the risk for precancerous cervical lesions. For women with positive high risk HPV test and ASC-US cytology, colposcopy is recommended because they have a 2-years risk of CIN3 and an invasive cervical cancer of 10% . Studies have demonstrated that for women with ASC-US and negative high-risk HPV test, the risk for precancerous cervical lesions is very low, similar to women with negative cytology and HPV test. The absolute risk of CIN3 for women with ASC-US and negative HPV is of 0,28% at that moment and of 0,54% at 5 years . Because this risk is less than 1% for these women, it is recommended to return to routine cervical screening [9].An ASC-US interpretation does not represent a specific cytological interpretation. Because of its morphologically equivocal nature, the inter- and intra-observer reproducibility of an ASC-US interpretation is less than that for the reliable, unequivocal cytological categories of LSIL and high-grade squamous intraepithelial lesion (HSIL) [Table 1).198 patients with ASC-US cytology have been analyzed in Micomi Clinic, between 2008 and 2013. All the patients included in the study have subsequently had a high oncogenic risk HPV testing and colposcopy. 103 (52%) patients tested positive for high risk HPV and 21  had associated colposcopy changes and precancerous and cancerous lesions were identified on biopsy. 95 (48%) patients tested negative for high risk HPV and none of these women had identifiable lesions at colposcopy . In this study, HPV testing had a negative predictive value of 100%, which would uselessly render a further colposcopy evaluation. HPV testing for women with ASC-US is not specific in identifying women with cervical lesions (Specificity 53%) and these results from a high prevalence of limited HPV infections in the age group of less than 30 years old. HPV infections usually clear up without any intervention within a few months after acquisition, and about 90% clear within two years [The statistical significance of these findings is limited by the small number of women included in the study. Also clinicians should exercise caution when extending these findings to the general population as the sample analyzed in this study has a higher economic status being formed by women who attended private medical care. High risk HPV testing for women with ASC-US cervical cytology is useful in determining the risk for precancerous and cancerous cervical lesions. A positive result is associated with a high risk for cervical lesions (20%) and for these patients colposcopy is necessary. For women with a negative result the risk for cervical lesions is practically null so colposcopy is not required and these women can return to routine screening. HPV testing for women with ASC-US reduces the number of colposcopies that require highly trained physicians and can be uncomfortable for women.


Working cell banks (WCB's) are commonly applied to initiate cell culture manufacturing campaigns for production of recombinant or therapeutic proteins. These campaigns typically begin with inoculation of cells previously cryopreserved in cryovials or ampoules. The baseline process proceeds from thaw of cell bank cryovials to scaling up through the seed train and inoculum train, followed by fed-batch production. While cryovials are typically used in the development of WCB's and initiation of manufacturing campaigns, they are not optimal for the growing demands of commercial production.Cryovials are small on average  and filling/removal is performed through an open screw cap. This process results in manual operations and the use of many culture vessels, resulting in the risk of contamination and potential campaign-to-campaign variability. Single-use (disposable) bags have been investigated more recently as a possible solution to minimize open handling steps and to shorten seed train scale-up. Bags offer larger storage volumes, but also come assembled with thermoplastic tubing for sterile connections. Wide-spread adoption of single-use bags for WCB applications has not been observed to this point as currently available tubing and connections can't hold up to the demands (break or can't be welded) when stored/transported at frozen or cryogenic (-196°C) temperatures.To overcome these challenges, novel thermoplastic tubing was developed to balance both the flexibility and robustness demands of cryogenic storage and tube welding characteristics necessary for sterile closed-system processing. The new FP-FLEX™ tubing can be frozen and maintained at cryogenic temperatures, thawed and sterile welded to other thermoplastic tubing (C-Flex®).A selection of different thermoplastic elastomer combinations were investigated in an effort to discover the optimal blend of materials to support the flexibility and robustness requirements during frozen storage and sterile welding needs for processing. A novel manufacturing method was also designed to enable welding of the FP-FLEX™ tubing directly to the single-use bags. This was critical to support a completely unitized, closed design. Studies were then carried out to determine both durability and functional utility of the FP-FLEX™ tubing for frozen storage and processing applications.Testing was carried out using 500mL nominal fill bags having (1/4" OD × 1/8" ID) FP-FLEX™ lines (8" L) and compared to bags having PVC tubing (standard application). For LN2 studies, all bags were filled to 140mL (+/- 5mL) with water and placed into aluminum cassettes prior to storage with tubing attached. Simulated drop tests were performed to test overall durability. Each Bag was pulled from the LN2 tank and immediately dropped horizontally 4X from a distance of 1ft, thawed and evaluated for damage. 10 of 10 bags with FP-FLEX™ tubing were completely intact while 10 out of 10 bags with PVC tubing were broken. A single drop (FP-FLEX™ bags only) from 2ft and 3ft resulted in a similar outcome (Table Finally, post freeze/thaw weld strength and integrity testing was performed to evaluate sterile welding capabilities of FP-FLEX™ tubing post-thaw Table . Tubing WCB's are commonly used for seed train manufacturing of therapeutic products. Traditional vials represent an open manufacturing process and are also limited to small volumes which contribute to lengthened production campaigns. The new FP-FLEX™ tubing has been designed and shown herein to meet the critical processing requirements for WCB's Figure .Freezing/Storage/transport to as low as -196°CWeldable to C-Flex® post freeze/thawCompatible with standard tube welding and sealing devicesClosed-system aseptic transfer via tube-to-tube connectionThe FP-FLEX™ tubing and Freeze-Pak™ bag represent a closed-system solution enabling frozen storage, sterile connection and reduced scale-up time for therapeutic production.


Each year approximately 6,000 new cases of head and neck cancer are registered in Poland. Human papillomavirus (HPV) and Epstein-Barr virus (EBV) have been associated with tumour formation. Cytokines have been shown to play an important role both in inflammation and carcinogenesis and they can be detected in saliva and serum with ELISA assays. Salivary biomarkers may be used as markers of early cancer detection.The aim of this study was the analysis of the serum and salivary levels of IL-10, TNF-α, TGF-β and VEGF in patients with oropharyngeal cancer and in healthy individuals. The level of these biomarkers was also analyzed in HPV- and EBV-related cases.p < 0.05 were considered statistically significant.The study involved 78 patients with histopathologically confirmed oropharyngeal squamous cell carcinoma and 40 healthy controls. Serum and salivary levels of IL-10, TNF-α, TGF-β and VEGF were analyzed both in patients and in healthy individuals by ELISA method using Diaclone SAS commercially available kits (France). EBV DNA was detected by the nested PCR for amplification of EBNA-2. HPV detection and genotyping was performed using the INNO-LiPA HPV Genotyping Extraassay . The obtained results were subjected to statistical analysis using Mann–Whitney and Kruskal Wallis tests. Test values of p = 0.0003; TGF-β: 11.3 ng/ml vs 7.8 ng/ml, p = 0.0005; VEGF: 614 pg/ml vs 210 pg/ml, p = 0.0004; TNF-α: 15.0 ng/ml vs 12.90 ng/ml, p = 0.1397) as well as in saliva .The level of tested cytokines was higher in patients than in controls both in serum (IL-10: 2.3 pg/ml vs 1.65 pg/ml, EBV DNA was detected in 51.3 % of patients and 20 % of controls, HPV DNA was present in 30.8 % of patients and 2,5 % of controls.The level of IL-10 was statistically higher in patients infected with EBV, HPV and co-infected with EBV/HPV. The level of TNF-α was significantly higher in patients infected with EBV, while TGF-β in patients with HPV infection and EBV/HPV co-infection.Detection of salivary cytokines may be very helpful in early diagnosis, treatment and prognosis of OSCC. Oral Squamous Cell Carcinoma (OSCC) as the most frequently diagnosed cancer in the head and neck region is a very important world-wide health problem . About 9It has been shown that inflammation plays a key role in different stages of carcinogenesis. Cytokines are a group of host immune products involved in inflammation, immunity and defence against HPV infection, and modulation of HPV clearance plays an important role in oncogenesis . Of partThis research was approved by the Ethics Committee and is in accordance with the GCP regulations (no. KE-0254/133/2013).The study was conducted among 78 patients with diagnosed and histopathologically confirmed OSCC hospitalized at the Otolaryngology Division of the Hospital in Radom, Poland. The patients had neither previous radiotherapy nor chemotherapy. The results were compared to 40 healthy controls. The control group involved persons hospitalized at the Otolaryngology Division due to diseases other than cancer. There were no statistically significant differences between the patients and the control group  Table . Male paFragments of fresh frozen tumour tissue (20 mg) both from the patients with OSCC and from the control subjects were cut and homogenized in a manual homogenizer Omni TH/Omni International/ Kennesewa/ Georgia /USA. DNA was extracted using a protocol as described in the DNeasy Tissue Kit Handbook . Purified DNA was quantified by spectrophotometer . The isolates were kept at −20 °C until the test was conducted. To verify the quality of the obtained DNA (presence of inhibitors of Polymerase Chain Reaction), assay of β-globin was performed.Venous blood samples from patients and control group were centrifuged at 1500 rpm for 15 min at room temperature and the serum was collected and frozen at −80 °C until analysis.About 5 ml of unstimulated whole saliva was collected. The saliva samples were centrifuged at 1500 rpm at room temperature for 10 min and diluted (1:1) in PBS and frozen at -80 °C until analysis.2, 0.2 mM dNTPs, 0.5 μM each forward and reverse primers and 0.5 U of HotStart Taq polymerase. During each run the samples were tested together with one negative (nuclease-free water) and one positive control .EBV DNA detection: All polymerase chain reaction (PCR) reactions were carried out in the final volume of 25 μl using HotStartTaq DNA Polymerase . Concentrations of PCR reaction components were prepared as follows: 2.0 mM MgClAmplification of EBNA-2 gene: The nested PCR was carried out for amplification of Epstein-Barr nuclear antigen 2 (EBNA-2). The sequence of primers used for PCR was as follows: outer pair 5’ – TTT CAC CAA TAC ATG ACC C – 3’, 5’ – TGG CAA AGT GCT GAG AGC AA – 3’ and inner pair 5’ – CAA TAC ATG AAC CRG AGT CC – 3’, 5’ – AAG TGC TGA GAG CAA GGC MC – 3’. 2 μl of extracted DNA was subjected to the PCR mixture with the concentration as described above. The first-round amplification consisted of activation of polymerase 95 °C for 15 min, 35 cycles of 94 °C for 1 min, 55 °C for 1 min, 72 °C for 2 min and the final extension at 72 °C for 5 min. The second-round amplification was performed with 1 μl of first round PCR product in 30 cycles with an annealing temperature at 60 °C. The amplicons 368 bp, 473 bp in length  were separated on 2 % agarose gel and purified using Gel-Out kit  for further analysis. Purified PCR products were sent to Genomed Warsaw company for sequencing.HPV DNA detection: HPV detection and genotyping was performed using the INNO-LiPA HPV Genotyping Extraassay  according to the manufacturer’s protocol. The kit is based on the amplification of a 65 bp fragment from the L1 region of the HPV genome. PCR products are subsequently typed with the reverse hybridization assay. The assay covers all currently known high-risk HPV genotypes and probable high-risk HPV genotypes  as well as a number of low-risk HPV genotypes  and some additional types .The levels of IL-10, TNF-α, TGF-β and VEGF were established in the saliva and sera of patients and healthy subjects by ELISA (enzyme-linked immunosorbent assay) using commercially available kits Diaclone SAS, France. The cytokine level of each serum sample was tested three times and the results are the means of these. The level of tested cytokines are expressed in pg/ml or ng/ml. The minimum detectable dose of IL-10 is less than 0.98 pg/ml (IL-10 HS ELISA kit) cat. no. 850.880.096; TNF-α – less than 8 pg/ml cat. no 950.090.096; TGF-β – 8.6 pg/ml cat. no. 650.010.096 and VEGF – 7.9 pg/ml cat. no. 650.080.096.p <0.05.To examine differences between the patients and controls in the distributions of demographic variables, smoking and drinking status, HPV and EBV status Pearson’s chi-square test was used. The Mann-Whitney test was used to compare the concentration of cytokines in patients and controls. The Kruksal-Wallis test was used to determine the association of the concentration of studied cytokines with such variables as smoking, alcohol abuse, histological grade (G) of the tumour, tumour (T) and node (N) stage according to TNM  classification and presence of viruses. The observed differences were considered statistically significant at p < 0.001). HPV DNA was detected in 30.8 % of patients, EBV DNA in 51.3 %, while in control group in 2.5 % and 20.0 %, respectively. Epidemiological, clinical and virological characteristics of patients are shown in Table The prevalence of HPV and EBV was higher in patients than in controls  and the size of tumour (T stage), while TGF-β level was related to N stage. The level of the TNF-α was higher in patients infected with EBV, whereas TGF-β was higher in HPV and HPV/EBV co-infected patients. IL-10 concentration was higher in HPV, EBV as well as in HPV/EBV co-infected patients.Various cytokines and growth factors play a significant role both in inflammation and carcinogenesis. Some cytokines are considered as pro-inflammatory , whereas other are associated with anti-inflammatory effects (TGF-β) . To the Interleukin-10  is an important cytokine produced by several cells such as normal and neoplastic B cells, macrophages, T cells and some cancer cells , 20. TheTumour necrosis factor α (TNF-α) is a cytokine involved in systemic inflammation and is one of the cytokines that make up the acute phase reaction. TNF-α is secreted by macrophages, monocytes, neutrophils, T-cells, NK-cells following their stimulation by bacterial lipopolysaccharides . It has TGF-β is a cytokine involved in various biological processes. The TGF-β pathway regulates many of the cellular processes and alterations in the signalling cascade may promote tumour development. The anti-proliferative activity of TGF-β1 is essential for maintaining normal tissue homeostasis and this activity of TGF-β1 is lost in many types of tumours , 31. SomBecause angiogenesis is essential for the growth and metastasis of tumours, we also investigated serum and salivary levels of VEGF , which is the main regulator of this process. In vitro experiments showed that VEGF stimulates mitosis of endothelial cells and their migration. VEGF may be also secreted by tumour cells and it acts in an autocrine manner to promote cell growth , 34. In As it is well documented, the main risk factors for head and neck cancer are tobacco, alcohol abuse and betel quid chewing but some types of head and neck SCC are linked to viral infections such as HPV and EBV.HPV infection has been recognized as a necessary but not the only one cause of certain types of human cancers because cancer eventually develops only in a small group of individuals infected with HPV. Other factors are, therefore, necessary for malignant transformation of HPV-infected cells , 40. ChrVEGF is also a cytokine investigated toward its prognostic value in patients infected with HPV. Jo et al.  observedMany researchers found that HPV(+) and HPV(−) OSCC are two distinct forms of the disease. In general, HPV(+) cancer patients are younger, have a much better prognosis than HPV(−) , 43, 45.Shi et al.  suggest BZLF1 stimulates expression of both TGF-β and IL-10 . EpsteinMany questions are unclear and will need to be answered by future studies. All the above-mentioned cytokines and growth factors act on the tumour growth. Some of them were definitely studied and explained long ago, some are constantly being investigated. Explaining the reasons for this influence is very difficult because many cytokines interact in various ways on cells, often in an antagonistic manner, depending on the concentration. There is no doubt that viruses which have the ability to connect with the genetic material of the host cells can significantly disrupt gene expression for cytokines and growth factors.It has been confirmed in numerous studies that HPV and EBV have the ability to cause various cancers, but there is a lack of studies which determine variability in the levels of cytokines in patients with OSCC with EBV infections.Our results clearly indicate increased levels of IL-10, TNF-α, TGF-β and VEGF both in serum and saliva and they suggest that these cytokines may constitute valuable tools in diagnosis, treatment and prognosis in cancer disease. However, further research is still required to prove the role and mode of action of potential biomarkers.CSIF, cytokine synthesis inhibitor factor; DNA, deoxyribonucleic acid; EBNA-2, Epstein-Barr nuclear antigen 2; EBV, Epstein-Barr virus; ELISA, enzyme-linked immunosorbent assay; GCP, good clinical practice; HPV, human papillomavirus; IARC, International Agency for Research on Cancer; IL-10, interleukin 10; mRNA, messenger ribonucleic acid; OSCC, oral squamous cell carcinoma; PCR, polymerase chain reaction; SCC, squamous cell carcinoma; TGF-β, transforming growth factor beta; TNF-α, tumour necrosis factor alpha; TNM, tumour, node, metastasis; VEGF, vascular endothelial growth factor


Human society has witnessed disasters and wars that left many consequences on families as well as social and individual life of the victims.In this research, we compared the psychological health and marital adjustment in Iranian employed veterans with veterans receiving disability pension.The study participants were all of the veterans of Isfahan city registered in Veterans and Martyr Foundation who were receiving disability pension, were still working, or had not received any disability pension yet. A total of 330 veterans were selected by randomized systematic sampling. The Symptom Checklist-90-Revised (SCL-90-R) questionnaire and Dyadic Adjustment Scale (DAS) were completed by the participants. The data were analyzed by Chi square test, independent samples t test, and Mann-Whitney U test.Almost half of the veterans did not demonstrate any psychopathology and half of them were diagnosed with borderline or serious psychopathology. Veterans receiving disability pension had more mental problems in comparison with the employed veterans. Veterans receiving disability pension had higher scores in psychosomatic disorders, obsessive-compulsive disorder, depression, anxiety, phobias, psychoticism, and total scales  in comparison with the employed veterans. Employed veterans and veterans receiving disability pension did not differ significantly regarding DAS scores.Occupational condition has an important effect on mental health of veterans. Mental health disorders are frequent among soldiers. Approximately 56% of soldiers returning from Iraq and Afghanistan have had two or more mental disorders . VeteranStudies showed that lack of suitable job is one of the most important factors affecting the psychiatric problems of veterans . When veIndividual occupation also affects other areas of life. Research in the areas of family and employment showed transition of psychological states from one role to another . The solIn this research, we compared the psychological health and marital adjustment in Iranian employed veterans with veterans receiving disability pension.This cross-sectional study was approved by Veterans and Martyr Foundation of Isfahan (No 5809/30/26220). The participants of the study were all employed veterans and veterans receiving disability pension registered by Veterans and Martyr Foundation of Isfahan up to the first half of 2010. This study was done from the second half of 2009 through the end of the year. All participants signed an informed consent form before entering the study. The purpose of study was explained to the participants. If they were unwilling to participate in the study, they were excluded. To determine the sample size, 30 employed veterans and veterans receiving disability pension were selected randomly and the study questionnaires was completed by them. Based on Cohen formula (1988) the total sample size was calculated at 261. With regard to the possible loss of subjects, the size of each group, namely, employed veterans and veterans receiving disability pension, was determined as 150. Sampling was done systematically and randomly using a list prepared by the Veterans and Martyr Foundation including veterans of more than 25% disability from three areas of the Isfahan City. A total of 300 veterans who met the inclusion criteria were recruited. The inclusion criteria were being married and having the physical ability to attend the meeting to respond to the questionnaires; the exclusion criteria were being single or divorced.SCL-90-R included 90 items to assess psychological symptoms . The scaThe purpose of Dyadic Adjustment Scale (DAS) is to assess the quality of the marital relationship or similar reciprocal relationships . The tesChi square test was used to determine the significance of differences between educational level and disability percent. The independent samples t-test was used to determine differences between groups regarding age, age at injury, and duration of injury. Test of normality showed that some variables had not normal distribution and Mann-Whitney U test was used to analyze them. Mann-Whitney U was performed to detect any significant difference in psychological symptoms and marital adjustment of employed veterans and veterans receiving disability pension. Evaluation of the correlation between variables such as age, age at time of injury, duration of injury, and type of injury with nine scales and total scores of SCL-90-R and DAS showed that only type of injury had significant correlation with some scales of the SCL-90-R.From 330 participants, 263 veterans filled out DAS property (110 of employed veterans and 162 of veterans receiving disability pension). In addition, 279 veterans filled out SCL-90-R property (110 of employed veterans and 169 of veterans receiving disability pension). The questionnaires with more than 25% unanswered questions were excluded from the final analysis. We evaluated the mental and marital conditions of employed veterans and veterans receiving disability pension. Based on the scales of the SCL-90-R questionnaire, 47.3% of veterans had no mental disorders  and 52.7% had some kind of psychopathology . The study showed that the employed veterans had a better mental status than those receiving disability pension.This study showed that the veterans receiving disability pension had higher scores in somatization, OCD, depression, anxiety, phobia, psychoticism, and total scales (GSI) in comparison with the employed veterans. Several studies in veterans had reported similar findings -11. PeopIn this research we found that somatization and anxiety scales had the greatest variance between SCL-90-R scales. Somatization and hypochondriasis are amongst the most common disorders in veterans , 8, 12. In our study, the employed veterans and the veterans receiving disability pension did not differ in subscales of DAS. Many studies showed that soldiers who experienced combat situations had problems in all the areas related to DAS -24, 35. This study showed that the veterans receiving disability pension had more psychological problems than the employed ones but the marital adjustment of two groups did not differ. Most of the Iranian veterans had experienced Iraq prisons too. Although most of the Iranian veterans participated in Iran-Iraq war voluntary and studies showed less stressors in volunteer combatant prisoners than army prisoners of war , combat Based on the results of the present study, we recommend implementing these reforms by Veterans and Martyr Foundation: encourage veterans to find jobs and creating jobs adapted to their abilities; finding and recording the veteran's abilities in the past; suitable use of the existing veterans facilities, e.g. land, repair shops, bank capital, etc; lending low-rental offices to veterans; providing "the gift of employment" for veterans who received disability pension but still remain in their jobs; and assistance for part-time or hourly employment of veterans with job-state conditions.


Head and neck squamous cell cancers are among the most aggressive. Their incidence and mortality rates are relatively lower in Middle Africa than worldwide, but in Gabon, these rates tend to be 2–3 fold higher than in neighboring countries. The main risk factors are alcohol and tobacco consumption. However, in the last decades, there was cumulated evidence that human papillomaviruses were a significant risk factor, particularly for oropharyngeal squamous cell cancer. In Gabon, as elsewhere in Africa, assessment of these 3 risk factors need to be improved to determine their respective role in the development of head and neck squamous cell cancers. The potential differences in alcohol/tobacco consumption habits as well as in infectious ecology between developing and developed countries can make it difficult to transpose current data on this issue. Determining the respective role of alcohol/tobacco consumption and human papillomaviruses in the development of head and neck squamous cell cancers is crucial for the management of these cancers that could become a serious public health issue in Gabon. Human papillomaviruses are not only a risk factor but also a biomarker with promising clinical potential for the follow-up of head and neck squamous cell cancers potentially able to select an adequate treatment. Then, assessing the epidemiological impact of human papillomaviruses in Gabon and in all of Africa would prove useful for the clinical follow-up of head and neck squamous cell cancers, and would also provide essential data to plan a global prevention strategy against head and neck squamous cell cancers due to human papillomaviruses. Head and neck  cancers, most frequently squamous cell carcinoma, are among the most aggressive worldwide with 599,637 new cases diagnosed and 324,834 associated-deaths in 2012 . This raAlcohol and tobacco consumption habits vary according to regions Table  and makehow to manage HNSCC, which could become a real public health problem, in Gabon?In Middle Africa, the incidence of HNSCC  is lower than the worldwide rate (respectively 4.5 versus 8.0), except in Gabon where reported rates are 2–3 fold higher (9.4)  was 26.50 l per capita of pure alcohol in 2010  for a global rate of 9.0 % in this population. These rates are lower for daily smokers: global rate of 7.0 % with 12.0 % of men and 1.0 % of women  is the most frequently diagnosed and the main cancer-related cause of death in the female population in Africa as well as in Gabon, , 11, 12.et al. reported that even the presence of specific HPV 16/18 antibodies could predict HNSCC development, stressing the potential clinical value of HPV status [i) to plan an adapted prevention strategy, ii) as a biomarker for a better follow-up of HNSCC patients, particularly in non-smokers and non-alcohol consumers [OPSCC are the most frequent HPV-attributable HNSCC , 21. TheV status . Thus, tHNSCC is becoming a matter of public health concern in Gabon. It is necessary to control the main risk factors for their prevention: tobacco, alcohol and HPV. First, it is imperative to determine accurately the rate of these risk factors in Gabon. Therefore, local smoking and drinking habits should be documented to implement adapted programs to raise the awareness of the Gabonese population. Moreover, extending HPV research to the whole Gabonese population is also essential. Numerous ongoing studies are focused on the female population to determine the HPV infection profile in a context of CC. Extending this research to HNSCC would be a valuable move to assess the HPV relative risk. This would ultimately allow for defining the global epidemiological impact of HPV to elaborate an effective vaccine anti-HPV strategy thus preventing the occurrence of HPV-attributable HSNCC.