The incidence of cervical cancer is expected to rise sharply in China. A reliable routine human papillomavirus (HPV) detection and genotyping test to be supplemented by the limited Papanicolaou cytology facilities is urgently needed to help identify the patients with cervical precancer for preventive interventions. To this end, we evaluated a nested polymerase chain reaction (PCR) protocol for detection of HPV L1 gene DNA in cervicovaginal cells. The PCR amplicons were genotyped by direct DNA sequencing. In parallel, split samples were subjected to a Digene HC2 HPV test which has been widely used for “cervical cancer risk” screen. Of the 1826 specimens, 1655 contained sufficient materials for analysis and 657 were truly negative. PCR/DNA sequencing showed 674 infected by a single high-risk HPV, 188 by a single low-risk HPV, and 136 by multiple HPV genotypes with up to five HPV genotypes in one specimen. In comparison, the HC2 test classified 713 specimens as infected by high-risk HPV, and 942 as negative for HPV infections. The high-risk HC2 test correctly detected 388 (57.6%) of the 674 high-risk HPV isolates in clinical specimens, mislabeled 88 (46.8%) of the 188 low-risk HPV isolates as high-risk genotypes, and classified 180 (27.4%) of the 657 “true-negative” samples as being infected by high-risk HPV. It was found to cross-react with 20 low-risk HPV genotypes. We conclude that nested PCR detection of HPV followed by short target DNA sequencing can be used for screening and genotyping to formulate a paradigm in clinical management of HPV-related disorders in a rapidly developing economy. Since opening up to the outside world, China has undergone tremendous changes both economically and socially. Many sexually transmitted diseases which were fairly rare some 30 years ago are now common occurrences . Health Cervical cancer is a major fatal malignancy among women, causing about 275,000 deaths annually worldwide, most in developing countries . The curIn resource-constrained settings, community outreach, education, and advocacy are the key components in advancing cervical cancer prevention initiatives , and theVarious HPV DNA tests have been introduced to meet the need for effective cervical cancer prevention screening in China , 14 and Since DNA sequencing is the generally accepted standard for molecular identification and genotyping of HPV , an HPV-This study was approved by the Institutional Review Boards of the Shanghai Institute of Biochemistry and Cell Biology (SIBCB), Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Changning District Maternal and Children Health Care Hospital (CMCHCH), and Jiading District Maternal and Children Health Care Hospital (JMCHCH).The clinical samples used for this study were from 1826 women visiting the gynecology clinics of the two above-referenced hospitals. The median age of the patients was 39.8 years (range from 20 to 65 years old). CMCHCH is a hospital in a peripheral district and JMCHCH in the suburb of Shanghai. Both hospitals serve an economically rapidly developing community. The patients included in this study were those who visited the clinics for a variety of minor gynecologic symptomatic disorders other than neoplastic diseases. The majority of the patients did not have prior regular gynecologic care, but were generally aware of the value of a “screen test” for cervical cancer prevention, and were willing to pay for a DNA “cancer screen” approved by the U.S. Food and Drug Administration (FDA) at the cost of about U.S. $50 per test to determine a cervical cancer risk. They were not willing to pay for an additional Pap cytology screen.According to the protocols of practice, the cervicovaginal cells at the transformation zone of the uterine cervix were collected by a gynecologist or a trained gynecologist assistant with a standard cytobrush (with spatula), and suspended in PreservCyt® or Surepath® fixative for routine Digene HC2 assays  to detect high-risk HPV DNA. With the patient's informed consent, an aliquot of the collected cell suspension from each double-blind number-coded sample was used for the HPV DNA nested PCR amplification followed by HPV genotyping by DNA sequencing if found to be HPV positive.The HC2 test was performed in the pathology laboratories at CMCHCH and JMCHCH according to instructions provided by Digene Corporation. Nested PCR HPV DNA detection followed by genotyping with short target DNA sequencing, using the MY09/MY11 degenerate primer pair for primary PCR and the GP6/MY11 primer pair for nested PCR, was performed at the SIBCB laboratory according to a low temperature PCR program as reported in the literature . Each neWhen more than one HPV genotype was present in a sample, the routine DNA sequencing on the GP6/MY11 nested PCR products was unable to generate a readable base-calling electropherogram because more than one HPV DNA template had been amplified by the degenerate and consensus general PCR primers. In order to determine accurately the prevalence of genotypes involved in these specimens, the ∼450 bp primary PCR products of 50 randomly selected samples with multiple HPV infections were reamplified, each by 24 individual genotype-specific nested PCR primer pairs listed in A β-globin primer pair for human genomic DNA amplification was used to control specimen adequacy. Specimens negative for HPV DNA with no companion β-globin gene amplification were excluded as insufficient. However, specimens found to be positive for HPV DNA by nested PCR and confirmed by DNA sequencing were considered sufficient even when no β-globin gene amplification was observed.All visualized GP6/MY11 primer-defined nested PCR amplicons were confirmed by direct automated fluorescent dye-terminator Sanger method to contain HPV DNA sequences. Of the 1826 liquid-based cervicovaginal specimens tested by both nested PCR and the HC2 kit on split samples in parallel, 171 were found to be negative for β-globin gene amplification and negative for HPV DNA by either nested PCR or by the HC2 kit, and were excluded as insufficient for evaluation. Of the remaining 1655 specimens tested by both methods, the PCR/DNA sequencing protocol showed 674 infected by a single high-risk HPV, 188 by a single low-risk HPV, and 136 by multiple HPV genotypes, 657 samples were negative by HPV DNA PCR, and positive for β-globin gene amplification, and therefore accepted as “true negative” for HPV infection .In comparison, the HC2 test classified 713 specimens as infected by high-risk HPV, and 942 as negative for HPV infections. Correlation of the results generated by these two methods on split samples showed that the HC2 test detected 388 (57.6%) of the 674 high-risk HPV isolates in clinical specimens, mislabeled 88 (46.8%) of the 188 low-risk HPV isolates as high-risk genotypes, and classified 180 (27.4%) of the 657 “true-negative” samples as being infected by high-risk HPV . The senOf the 50 samples infected by more than one HPV genotype selected for individual genotype-specific nested PCR amplifications, 34 (68%) samples were found to contain two HPV genotypes, 11 (22%) samples three HPV genotypes, and four (8%) samples four HPV genotypes. One (2%) sample contained five HPV genotypes .Base-calling invariably failed to resolve mixed DNA sequences generated from more than one nested PCR amplicon in a single Sanger reaction . HoweverAlthough cervical cancers are initiated by persistent infection of high-risk HPV, less than 10% of new HPV infections result in persistent HPV infections and precancerous pathologies, typically within 5–10 years. Invasive cancer only develops in a very small percentage of women with precancerous cellular changes over one to three decades [In developed countries, women with cervical precancer usually have a positive Pap cytology screening result of high-grade squamous intraepithelial lesion (HSIL), then are referred to colposcopic biopsy for a histological diagnosis of cervical intraepithelial neoplasia grade 2 or grade 3 (CIN2 or CIN3) before an ablative procedure is performed. Medical practice guidelines are against unnecessary colposcopic biopsies and needless traumatic surgical procedures which may have undesirable health consequences .In developing countries, quality cytology screen, colposcopic biopsies and histopathological diagnostic services are not readily available. The HC2 test kit or its cheaper version has been recommended for cancer screening in place of Pap cytology in China , 31. TheA clinically useful HPV nucleic acid test must be able to reliably answer two questions: (1) Is there HPV DNA in the specimen? (2) What is the genotype of the HPV detected?Since persistent HPV infection is a necessary factor for cervical cancer development, a truly negative HPV test result practically rules out the presence of cervical precancer or cancer in the specimen. As the pathology in persistent HPV infection advances to precancer and cancer, the viral load per abnormal cell tends to decrease while the HPV-rich koilocytes are progressively replaced by dysplastic cells with a higher nuclear/cytoplasmic ratio in the epithelum –35. The The present study was performed using DNA sequencing as the gold standard for HPV screening , revealiAt the analytical level, we confirm that the high-risk probe B cocktail of the Digene HC2 kit cross-reacts with the sequences of HPV-6, -11, -13, -32, -40, -42, -43, -53, -54, -55, -61, -62, -66, -67, -69, -70, -73, -81, -82, and -84. Cross-reaction causes 46.8% of the untargeted low-risk HPV genotypes to be mislabeled as high-risk HPV, which may lead to further unnecessary confusion. Compared with a nested PCR/DNA sequencing technology, the HC2 kit detects only 57.6% of the single high-risk HPV and 44.2% of HPV-16 infections. Since it cannot perform genotyping, the HC2 test does not offer the needed information for follow-up of persistent HPV infections.Nested PCR is a recognized highly sensitive technology for HPV DNA detection and for preparing a template for DNA sequencing. Over 50% of the cervical HPV infections depend on a nested PCR DNA amplification for detection and subsequent validation . The DigWe designed a set of specific PCR primers for the 24 commonly encountered HPV genotypes to analyze samples infected with multiple HPV genotypes. For the samples containing more than one HPV type, the routine DNA sequencing was unable to generate a readable base-calling electropherogram as there was more than one type of PCR product presenting in PCR amplification mixtures. Theoretically, there exist variants in one genotype, so some of these genotype-specific primers listed may as well be “variant-specific”.Our understanding of the clinical significance of multiple HPV infections is incomplete. For the first time, we have introduced a protocol of using 24 individual-specific nested PCR amplifications for each specimen containing more than one HPV for final genotyping by DNA sequencing. Preliminary observation indicates that each specimen with multiple HPV infections seems to contain at least one high-risk HPV genotype. Although this labor-intensive procedure requiring 24 nested PCRs for each sample with multiple HPV infections is cost-prohibitive in routine diagnostic practice, it may provide a valuable tool for further investigation of the evolvement of multiple HPV infections.


A highly divergent human papillomavirus (HPV) strain, HPV-L55, was identified in fecal samples from children hospitalized with diarrhea in China. The L1 gene of HPV-L55 shares <75% identity with previously reported HPVs, indicating that this virus represents a novel type of HPV. Phylogenetic analysis classified this virus as a member of the gammapapillomaviruses. Papillomaviridae  are a diverse class of small, circular, double-stranded DNA viruses in the family aviridae . Papilloomavirus . Recent omavirus , HPV-159omavirus , and HPVUsing the customized informatics pipeline VirusHunter , 7, we iThe full HPV-L55 genome was sequenced using the genome walking method . The primers were designed based on the original 454 pyrosequencing read, along with newly amplified HPV-L55 sequences; long fragments were further amplified using Ex Taq DNA polymerase  for final sequence confirmation. The sequences were then aligned and manually edited to produce the complete viral genome assembly. The complete circular genome of HPV-L55 is 7,277 bp in length with a G+C content of 37.47%.29CxxC separated by 36 amino acids in the E6 protein, and a modified zinc-binding domain CxC(x)29CxxC in the E7 protein  and a polyadenylation signal .The HPV-L55 genome encodes seven open reading frames (ORFs) , of which the putative E4 ORF contains a start codon and completely overlaps the E2 ORF. The genome contains a nucleotide-binding helicase domain (GPSNTGKS [G-X4-GKT/S]) in the E1 protein, two conserved zinc-binding domains of CxxC(x) protein , 9. In aGammapapillomavirus of Papillomaviridae, which currently includes >10 viral species known to infect humans . HPVs have traditionally been classified into different types based on the criterion that each type exhibits <90% similarity to the L1 nt sequence of other HPVs (Further studies will be necessary to determine the prevalence of this virus in the general population. Although a direct association between HPV-L55 and human disease has yet to be established, the full-genome sequence of HPV-L55 described here will be important for determining the full picture of genetic diversity in HPVs.KF482069.The full HPV-L55 genome sequence was deposited in GenBank under the accession no.


The authors describe the first case of eye amelanotic cellular blue nevus reported in literature and discuss the main differential diagnosis. The blue nevus is a benign, usually solitary lesion which is characterized by a localized proliferation of melanocytes and is believed to represent an abnormal arrest in embryonal migration of neural crest melanocytes .Blue nevi are most commonly found on the skin. Rare cases of common blue nevi have been reported in the vagina , the uteWe report an unusual case of iris amelanotic cellular blue nevus in a young woman.A 37-year-old woman presented herself at the Ophthalmological Department for a left eye pupil deformity. The slit lamp microscopy revealed an iris neoformation in the inferior temporal quadrant of the iris. This neoformation caused pupil dyscoria and corectopia and uveal ectropion. The color of the neoformation was light pink and it was richly vascularized Figures . The UltIris fluorescent angiography showed new formed vessels with abundant leakage in the neoformation.After a complete workup, in the suspicion of an uveal amelanotic melanoma, surgical excision of the neoformation was programmed. Before surgery, a laser photocoagulation of the temporal inferior retinal quadrant was performed to prevent retinal detachment.Under general anesthesia a sector iridociclectomy and a pupilloplasty were performed.Histologically the lesion was composed of a dense and diffuse proliferation of amelanotic cells nested in a nevoid pattern and surrounded by a dense fibrous stroma .The cells varied in morphology from short spindle-shaped to dendritic one. The neoplastic proliferation involved the iris stroma and, focally, the contiguous ciliary body . All celOn the basis of morphologic and immunohistochemical features, the tumor was diagnosed as amelanotic cellular blue nevus.Blue nevus was first described by Tièche in 1906 . EarlierDifferential diagnosis included all nonmelanocytic tumors with spindle cells . In particular, amelanotic cellular blue nevus must be distinguished from malignant cellular blue nevus  and otheOur case is the first description of a blue amelanotic nevus involving the eye. Ophthalmologists must be aware of the possibility of this entity in the differential diagnosis of eye neoformations.


Cervical cancer and its precursor lesions represent a significant public health problem for developing and less-developed countries. Cervical carcinogenesis is strongly correlated with persistent high-risk human papillomavirus (HPV) infection, which is mostly associated with expression of the p16 and E6 HPV-related proteins. The aim of this present study was to determine the expression of the p16 and E6 proteins in females with high-grade lesions treated with conization, and to discuss the role of these proteins as prognostic markers following treatment. In total, 114 females were treated for high-grade cervical intraepithelial neoplasia  by conization with large loop excision of the transformation zone (LLETZ). Following surgery, the patients returned within 30–45 days for post-operative evaluation. A follow-up was conducted every 6 months for 2 years. At each follow-up appointment, a Pap smear, colposcopy and HPV DNA test were performed. E6 and p16 immunohistochemical tests were conducted on the surgical specimens. The positive expression of p16 was correlated with the presence of lesions with increased severity in the surgical specimens (P= 0.0001). The expression of E6 did not demonstrate the same correlation (P=0.131). The HPV DNA hybrid, collected in the first post-operative consultation as a predictor of the cytological abnormalities identified at the 24-month follow-up assessment, presented a sensitivity of 55.6%, a specificity of 84.8%, a positive predictive value of 33.3% and a negative predictive value of 93.3%. The role of p16INK4A as a marker of CIN was also demonstrated; the expression of p16 and E6, however, did not appear to be of any prognostic value in predicting the clearance of high-risk HPV following conization. A negative hybrid capture test was correlated with a disease-free outcome. Human papillomavirus (HPV) infects numerous females worldwide and is generally transmitted through sexual contact . The majThe high-risk HPV types encode two oncoproteins, E6 and E7. The E6 oncoprotein binds to the p53 tumor suppressor, resulting in its inactivation and the prevention of cellular apoptosis . The E7 The aim of the present study was to characterize p16 expression in patients treated by conization with large loop excision, and to compare the p16 performance with the E6 immunohistochemical and HC2 test results in combination with the Pap smear examination.Between March 2006 and May 2009, 114 females were treated for high-grade cervical intraepithelial neoplasia (CIN 2/3) by conization with large loop excision of the transformation zone (LLETZ) at the Department of Gynecology, Faculty of Medicine, São Paulo University . Following surgery, the patients returned within 30–45 days for post-operative evaluation. A follow-up was conducted every 6 months for 2 years. Each follow-up appointment comprised a Pap smear, colposcopy and HPV DNA test.The procedure was explained to all the patient and written informed consent was provided. The study was approved by the ethics committee of the Clinics Hospital, Faculty of Medicine, São Paulo University. Immunohistochemical examinations, including E6 and p16 staining, were performed on the surgical specimens.The HC2 test was conducted according to the manufacturer’s instructions . Only high-risk HPVs were examined and the carcinogenic types included types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68.® Histology kit according to the manufacturer’s instructions . Briefly, subsequent to the use of retrieval solution at 125°C for 3 min and 90°C for 20 min, the slides were cooled at room temperature and washed in wash buffer (1:10 dilution), for 5 min. Endogenous peroxidase was blocked with peroxidase-blocking reagent at a volume of 30 ml per slide for 5 min. Primary ready-to-use p16 antibody was added after 30 min at room temperature. Visualization reagent was utilized for signal amplification. The revelation with diaminobenzidine (DAB) was performed with 15 ml 3,3′-diaminobenzidine (DAB) chromogen and counterstained with hematoxylin.The immunohistochemical reaction for p16 was performed with the CINtec2O2). The primary E6 monoclonal antibody (C1P5) sc460, from mouse E6 HPV 16 and HPV 18  was used at a 1:200 dilution. The revelation was performed with an ADVANCE™ horseradish peroxidase (HRP) kit .Antigen retrieval was conducted using a microwave and a solution of 10 mM citric acid  for three minutes at 125°C. Endogenous peroxidase blocking was performed with 6% hydrogen peroxide , the follow-up Pap smear was adopted as the gold standard. In all statistical tests, P<0.05 was considered to indicate a statistically significant difference.The HC2 HPV DNA test  was performed in 112 of the included patients prior to the surgical procedure. A total of 108 patients tested positive for HPV DNA and four tested negative prior to the procedure. Two cases had no HC2 HPV DNA test performed. The cytological results prior to the surgical procedure were as follows: 71 patients presented with HSIL, 2 with HSIL and AGC and 14 with low-grade squamous intraepithelial lesions (LSIL). Another 6 patients exhibited ASC-H, 1 exhibited ASC-H + AGC, and 6 exhibited ASC-US. Only 1 patient presented with ASC-US + AGC, 4 presented with AGC and 9 were classified as having normal cytology. The patients who had normal, ASC-US or LSIL cytology presented with CIN 2/3 in their biopsy samples. The cytological and histological findings prior to treatment are listed in in situ (AIS), and one (0.9%) with micro-invasive carcinoma. The pathological examination of the excised cervical specimens revealed the following diagnoses: 18 (15.8%) patients with chronic cervicitis; 11 (9.6%) with CIN 1; 19 (16.7%) with CIN 2; 64 (56.1%) CIN 3; one (0.9%) with CIN 3 and adenocarcinoma The results of the p16 and E6 immunohistochemical reactions, the HC2 HPV DNA tests prior to the surgical procedure and the histopathological findings in the surgical specimens are presented in The accuracy values of the HC2 test in predicting cytological abnormalities over the 2-year follow-up are shown in According to the results of the HPV DNA hybrid, collected in the first post-operative follow-up as a predictor of the cytological abnormalities found in the 24-month follow-up period, a sensitivity of 55.6%, a specificity of 84.8%, a positive predictive value of 33.3% and a negative predictive value of 93.3% were recorded. In comparison, + biopsy-diagnosed cases, which reinforced its potential use as an accurate marker of high-grade cervical lesions.In our previous study, containing partial information from the present study, we identified that patients with a combination of negative cytology and negative hybrid capture test results did not exhibit high-grade lesions at the conization follow-up examination . The res+ cases (43.5%) were E6-positive. It has been suggested that differences in E6 variants prevalent in cervical carcinoma are not correlated with the carcinogenic potential of the E6 protein. Moreover, E6 variants have revealed comparable abilities in preventing growth arrest and inhibiting the induced p53 elevation. Differences were detected in the ability to deregulate stratification and differentiation, as well as in modulating apoptosis and hyperactivating the Wnt signaling cascade (p16 changes in the methylation profile of cervical HPV-induced lesions have been implicated in transcription and replication control, potentially triggering the neoplastic transformation . This is cascade . The abs cascade .The present study demonstrated the predictive potential of the negative values in the hybrid capture test during the follow-up of the patients that underwent conization. In addition, specificity was observed in each clinical visit. Repeated detection of high-risk HPV was demonstrated to be significantly more specific, but less sensitive, in identifying females at risk for CIN 2/3 as compared with a single time-point measurement. Moreover, sensitivity has been estimated to decrease and specificity to increase when the testing intervals were increased from 12 to 24 months . The varIn conclusion, the current study supported the critical function of p16INK4A as a highly specific marker of CIN. However, the immunohistochemical expression of p16 has been previously demonstrated to have no prognostic value in predicting the clearance of high-risk HPV following conization . Overexp


Human papillomavirus (HPV) currently represents an important risk factor for cancer development and infertility in humans. Whilst binding of HPV to spermatozoa has been associated with male infertility, an investigation about the presence of HPV-DNA in non-spermatozoal semen cells is lacking. Previous findings documented the presence of HPV in peripheral blood leukocytes. The aim of this study was to investigate the expression of HPV markers in semen and blood leukocytes during HPV-16 infection.A total of 32 subjects, 16 patients affected by HPV-16 semen infection and 16 controls, were evaluated in our andrological centre and enrolled in the study. Semen non-spermatozoal cells from all subjects were isolated and evaluated for the expression of HPV-16 markers  and further characterized for their molecular phenotype. Analogue determination was performed on peripheral blood mononuclear cells.The presence of HPV-DNA by FISH analysis in a round cell population from semen, confirmed to be CD45+ leukocytes, was observed. These HPV-DNA containing-cells also displayed HPV-16-E6 and HPV-16-L1 viral proteins and, upon further investigation, were found to be CD20+ and CD56+, likely phenotypes of B cells and natural killer cells (NK) respectively. In 25% of the patient group, a very small population of peripheral blood mononuclear cells was found to be positive for HPV-DNA via FISH. These cells displayed the CD20+ and CD56+ phenotype alike. None of the control subjects displayed HPV-DNA in either semen or peripheral blood.Considering the role of CD20+ and CD56+ cell populations in the antiviral immune response, the detection of HPV markers on leukocytes may reflect the presence of virus particles within the endosomal compartment. However, the presence of HPV markers in circulating mononuclear cells raise concerns about the risk of developing cancers to distal organs. Samples were finally counterstained with DAPI and mounted in antifade-buffer. Slides were analyzed with a Video-Confocal (VICO) fluorescence microscope . Evaluation of nuclear hybridization signals was performed in triplicate by different investigators. Positive controls staining for HPV-E6 and L1 proteins, performed on Caski cell line infected by HPV-16, are reported in Additional file t-test. Comparisons between frequencies were performed using the χ2 test.Differences in seminal parameters between patients and controls were evaluated by an unpaired two-sided Student’s Differences were considered significant when P < 0.05. Variables are presented as mean ± SD.The seminal parameters of the HPV16-infected patients and control subjects assessed are reported in Table 3 cells/ml and 0.002 ± 0.001 103 cells/ml in infected and non infected samples respectively, P > 0.05; Table No difference between the number of round cells was found comparing the two groups  was initially assessed by FISH. As reported in Table in vitro studies that cytotoxic activity and cytokine production by NK cells appeared to be linked to rapid HPV-VLP entry into these cells by macropinocytosis. Since CD16 blockade inhibited this process, the authors concluded that CD16 is necessary for HPV–VLP internalization [The results of this study have identified, for the first time, the presence of HPV16 in CD45+ leucocytes in the semen of infected patients. Furthermore, HPV was also detected in a very restricted subpopulation of circulating PBMC bearing a similar immunophenotype. Although these findings are in contrast with the known tropism of HPV for epithelial cells , other alization , even iflization . Howeverlization ,35. ThusIt has been postulated that HPV in polymorphonuclear cells may be involved in vertical transmission of the virus, following the detection of HPV-DNA in reproductive and placental cells, as well as in virgins, infants and children ,37. IndeIn addition to the low number of patients enrolled, the main drawback of this study is represented by the extremely low percentage of HPV positive leukocytes recovered in seminal fluid and peripheral blood. A possible reason for these findings is that semen immune cells can become unproductively infected with HPV16 when trafficking through the seminal compartment in HPV16-infected men, and these cells may traffic back into circulation and be detected at low frequency in peripheral blood. This condition limited our ability to isolate HPV bearing leukocytes and further evaluate their infectiveness. Thus a mechanistic explanation about how virus enters circulating mononuclear cells remains an open question. Moreover, the assessment of HPV DNA on peripheral blood performed by INNO LiPA was not able to confirm the results obtained by FISH and Single-step PCR analysis (data not shown). A possible explanation of this discrepancy may reside in the different method sensitiveness.In conclusion, this is the first study reporting the presence of HPV-antigens together with HPV-DNA in circulating PBMC from male patients with HPV-16 infection of the genitourinary-tract. This evidence raises major concerns about the risk of developing cancers to distal organs.The authors declare that they have no competing interest.CF supervised the study; DP performed outpatients evaluation; AB performed laboratory evaluation; SM carried out the molecular genetic studies; AL supervised manuscript editing; CF, LDT, AG, DP drafted 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/1471-2334/13/593/prepubA: Positive FISH staining for HPV-DNA and immunofluorescence staining for HPV-16 E6 an L1 proteins in cultured Caski cells infected with HPV 16. B: Positive immunofluorescence staining for human CD45, CD20, CD4, CD8 and CD56 antigens performed in isolated peripheral blood mononuclear cells.Click here for file


INK4a and reduced expression of pRb, two cellular markers for HPV-induced cell transformation, were observed. Exons 4–10 of TP53 showed no mutations or polymorphisms. The presence of HPV58 as single HPV infection in combination with a broad variety of direct and indirect markers of HPV transformation provides comprehensive evidence that this oropharyngeal SCC was driven by HPV58.Persistent human papillomavirus infection (HPV) is recognized as an important etiologic factor for a subset of head and neck squamous cell carcinomas (SCC), especially those arising from the oropharynx. Whereas HPV16 accounts for the majority of HPV DNA-positive oropharyngeal SCC, infections with other mucosal high-risk HPV types are quite rare and biological data demonstrating their causal involvement are insufficient. Here we present the first case of an oropharyngeal SCC driven by HPV type 58. A 69-year-old Caucasian woman presented with an enlarged and firm left tonsil. A computed tomography scan showed a left tonsillar mass, extending to the soft palate and the glossotonsillar sulcus. The patient underwent extended radical tonsillectomy and ipsilateral selective neck dissection. Pathology confirmed an infiltrating, poorly differentiated SCC of the left tonsil with node metastasis (pT2N1). Adjuvant external beam radiation therapy (60 Grays (Gy)) was administered. After 1 year of follow-up, the patient is well with no evidence of cancer recurrence. HPV analyses of the tumor tissue by BSGP5+/6+ −PCR/MPG, targeting 51 mucosal HPV types, showed single positivity for HPV type 58. Presence of HPV58 E6*I RNA demonstrated biological activity of the virus in the tumor tissue, and presence of serum antibodies to HPV58 oncoproteins E6 and E7 indicated presence of an HPV58-driven cancer. Overexpression of cellular protein p16 Oropharyngeal squamous cell carcinomas (OPSCC) are categorized as head and neck squamous cell carcinoma (HNSCC) together with squamous cell carcinoma of the oral cavity, larynx and hypopharynx. OPSCC account for approximately 50,000 incident cases ,2, and tAmong the 51 mucosal HPV types known so far, 12 have been classified as carcinogenic (class I) for cervical cancer (CxCa) . While HIn contrast to CxCa, an even larger majority of HPV DNA-positive OPSCC are associated with HPV16 89% - 97%), and DNA of other carcinogenic non-HPV16 types has been detected only rarely in OPSCC tissues 9% - 97%,. A recenIntriguingly, only a subset of HPV16 DNA-positive OPSCC display HPV16 carcinogenic activity in the tumor tissue, i.e. are HPV-driven (HPV DNA-positive RNA-positive) OPSCC, particularly in populations with low HPV DNA prevalence in OPSCC such as Western Europe. This indicates that presence of HPV DNA alone is not sufficient proof for causal involvement of any HPV DNA found in an OPSCC tissue. For non-HPV16 types found in OPSCC molecular proof of causality is largely lacking.INK4a and downregulation of tumor suppressor proteins pRb and p53. In patients with invasive HPV-driven cervical, penile and oropharyngeal SCC overexpression of E6 and E7 oncoproteins frequently leads to strong antibody responses against these viral proteins [Over the last 25 years a rather detailed model of HPV-driven transformation of human tumor cells has been established. Truly HPV-transformed tumor cells contain at least one viral genome copy per cell and express the viral oncogenes E6 and E7. Interaction of the E6 and E7 oncoproteins with key regulators of cell cycle and apoptosis leads to upregulation of cellular protein p16proteins .This model has been extensively verified in cervical carcinoma for all HPV types classified by IARC/WHO as carcinogenic, probably and possibly carcinogenic . It has Here, we present the first case of an OPSCC driven by HPV58, a carcinogenic HPV type with low prevalence in CxCa, as demonstrated by a broad variety of HPV transformation markers. The present case is part of an ongoing prospective multicentric study of head and neck cancer patients from North-East Italy.A 69-year-old Caucasian woman presented with a 2 months history of foreign-body sensation in the throat. She had a negative history for tobacco and alcohol use. The patient underwent regular gynecological examinations and cytological cervical cancer screenings until the age of 64, and no HPV-associated genital lesions or cytological alterations were found. No anogenital tumor had become symptomatically apparent since then and extending also into the follow-up period after treatment of the index throat condition. The patient got infected with hepatitis B virus (HBV) by blood transfusion at the age of 26, and had been treated for lichen ruber planus in the oral cavity at the age of 34. The ENT (Ear Nose and Throat) physical examination revealed an enlarged left tonsil without ulceration. On palpation, the left tonsil was found to be firm to hard in consistency. She did not have any palpable cervical lymphadenopathy. A computed tomography (CT) scan with contrast of the neck confirmed a left tonsillar lesion of 20 × 23 × 26 mm in dimension, extending up to the soft palate, without crossing midline, and down to the glossotonsillar sulcus, and revealed two homogeneously enhancing lymph nodes, with maximum transverse diameters of 13 mm, in the left submandibular triangle. Baseline investigations including complete and differential blood cell count, serum electrolytes, liver and renal function tests, electrocardiogram, and X-ray chest, were well within normal limits. The patient underwent wedge biopsy of the left tonsillar mass. Pathologic review revealed the presence of a poorly differentiated SCC with basaloid features.The patient underwent left side mandibulotomy, extended radical tonsillectomy and ipsilateral selective neck dissection (levels I-II-III-IV). The surgical defect was reconstructed with a microvascular ulnar forearm flap. Final pathology confirmed an infiltrating, poorly-differentiated SCC of the left tonsil, with tumor measuring 28 mm in greatest dimension and a thickness of 14 mm, abundant comedo-type necrosis and the presence of both vascular and perineural invasion. Histopathological examination of the neck dissection tissue showed an enlarged lymph node with intracapsular metastasis at level II (pT2N1). To minimize the risk of locoregional recurrence the treatment was completed with adjuvant external beam radiation therapy (EBRT), which started 7 weeks after surgery. Radiation treatment targeted the tonsillar bed and the left cervical region. A dose of 60 Gy was delivered with conventional fractionation . The HPV molecular data were not available at the time of clinical diagnosis and tumor treatment. After 1 year of follow-up, the patient is well with no evidence of recurrent cancer.Analyses were performed in fresh-frozen tumor biopsy, formalin-fixed paraffin-embedded (FFPE) tumor tissue and serum. All samples were collected at the time of initial tumor diagnosis before surgery and radiotherapy.Genomic DNA was extracted from sections of fresh-frozen and FFPE tissues following previously described procedures ,25,30. MHPV genotyping was performed by BSGP5+/6+ −PCR/MPG -33, an aGenomic DNA extracted from both fresh-frozen and FFPE tumor tissues contained HPV58 as single mucosal HPV infection, as shown by both mucosal HPV genotyping methods.Total RNA was obtained from FFPE tissue sections using the Pure-Link FFPE Total RNA Isolation Kit  following the manufacturer’s protocol. Viral RNA expression was analyzed by E6*I reverse transcription (RT)-PCR . BrieflyThe HPV antibody status was analyzed by a glutathione S-transferase (GST) capture immunosorbent assay using as antigens full-length HPV proteins bacterially expressed as GST fusion proteins ,36 in coINK4a and pRb, markers for HPV transformation, were evaluated by immunohistochemistry (IHC). The monoclonal antibodies CINtec  and NCL-RB , were used for p16INK4a and pRb, respectively. Well characterized sections from CxCa and a healthy mucosa were used in each staining batch as reference for scoring of the protein expression levels for both markers. IHC was evaluated independently by two investigators. The tumor cells of the sections showed strong nuclear and diffuse cytoplasmic staining of p16INK4a (>95%) and low staining of pRb (<25%), while proliferating basal and parabasal cells of the tumor-adjacent mucosa showed low p16INK4a (<5%) and high pRb (>25%) expression  presence of HPV58 E6*I RNA encoding the viral oncoprotein E7, which in HPV58-positive cervical carcinoma is a consistent marker of biological activity of the virus ; (ii) sted OPSCC  and alsoed OPSCC ,48, and ed OPSCC . We furtThis HPV58-driven oropharyngeal carcinoma is the first well-documented case of HPV transformation in the oropharynx by a non-16 HPV-type.Written informed consent was obtained from the patient for publication of this Case Report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.CxCa: Cervical carcinoma; ENT: Ear, nose and throat; FFPE: Formalin-fixed paraffin-embedded; GST: Gluthatione S-transferase; Gy: Gray; HPV: Human papillomavirus; IARC: International agency for research on cancer; IHC: Immunohistochemistry; OPSCC: Oropharyngeal squamous cell carcinoma; PCR: Polymerase chain reaction; pRb: Retinoblastoma protein; UbC: Ubiquitin C; WHO: World Health Organization.The authors L. Baboci, D. Holzinger, P. Boscolo-Rizzo, A. Del Mistro, G. Spinato, R. Bertorelle, L. Biasini, A. Michel, R. Bussani, L. Alemany, G. Tirelli and M.C. Da Mosto disclosed no potential conflicts of interest. M. Schmitt and M. Pawlita have received research support through cooperation contracts of DKFZ with Roche and Qiagen in the field of development of HPV diagnostics.LB carried out the molecular analyses and drafted the manuscript, MP conceived the study, participated in its design and coordination, and supervised to draft the manuscript, PBR, GS, GT, RB and MCDM participated in the management of the patient, provided the samples and contributed to draft the clinical part of the manuscript, ADM contributed and supervised to draft the manuscript, DH supervised the molecular analyses and contributed to draft the manuscript, LB and RB performed the TP53 sequencing analyses, AM provided the serological data. MS contributed to draft the manuscript. LA provided unpublished metanalyses data on HPV type 58. All authors read and approved the final manuscript.


Multiple viruses, including human immunodeficiency virus, Epstein Barr virus (EBV) and mouse mammary tumour virus have been identified in human milk. High risk human papillomavirus (HPV) sequences have been identified in breast cancer. The aim of this study is to determine if viral sequences are present in human milk from normal lactating women.in situ polymerase chain reaction (PCR) techniques were used to identify HPV and EBV in human milk samples from normal lactating Australian women who had no history of breast cancer.Standard (liquid) and High risk human papillomavirus was identified in milk samples of 6 of 40 (15%) from normal lactating women - sequencing on four samples showed three were HPV 16 and one was HPV 18. Epstein Barr virus was identified in fourteen samples (33%).The presence of high risk HPV and EBV in human milk suggests the possibility of milk transmission of these viruses. However, given the rarity of viral associated malignancies in young people, it is possible but unlikely, that such transmission is associated with breast or other cancers. High risk human papillomavirus (HPV) is primarily considered as a sexually transmitted infection . HoweverHere we report the identification of high risk HPV types 16 and 18 plus Epstein Barr virus (EBV) in milk from normal lactating Australian women. These findings have in part, been previously reported .This project was formally approved by the University of New South Wales, Australia, Human Research Ethics Committee (HREC 05163). All participants gave written, informed, prior consent.oC for 1-3 hours, after which a phenol extraction followed by 2 chloroform washes were performed before an alcohol precipitation. The DNA pellet was resuspended in Tris-EDTA (TE) and checked with a β -globin PCR analysis. A reagent blank of bovine skim milk was used for each set of milk extractions.Samples of human breast milk were collected from 40 lactating Australian women. None of these women had a history of breast cancer. The milk was expressed into sterile 50ml falcon tubes. To prevent contamination, the milk samples were then transferred into 1.5ml eppendorf tubes using filtered tips for all procedures. The 1.5ml tubes were centrifuged at 2,000 rpm for 10 minutes. The whey and fat were removed and the pellet was washed twice with phosphate buffered saline (PBS). The pellet was then resuspended in 200μl of PBS. 10μl 1M Tris pH8 and 10μl of 0.5M ethylenediaminetetraacetic acid (EDTA) were added to 100ul of the PBS based solution, the mixture vortexed and 2μl 2% sodium dodecyl sulfate (SDS) and 1μl proteinase K (10mg per ml) added. The tubes were heated at 55β-globin primers G073 (5′-GAAGAGCCAAGGACAGGTAC-3′) and G074 (5′-CAACTTCATCCACGTTCACC-3′). HPV screening used nested PCR with outer primers, PGMY, and inner primers GP as per Heng et al [DNA integrity was confirmed by standard PCR using ng et al . For EBVng et al . The cycin-situ PCR, drops of the resuspended cell pellet in PBS were dried on glass slides followed by fixation in ice cold methanol for 5 minutes. An H&E stain of the slides identified the milk samples which contained cells. Selected samples were treated with 0.1% triton X digestion for 10 minutes at room temperature. The primers used for HPV in situ PCR were the outer primers used for the HPV screening. 75 μL of PCR mixture was prepared for each slide with the concentration of the reagents as follows: 7.5 μL 10X PCR buffer (Roche Diagnostics), 0.375 μL dNTP mixture , 0.33 μL digoxigenin (DIG) – 11 – dUTP , 6 μL MgSO4 , 1 μL (10pM) forward primer and 1 μL (10pM) reverse primer, 1 μL Taq Polymerase (Roche Diagnostics) and sterilized water. The thermocycling conditions were as follows: initial denaturation at 95°C for three minutes; 35 cycles at 95°C for one minute, 55°C for one minute and 72°C for one minute and a final extension at 72°C for five minutes.In situ PCR was attempted on a different set of milk samples. To prepare the milk samples for Six of the forty milk samples 15%) were positive for HPV. Sequencing of four samples showed three were positive for HPV type 16 and one was positive for HPV 18  were positive for EBV. Sequencing of two samples showed identical sequences to an EBNA 1 region of EBV strain B95-8. One of these samples was also positive for HPV 18.In-situ PCR on a centrifuged milk sample showed high risk HPV in the nuclei of epithelial cells , mouse mammary tumour virus and Epstein Barr virus have previously been identified in human milk -14. TranHigh risk HPVs have been identified in both normal breast tissues and breast tumours of Australian women . TherefoThe identification of viral sequences does not prove that these viruses are biologically active. The identification of HPV viral transcripts in human milk would be of future value. With respect to HPV such identification can indicate whether the HPV E6:L1 mRNA expression ratio is high (high E6 low L1) which is an indication that HPV is integrated and probably transforming, that is, the HPV is biologically active within the breast milk epithelial cells.It is not known whether HPVs have long latency periods . It is aEDTA: Ethylenediaminetetraacetic acid; H & E: Haematoxylin and eosin; HPV: Human papilloma virus; HIV: Human immunodeficiency virus; PBS: Phosphate buffered saline; PCR: Polymerase chain reaction; SDS: Sodium dodecyl sulfate; TE: Tris-EDTA.There are no conflicts of interest for any of the authors.in situ PCR, preparation of the manuscript. NJW: supervision and quality assurance of the laboratory work, preparation of the manuscript. JSL: conceptualisation, collection of milk samples, analysis of data, manuscript preparation. All authors read and approved the final manuscript.WKG: collection of milk samples, conduct of standard and


Since the introduction of the HPV vaccine, questions have been asked about its efficacy in preventing cancer linked with HPV. Concerns about the HPV vaccine safety profile have also been raised. This paper highlights the rapidly growing body of evidence  illustrating both the safety of the HPV vaccine, through a detailed investigation of reported adverse events, and its efficacy in reducing both HPV infections rates and the resulting drop in cervical lesions, which have been demonstrated to be good predictors of cervical cancer risk. The first human papillomavirus (HPV) vaccine, Gardasil®, was registered in Australia in 2006 and was followed in 2009 by Cervarix®. However, since the introduction of these HPV vaccines both their safety and efficacy have been questioned . These iin situ (AIS) associated with HPV ; 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, and 82 . ObviousSchiller and colleagues  reviewedAs outlined previously, one of the major outcomes examined by clinical trials of these vaccines is the prevalence of HPV positive AIS and CIN lesions. The PATRICIA study  found CeOther studies also provide evidence for vaccine cross-protection against non-vaccine HPV types in a variety of conditions, such as reduced HPV35  infectioWhile both vaccines are very effective against HPV16/18 and appear to provide cross-protection against some non-vaccine oncogenic HPV types it is worth specifically investigating whether vaccination is actually causing a net reduction in HPV associated AIS and CIN lesions. As Cervarix® had better cross-protective properties it is not surprising that the TVC displayed larger reductions is all HPV associated markers; CIN2+ (33.1%), CIN3+ (45.6%) and AIS 76.9%) [6.9% [7].One of the most frequently raised concerns about HPV vaccination is that one of the major outcomes of clinical trials, cervical intraepithelial neoplasia (CIN) are not good predictors for progression to cervical cancer, thus making it impossible to say on that basis alone whether HPV vaccines will reduce cervical cancer incidence. It is worth taking a moment to investigate if firstly there is a link between HPV and CIN in the first place. As described above, clinical trials have demonstrated HPV vaccination reduces CIN lesion incidence. This is not surprising, as a systematic review and meta analysis of over forty trials, and 22,000 women, found that, although there was a lot of variation in methodology, a persistent HPV infection was “consistently and strongly associated” with CIN2/3 lesions . It has A recent study demonstrated the importance of HPV as a determinant of (pre)invasive cervical cancer when they showed that 3.7% of the women, in a study of over 330,000 women, with normal cervical cytology (pap smear) and positive HPV status experienced 34% of the CIN3+, 29% of the cancers and 63% of the adenocarcinomas . There iIn terms of a biological mechanism we know that certain HPV types are strongly associated with different chromosomal changes, in particular those associated with sections of DNA containing tumor-suppressing genes ,26. ThesSince introduction, safety concerns have been raised about reported serious adverse reactions to HPV vaccination. A number of these concerns are about vaccine ingredients in general but the safety of these ingredients has been well established (reviewed in ). It is Adverse events have been reported following HPV vaccination Table  but clinEvidence from large scale clinical trials has been utilised to assess whether serious adverse events are more likely post HPV vaccination with a systematic review and meta-analysis having been undertaken to examine the combined results of 7 unique randomized clinical trials  of HPV vaccines . These 7Large scale clinical trials can provide information on adverse events prior to mass vaccination but their statistical power is limited by their participant numbers. The clinical trials described above included over 44,000 women and as such may not be expected to reliably detect rare  adverse events. Passive reporting systems such as the U.S. post-marketing safety surveillance program database VAERS can provide information that can help identify (rare) adverse events. As VAERS is an open system where any member of the general public can enter a vaccine reaction it is difficult to directly analyze the publically available data to assess causal relationship between notified events and vaccine administration without further investigation. Gold and colleagues  give an Neisseria meningitidis serogroup B caused meningoencephalitis and the final death was related to prescription drug abuse. The authors concluded that statistically  these results were not significantly (p=0.92) different from what you would expect from a similar sized unvaccinated population.A number of examinations of the VAERS data for HPV vaccination have shown a low rate of adverse reactions, and no link for any causal relationship between HPV vaccination and reports ,44. TherA recent study by an Australian group  systematIt has also been suggested that HPV vaccination can increase the probability of progression of established persistent infection to CIN2+ or higher. This was based on the report of a single small study , howeverOther specific concerns about serious adverse events following HPV vaccination such as the possibility of increased autoimmune conditions in the vaccinated, increased incidence of Guillain-Barré Syndrome (reviewed in ) or incrThe first HPV vaccine was introduced in 2006 and since its introduction it has been a topic of controversy, with a number of questions being asked about the vaccine; Did it work? How long would the protection last? Would there be an increase in HPV types not covered by the vaccine? Did it actually prevent cancer? Was it safe?In the seven years since the registration of the first HPV vaccine these and many other questions have been investigated by the scientific/medical research community. This review describes a large number of studies that have analysed the growing set of safety data, and have demonstrated the safety of HPV vaccines and answered the very specific concerns raised, particularly in regards to nervous system reactions, interactions with other vaccines, and HPV vaccine influencing the course of existing lesions. In terms of virology the current evidence shows that HPV vaccination is highly efficient at preventing vaccine associated HPV types and that protection is well over 90% if given to HPV naïve individuals -4. AdditHPV vaccination has been introduced for less than 7 years and as such it is difficult to quantitate the effect it will have on the incidence of cervical, vulvar/vaginal, penile, anal and other cancers. There is very strong, some say conclusive data, that HPV is the root cause of over 99% of cervical cancers -24,27. HThe goal of this review was to investigate the published scientific/medical literature to determine whether the oft repeated queries about HPV vaccination safety and efficacy have been examined. The rapidly growing body of research, including immunology, virology, public health, epidemiology and a number of other fields, can allow the whole community including doctors, medical researchers, parents and other interest groups to be more confident that the benefits of HPV vaccination far outweigh the risks and that mechanisms are in place to continue monitoring possible adverse events into the future.in situ.HPV: Human papillomavirus; CIN: Cervical intraepithelial neoplasia; MITT: Modified intention to treat; ITT: Intention to treat; TVC: Total vaccine cohort; AIS: Adenocarcinoma The authors have no competing interests and received no funding for this work.DH chose the topic and wrote and researched the initial version of the review. CEL and MJB were involved in the literature research and contributed to the writing and drafting of the manuscript. All authors have read and approved the final manuscript.


In Norway, Pap smears with atypical squamous cells of uncertain significance (ASCUS) and low-grade squamous intraepithelial lesions (LSIL) are triaged after 6 months. The aim of the study was to evaluate effects of implementing human papillomavirus (HPV) test (2005) in delayed triage of ASCUS and LSIL in a cohort of women from Western Norway. After a survey of 119,469 cervical Pap smears during 2005–2007, a total of 1055 women with an index ASCUS or LSIL were included in the study and followed up for 3–6 years with respect to progression into cervical intraepithelial neoplasia grade 2 or worse (CIN2+). Overall sensitivity for detection of CIN2+ with HPV testing and cytology was 96% and 72%, respectively. The sensitivity for detection of CIN2+ was not affected by age, but the specificity of the HPV test increased with age. Thus, for the age groups <34 years, 34–50 years, and >50 years, the specificity of a positive HPV test to detect CIN2+ was 47%, 71%, and 82%, respectively. Positive predictive values for CIN2+ in women with positive cytology, positive HPV test, negative cytology, negative HPV test, or negative HPV and cytology tests were 52%, 41%, 8%, 1.5%, and 0.4%, respectively. HPV testing resulted in a net 22% increased detection of CIN2+. Fifty-six percent of CIN2+ was detected at an earlier time point with HPV testing in triage. Implementation of HPV testing in delayed triage of ASCUS and LSIL improved the stratification of CIN2+ risk and increased CIN2+ detection and at an earlier time point than with triage by cytology alone. Uterine cervical cancer is the second most common cancer among women worldwide Most of the hrHPV infections regress spontaneously, whereas some progress to high-grade lesions and eventually into cancer A hrHPV infection can be detected through HPV testing. This method is more sensitive, but less specific than cytology to detect clinical relevant infections In 1995, an organized screening program for cervical cancer was introduced in Norway. Women with an index ASCUS or LSIL Pap smear were triaged with a control smear after 6 months. In 2005, HPV testing was introduced in the triage of ASCUS and LSIL within the program.In this study, we report on women with an index sample of ASCUS or LSIL and monitoring with cytology and HPV testing in delayed triage. A major aim of this study was to evaluate if HPV testing would recover more CIN2+ than cytology alone and enable a more differentiated follow-up screening algorithm in selected groups of women.In Norway, cervical screening is organized as a triennial screening to all women, aged 25–69 years, and with a written reminding letter to all non-attending women.A total of 119,469 smears were screened during a 3-year period from 2005 to 2007, and patients with ASCUS or LSIL were identified. Among these, 1055 patients were enrolled in the study. The patients were followed up over the next 3–6 years with respect to progression into CIN2+ based on diagnostic cervical biopsies and/or cervical resected cones. The more severe lesion was recorded. All samples were examined at Haukeland University Hospital, which is a regional hospital of Western Norway.Positive cytology means a Pap smear Negative cytology means a normal or reactive smear without suspicion of ASCUS+. Index sample refers to the initial Pap smear that showed ASCUS or LSIL and led to enrollment in the study. The first follow-up test refers to the first control test performed after the index sample.All women encountered with ASCUS or LSIL during the period 2005–2007 were evaluated for enrollment. Women with a positive cytology or positive HPV test during the previous 2 years were excluded. These women were not considered to have a de novo cytologically encountered cervical lesion. Lack of such information did, however, not exclude women from being enrolled in the study, as many of these women had never attended a screening program previously. Furthermore, the HPV test should be sampled together with the first follow-up smear or less than 1 month before or after the smear was taken.A total of 2172 women with ASCUS or LSIL were identified. Women with (1) prior abnormal smear(s) and/or positive HPV tests, (2) lack of follow-up information, and incomplete HPV follow-up of ASCUS/LSIL were omitted from the study  identifies the presence of hrHPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68 without notifying the presence of any single hrHPV subtype. The HPV test results were normally not available to the cytotechnicians screening the smears, but available to the pathologist who finally signed out the smears.A separate brush sample for HPV testing was normally taken in parallel with the Pap smear. The HPV samples were stored at 4°C until further processing. The HPV DNA test  versus cytology alone is that HPV-positive women will be referred for immediate colposcopy if cytology is positive. HPV-positive women with negative cytology are also followed up with new cytology smears and/or colposcopy until the HPV test is negative (regression of disease) or has progressed to CIN2+. All HPV-negative women are referred back to routine screening. In the triage modality with cytology alone, an index ASCUS sample is referred back to routine screening if the follow-up cytology is normal (negative). For index LSIL, two ensuing cytology smears must be normal before the woman is referred back to routine screening. A woman will be referred for colposcopy if two ensuing repeat control tests show persistent equivocal or low-grade lesions. In both screening modalities, immediate colposcopy will follow if cytology shows high-grade lesion or suspicion of its presence.http://www.kreftregisteret.no).An evaluation of the cervical test records in relation to the clinical management algorithms was done by three cytopathologists . A complete outline of the management guidelines of ASCUS/LSIL is available at The Cancer Registry of Norway  for detection of CIN2+ in cytology and with HPV testing refer to the first follow-up cytology and HPV test taken after index ASCUS/LSIL. The cytology test was considered correctly positive if ASCUS+ identified a woman with CIN2+ or one who developed CIN2+ during follow-up. This definition of true positivity in cytology has also been used by others Cumulative incidence rates were calculated using the method described by Kaplan and Meier Sixty-eight percent of the women in the study group Fig.  had theiMost women with LSIL were younger than 34 years, whereas most women with ASCUS were older than 34 years Table . A skeweThe significance of cytology and HPV testing for detection of women with CIN2+ was then evaluated in delayed triage. Among 1055 women with index ASCUS or LSIL, 214 CIN2+ were recovered during the observation period of 3–6 years  was observed by HPV testing in triage as compared to cytology alone . In index LSIL women, HPV testing led to only a moderate 8.5% increased detection of CIN II (P = 0.4).A delayed triage algorithm with cytology and HPV testing versus cytology alone was evaluated for recovery of CIN2+ in women with index ASCUS or LSIL. A net 22% increased CIN2+ detection . In the triage modality with cytology and HPV testing CIN2+ was detected at an earlier time point than in the triage modality with cytology alone  with ASCUS or LSIL at baseline did not have a HPV test at their first follow-up smear Fig. . In the In the ALT's study, triage of ASCUS with HPV test was clinically useful, but of limited value in triage of LSIL as most (82.9%) of these women were reported to be HPV positive The cumulative incidence rates of CIN2+ in the HPV-positive women were high in this study  for developing CIN2+ at baseline In this study, an effect of cytology and HPV testing versus cytology alone on detection of CIN2+ in triage of ASCUS and LSIL was based on the evaluation of all cytology and surgical pathology reports of each woman with CIN2+ included in the study. This approach will only give estimates for detection of CIN2+ with and without HPV testing in triage of ASCUS and LSIL, and is related to the assumption that women triaged with cytology alone fully adopt the clinical management algorithm. Also, the effect of implementing HPV testing in triage is based on results at the first follow-up after index ASCUS and LSIL. This means that the potential for a secondary catch up of a previously missed CIN2+ in the first screening round with cytology or HPV test is not accounted for by this approach.We report an estimated net 22% increased recovery of CIN2+ by the use of HPV testing versus cytology alone Fig. . More thIt was observed that in a screening modality based on delayed triage of ASCUS/LSIL very few women with an index ASCUS smear above 50 years of age were HPV-positive, and for those the specificity of a positive HPV test for CIN2+ approached the specificity of a positive cytology test. Furthermore, HPV testing led to an increased and more rapid detection of CIN2+ and to an improved stratification of CIN2+ risk. Thus, positive HPV tests showed high PPVs for CIN2+, indicating that the clinical relevant lesions were identified in this screening algorithm. A particular low risk for CIN2+ was observed in HPV test negative women indicating that a more differentiated routine screening protocol could be advocated for these women.None declared.


In addition a catch-up programme was implemented for older girls up to 18 years of age from 2009 to 2011, with an uptake rate of 49.4%. Information about future uptake of cervical screening according to vaccination statistics is important in order to understand the impact of the vaccination programme and implications for a national cervical screening programme. We analysed data on a cohort of women who had been offered the HPV vaccine in the catch-up programme and were invited for cervical screening between 2010 and 2012 in Wales  women had had HPV vaccination and 45.9%  women attended for cervical screening. Women who were unvaccinated were less likely to attend cervical screening ). Of those who attended for screening, 13.9% of vaccinated women had abnormal cytology reported compared to 16.7% of women who were unvaccinated. Women who lived in areas with high levels of social deprivation were less likely to be vaccinated ) or attend cervical screening ) compared to those who lived in the least deprived areas.In our cohort, 48.5% (These data highlight the need for new strategies to address inequalities in cervical screening uptake and can inform further mathematical modelling work to clarify the impact of the HPV vaccination programme on future cervical cancer incidence. Both vaccines prevent acquisition of HPV16 and 18 infections There are two commercially available Human Papillomavirus (HPV) vaccines licensed by the FDA for prevention of cervical cancer: Cervarix® was introduced in September 2008 in schools, with a recommended 3 doses administered to girls aged 12–13 years. A two-year catch-up vaccine arm was added for older girls who potentially would still benefit from the immune response induced by the HPV vaccine. Such a comprehensive national vaccination programme is expected to change the epidemiology of cervical cancer in the UK population. However, the impact of such a programme will depend on vaccine uptake, cervical screening uptake and the risk of exposure in women who are not vaccinated and not screened. If women who are unvaccinated choose not to attend for cervical screening, and have high risk of exposure to HPV, then the impact of the vaccination programme will be less than predicted, with potential to increase inequalities in cervical cancer incidence in the population.In the UK, a national HPV vaccination programme using the bivalent vaccine, CervarixIn order to understand the likely impact of the HPV vaccination programme for cervical cancer incidence it is important to understand the screening behaviour of women according to whether or not they have been vaccinated. In this study we report the factors associated with HPV vaccination uptake, cervical screening uptake and clinical outcome according to HPV vaccination status for the first cohort of women who had been offered the HPV vaccine and were invited for cervical screening within a national population-based cervical screening programme in Wales.22.1The study population comprised women who were born between 1 st September 1990 and 29th February 1992 who were resident in Wales on 1 st April 2012. These women would have been offered HPV vaccination as part of the catch-up campaign, and invited for routine cervical screening between 1st September 2010 and 29th February 2012 as they turned 20 years of age, or after moving into Wales.2.22.2.1The Centre for Improvements in Population Health through e-Records (CIPHeR) has established the Secure Anonymised Information Linkage (SAIL) databank, which brings together and anonymously links a wide range of person-based data HPV vaccination data (number of doses and dates administered) were extracted from both the CSW and NCCHD databases and triangulated to give a complete vaccination history for the cohort of women. Data on cervical screening uptake and clinical outcome were obtained from the CSW databases. Data on birth characteristics of the women such as maternal age at birth, gestational age at birth and childhood vaccination status (for any childhood vaccinations as per the recommended schedule for immunisations in the UK) were extracted from the NCCHD. Data on quintile of social deprivation was based on Townsend score calculated using data from the 2001 Census, based on the woman's area of residence on April 1st 2012.2.3All analyses were carried out using SPSS v19. Univariate binary logistic regression was used to describe the association between each variable  and (i) HPV vaccination uptake, (ii) cervical screening uptake and (iii) cervical screening abnormality. Multivariate binary logistic regression was used to obtain odds ratios for the association between HPV vaccination uptake and cervical screening uptake, adjusted for the variables listed above.Women were categorised as having been partially HPV vaccinated if only 1 or 2 of the recommended 3 doses were recorded, and fully HPV vaccinated if 3 or more doses were recorded.Childhood vaccination was defined as any childhood vaccination recorded on the NCCHD database (excluding HPV vaccination). A cervical screening cytological abnormality was defined as a result of borderline changes, mild/moderate/severe dyskaryosis or worse. Results reported as inadequate or negative were considered not abnormal for this analysis.Data were missing for some variables in the cohort: maternal age (29.7%); gestational age (33.9%); and childhood vaccinations (21.1%). We carried out a complete case analysis and analysis that included the missing data as a separate category. The results were similar in both models so we have presented the results with missing data as a separate category.The analyses were restricted to cases with available social deprivation data based on the Townsend score for deprivation quintile 3There were 33,601 women on the NHS AR for the study cohort and time period. Data were available for 30,882 women from the CSW and 24,351 women from the NCCHD . 14,966/n = 10,109/30,882; 32.7%); (ii) partial HPV vaccination with 1–2 doses ; (iii) not HPV vaccinated .There was a statistically significant relationship between uptake of the HPV vaccine and social deprivation quintile . Women fThe highest proportion of women not vaccinated was observed for the groups with maternal age under 20 years and 20–24 years  compared to groups whose mothers were older and this was statistically significant  and OR 0.80; 95% CI , respectively). There was no clear relationship between gestational age and HPV vaccination.Women who were fully vaccinated were more likely to have attended for cervical screening than women who had not been vaccinated and this was statistically significant ). In women who had attended cervical screening, 8006/14,164 (56.5%) had received at least one dose of the HPV vaccine. In women who had not attended for cervical screening, 6960/16,718 (41.6%) had received at least one dose of the HPV vaccine.Reported cervical screening cytological abnormalities in the study population are shown in 4This is the first UK study to investigate uptake of cervical screening following implementation of the HPV vaccination programme in the catch-up group. In contrast to concerns that vaccination would have a negative impact on a woman's decision to attend for cervical screening, uptake of the HPV vaccine was positively correlated to uptake of cervical screening. Social deprivation was the main factor affecting uptake of both the HPV vaccine and cervical screening, with the highest levels of non-participation observed in the most deprived quintile (59.2% unvaccinated and 58.7% unscreened compared with 41.3% and 49.9% in the least deprived quintile).In women who attended for cervical screening, HPV vaccination had a protective effect with the lowest proportion of cytological abnormalities detected  had neither attended for screening nor received the HPV vaccine. However, although social deprivation affected the initial engagement, once women engaged, at least in this age group, there was no significant difference in clinical outcome. Cervical cancer rates are higher in women from more socially deprived backgrounds In women offered HPV vaccination through the catch-up arm of the programme, this study shows a protective effect with a reduction in cytological abnormalities from 16.7% in unvaccinated women to 13.9% in vaccinated women. However, the level of abnormalities detected in the vaccinated women is still relatively high, potentially reflecting acquisition of the virus prior to vaccination. This data suggests that the catch-up arm of the vaccination programme has not had a substantial protective effect and a higher impact on cytological abnormalities is anticipated in the target group, who may not have been exposed to the virus prior to vaccination. Women who have chosen to receive the HPV vaccination and attend for cervical screening may be more health conscious, and this may be reflected in their sexual behaviours. It is therefore possible that they may be less likely to become infected with HPV, accounting for the reduction seen in the proportion of cytological abnormalities.The findings reported here emphasise the need to promote further engagement with health services in more socially deprived areas with a focus on younger age groups to enhance the potential benefit of prevention programmes in early diagnosis and treatment long term. The HPV vaccination programme represents an ideal opportunity to convey the benefit of prevention programmes and reinforcement of this message is needed.5Uptake of HPV vaccination was positively correlated with uptake of cervical screening, and cytology results indicate that vaccination has a protective effect against an abnormal result. Women from more socially deprived areas engage less with cervical cancer prevention healthcare services. New strategies to enhance uptake of screening services need to be directed at young women with a focus on areas classified as socially deprived.SP and SH conceived of the study. HB, SB and MAR collected the data for the study. HB, SH and SP contributed to the analyses of the study and all authors contributed to the interpretation of results and the writing of this paper and have approved the final draft.All authors declare no conflicts of interest that could have influenced this work.


OESCC cell lines which are p38δ-negative (KE-3 and -8) grew more quickly than cell lines (KE-6 and -10) which express endogenous p38δ. Re-introduction of p38δ resulted in a time-dependent decrease in OESCC cell proliferation which was exacerbated with p-p38δ. In addition, we observed that p38δ and p-p38δ negatively regulated OESCC cell migration in vitro. Finally both p38δ and p-p38δ altered OESCC anchorage-independent growth. Our results suggest that p38δ and p-p38δ have a role in the suppression of OESCC. Our research may provide a new potential target for the treatment of oesophageal cancer.Oesophageal cancer is an aggressive tumour which responds poorly to both chemotherapy and radiation therapy and has a poor prognosis. Thus, a greater understanding of the biology of oesophageal cancer is needed in order to identify novel therapeutic targets. Among these targets p38 MAPK isoforms are becoming increasingly important for a variety of cellular functions. The physiological functions of p38α and -β are now well documented in contrast to -γ and -δ which are comparatively under-studied and ill-defined. A major obstacle to deciphering the role(s) of the latter two p38 isoforms is the lack of specific chemical activators and inhibitors. In this study, we analysed p38 MAPK isoform expression in oesophageal cancer cell lines as well as human normal and tumour tissue. We observed specifically differential p38δ expression. The role(s) of p38δ and active (phosphorylated) p38δ (p-p38δ) in oesophageal squamous cell carcinoma (OESCC) was delineated using wild-type p38δ as well as active p-p38δ, generated by fusing p38δ to its upstream activator MKK6b(E) via a decapeptide (Gly-Glu) Oesophageal cancer is the seventh most common cancer worldwide  with its2-terminal kinase (JNK) and p38 MAPK families. The p38 MAPK family consists of four members; p38α (MAPK14) of which there are two splice variants (The mitogen-activated protein kinases (MAPKs) are serine/threonine kinases and include the extracellular-regulated kinase (ERK), c-jun NHvariants , p38β , enzymes from New England BioLabs  and primary antibodies from Cell Signaling Technologies , unless otherwise stated.The patient cohort consisted of ten patients with OESCC of both genders ranging in age from 44 to 81 years. Formalin-fixed, paraffin-embedded (FFPE) oesophagectomy specimens from ten patients consisted of ten paired samples of primary tumour and metastatic lymph nodes with 10 samples of non-tumour adjacent tissues (NAT). Patient features are summarized in μg/ml streptomycin and 100 U/ml penicillin. KE cell line features are summarized in μg/ml streptomycin and 100 U/ml penicillin. KYSE-450 cells  were maintained in 45% RPMI-1640/45% Ham’s F-12 nutrient mixture supplemented with 10% FCS, 100 μg/ml streptomycin and 100 U/ml penicillin.The KE oesophageal cancer cell lines  –17 as we4 cells/well in a 6-well tissue culture plate. Cell viability was assessed by trypan blue (0.4% w/v) exclusion assay at the indicated times  according to the manufacturer’s instructions.Nuclear and cytosolic fractions were isolated from 2×10SwaI restriction sequence using a QuikChange Lightening Site-Directed Mutagenesis kit (Agilent Technologies) . A PCR generated DraI-DraI fragment encoding FLAG-p38δ with a 5′ (Gly-Glu)5 linker  to facilitate folding as previously described (SwaI linearised pcDNA3-MKK6b(E) with T4 DNA ligase. The pcDNA3-MKK6b-FLAG-p38δ plasmid (inactive MKK6b) was created by the substitution of Glu151 and Glu155 with Ser and Thr respectively by site-directed muta-genesis . The pcDNA3-MKK6b(E)-FLAG-p38δDN (dominant negative) (p-p38δDN) plasmid was created by substituting Thr180 and Tyr182 of p38δ with Ala and Phe respectively by site-directed mutagenesis . DNA sequence analysis confirmed the integrity of all plasmids.p38δ (pcDNA3-FLAG-p38δ) and constitutively active MKK6b [pcDNA3-MKK6b(E)] plasmids were a kind gift from Professor J. Han  and have previously been described . To consescribed  was ligaμg of plasmid DNA according to the manufacturer’s instructions. Twenty-four hours following transfection cells were transferred to 100-mm diameter dishes and transfected cells were selected in growth medium containing 800 μg/ml Geneticin. After 4–8 weeks, individual cell colonies were transferred for clone expansion.KE-3 cells were transfected using Lipofectamine™ 2000 reagent (Life Technologies™) and a total of 4 2 antibody (Zymed Laboratories Inc.) have previously been described by us (® WestDura Extended Duration Substrate (Pierce Biotechnology) and bands were visualized using a Syngene G:Box ChemiXR5 Gel Documentation System.Supernatants used for immunoblotting with specific antibodies, p38α and -δ, phospho-p38 MAPK and MKK6 antibodies (New England Biolabs), p38γ (Upstate) and p38βed by us ,21. ChemThis was performed as previously described by us . Briefly® IC Human phospho-p38δ (T180/Y182) sandwich ELISA (DYC2124-5) according to the manufacturer’s instructions. Absorbance was read at 450 nm on a Tecan Sunrise spectrophotometric plate reader and analysed using the XRead software program.Cell lysates were analysed for p38δ phosphorylation at T180/Y182 using the R&D Systems DuoSet4 cells/well into a 96-well plate of the upper chamber. The bottom chamber contained 10% FCS as the chemoattractant. Cells were left migrate for 24 h through the matrigel filter (8 mm). Migrated cells were treated with MTT -2,5-diphenyltetrazolium bromide) (5 mg/ml) and absorbance read at 540 nm to calculate viable cell numbers as previously described  agar on an underlay of 0.8% (w/v) agar. After a 21-day incubation colonies were stained with MTT (5 mg/ml) overnight and counted.The role of p38δ in anchorage-independent growth was assayed using a soft agar colony-forming assay as previously described . Cells wKE-6 cells at 75% confluency in antibiotic-free media were transfected with 100 nM p38δ MAPK siRNA or control siRNA-A  according to the manufacturer’s instructions and as recently described .® VILO™ cDNA Synthesis kit (Life Technologies) from total RNA isolated from cells using an Illustra RNASpin Mini kit  according to the manufacturer’s instructions. p38δ mRNA was amplified from cellular cDNA under the following conditions: ddH2O, 1X DreamTaq buffer, 0.2 mM dNTPs, 0.25 μM p38δ forward primer: 5′-CCACGTTAAACTGCCCATCT-3′, 0.25 μM p38δ reverse primer: 5′-CCGCCACAAGCTAAAAAGAG-3′, 1 μl cDNA and 1 U DreamTaq DNA polymerase . RT-PCR products were analysed by agarose gel electrophoresis.First-strand cDNA was synthesised using SuperScriptThe relative levels of phosphorylation of 26 kinases was examined in cell lysates using a Proteome Profiler Human Phospho-MAPK array  according to the manufacturer’s instructions. Following chemiluminescent detection, pixel density of each spot was analysed using Scion image software.The research was approved by the Clinical Research Ethics Committee of the Cork Teaching Hospitals.Results are expressed as mean ± SE. Statistical comparisons were made by using analysis of variance with subsequent application of Student’s t-test, as appropriate. GraphPad InStat 3 software was used also for statistical analysis.2, -γ and -δ as previously described by us . Upstream MKK3 and -6 are thought to be the major protein kinases responsible for p38 activation  in FFPE oesophagectomy specimens from ten patients with squamous cell carcinoma. Samples consisted of ten paired primary tumour and metastatic (lymph nodes) as well as corresponding non-tumour adjacent tissues (NAT) as outlined in To investigate whether the p38 isoform expression pattern we observed The results obtained for differential p38δ expression in both the oesophageal cell lines and the human samples prompted us to investigate further the effect(s) if any this particular isoform may have on the tumourigenicity of OESCC. Firstly, we examined whether the absence or presence of endogenous p38δ expression could have an effect on the proliferation rate of our OESCC cell lines. Using the trypan blue exclusion assay we compared the proliferation rate of KE-3 and -8 cell lines (which do not express p38δ) versus KE-6 and -10 (which express p38δ). We observed that at all time-points studied (24–120 h) both cell lines KE-3 and -8 proliferated faster than KE-6 and -10 cells .5-FLAG-p38δ (data not shown) as well as pcDNA3-MKK6b(E)-(Gly-Glu)5-FLAG-p38δ both produced a single polypeptide with a molecular mass of 82 kDa as expected when using p38δ, p-p38 and MKK6 antibodies, respectively  (data not shown). As expected we did not observe phosphorylation of p38δ in cells transfected with p-p38δDN (To investigate whether p38δ or active (phosphorylated) p38δ (p-p38δ) drives the observed anti-proliferative phenotype  we re-inectively . As bothtibodies  but did tibodies . Of notetibodies . This istibodies  and humatibodies . A 4-foltibodies . This lep-p38δDN . We alsop-p38δDN .To ensure specific phosphorylation of p38δ only and not the other three p38 isoforms  we performed a human phospho-MAPK antibody array (R&D Systems). We did not observe phosphorylation of p38α, -β or -γ in nontransfected KE-3 cells or cells stably transfected with p38δ or p-p38δ . We did,μM). To confirm our immunohistochemical findings cytosolic and nuclear extracts were prepared from transfected and non-transfected KE-3 and KE-6 cells and examined by western blot analysis. The use of PARP as a nuclear-restricted marker and Paxillin as a cytosolic marker ensured that there was no cross contamination between the subcellular fractions  or a nuclear export signal (NES) but their subcellular localisation can be regulated in part by their interacting proteins . We compractions . Similarractions .DN demonstrated the same proliferation rate as non-transfected cells or cells transfected with pcDNA3 only  (A key characteristic of cancer cells is their ability to migrate and progress from primary tumours to metastases in distant organs. A recent report summarizes the roles of p38 MAPKs in cancer invasion and metastasis . This re(177±21) . In cont(177±21) .Oesophageal cancer is a highly aggressive treatment-refractory disease with a high mortality rate ,5,6. As in vivo. The loss of p38δ expression provides a survival advantage for OESCC which demonstrates increased cell proliferation, migration and contact inhibition. Re-introduction of p38δ, however, leads to reversal of these tumourigenic effects. Thus, recent evidence (Emerging role(s) for p38 MAPKs in different aspects of cancer has recently been outlined. To-date the best studied and reviewed isoform in cancer is p38α. It has been characterized as both a potential tumour suppressor ,37–39 anevidence  as well The fusion of p38δ to its upstream kinase MKK6b or active MKK6b [MKK6b(E)] generated a constitutively active p38δ which was used as a tool to study its specific effect(s) in OESCC. Re-introduction of p38δ (with subsequent activation) or active p-p38δ into KE-3 OESCC attenuated cell proliferation, migration and anchorage-independent growth. The strength and duration of p38 activation has been shown to play a crucial role in determining cell fate. Strong activation has been shown to induce apoptosis whereas lower levels results in cell survival ,39. In o−/− cardiomyocytes from transgenic mice , ERK1/2 or JNK1/2/3 expression (data not shown) or activation levels. This is in agreement with a recent bio-informatics analysis of MAPK pathways which specifically identified that persistent activation of p38δ is resistant to interaction with other MAPKs . This lanic mice . We did Reports of the involvement of p38 MAPKs in a variety of different pathological conditions is continuing to increase fuelling interest in the development of potent and specific drugs for modulating the activity of these kinases. Presently there are a number of p38 inhibitors undergoing clinical trials for the treatment of inflammatory diseases ,45. ResuIn conclusion, our results reveal previously undocumented p38δ differential expression and function in OESCC. We identified a subset of OESCC cell lines as well as human primary and metastatic tumour samples that exhibit downregulation of p38δ protein expression. We now provide evidence that loss of expression of this particular isoform may be a mechanism by which OESCC cells promote carcinogenesis. Re-introduction of p38δ into OESCC negative cell lines suppressed different aspects of tumourigenesis. Our data warrant further investigation to understand the important physiological and pathophysiological effects of p38δ in OESCC and is currently in progress. This knowledge should identify which pathways, substrates or regulators are affected specifically by p38δ in providing an antitumourigenic effect in OESCC. Armed with this information uncovering novel targets and the development of new therapeutics may be possible for this common cancer that continues to demonstrate a generally poor clinical outcome.


TSG101) in cervical cancer cells. To identify the mechanism responsible for TSG101 downregulation during cervical cancer development, we analyzed the TSG101 promoter using cis-element cluster finder software. One of the transcription factors whose binding site was detected in the TSG101 promoter was late SV40 factor (LSF). The aim of this study was to analyze the TSG101 protein and LSF expression levels during cervical cancer development. Immunohistochemical analysis confirmed a previously observed decreased expression of TSG101, whereas quantitative polymerase chain reaction (qPCR) and immunohistochemistry analysis revealed high expression of LSF in cervical, precancer and cancer cells compared with human papillomavirus (HPV)-negative non-cancer samples. High expression of LSF in cervical cancer HPV-positive cells suggests that this protein may be important in the regulation of TSG101 expression, as well as in cervical carcinogenesis. The role of LSF as a mediator in cervical cancer development must be confirmed in future studies.Our previous study demonstrated a decreased expression of tumor susceptibility gene 101 ( Human papillomavirus (HPV)-mediated transformation of human cervical epithelial cells has been recognized as a multi-step process in which not only viruses but also certain additional unknown factors and (epi)genetic events are required.TSG101)  software. One of the transcription factors, whose binding site was detected in the TSG101 promoter, was LSF  promoter . Late SVme 12q13 ,20. LSF me 12q13 –24.Human LSF is a 502-amino acid protein with a molecular weight of ~57 kDa . It consWith the exception of its important role in regulation of viral and cellular promoters, including SV40, and HIV-1 promoters, including fibrinogen and α-globin, LSF is involved in numerous other important biological functions, such as regulation of the cell cycle, cell growth and development, DNA synthesis, cell survival and Alzheimer’s disease. Moreover, LSF also functions as an anti-apoptotic factor ,29,30.The aim of the present study was to analyze TSG101 and LSF protein expression during cervical cancer development.Samples were collected from 116 patients  undergoing gynecological surgical procedures at the Department of Obstetrics and Pathology of Pregnancy, Medical University . The study group consisted of 29 HPV-positive cervical samples (carcinoma colli uteri/squamous cell carcinoma), 30 HPV-positive high-grade squamous intraepithelial lesion (HSIL) samples, 28 HPV-positive low-grade squamous intraepithelial lesion (LSIL) samples and 29 histopathologically normal, HPV-negative cervical tissues obtained from women undergoing treatment for reasons other than cervical cancer. The study was approved by the Ethics Committee of the Medical University of Lublin. For cancer and neoplastic localization, all specimens initially underwent hematoxylin and eosin staining followed by a pathological review. Cervical sections comprising ≥70% cancer cells were used as cancer samples. The tissue samples were frozen immediately in liquid nitrogen and stored at −80°C until further analysis. Patients provided written informed consent.Total DNA was isolated from study cells using a QIAmp DNA Midi kit  according to the manufacturer’s instructions.HPV DNA was identified by polymerase chain reaction (PCR) amplification of the HPV gene sequence using isolated DNA and primers: MY09, MY11  and LC1,Total RNA was isolated from normal, dysplastic (LSIL and HSIL) and cancer tissues using an RNeasy Mini kit (Qiagen) following the manufuacturer’s instructions. DNA was removed by DNase treatment .Total RNA (1 μg) was reverse transcribed to cDNA using the QuantiTect Reverse Transcription kit (Qiagen). PCR reactions were run under the following conditions: pre-denaturation at 95°C for 5 min, then 40 cycles at 95°C for 10 sec, 63.8°C for 20 sec, 72°C for 20 sec and a final extension at 72°C for 6 min. qPCR was performed on a Corbett Rotor-Gene 6000 .PCR was performed using SYBR Green PCR master mix  and appropriate primers . Total cDNA (2 μl) and primers were added to 8 μl Power SYBR Green PCR master mix.Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as reference gene . All reaImmunohistochemical analysis was prepared using a LSAB System-HRP visualization kit , mouse monoclonal anti-TSG101 antibody  and mouse monoclonal anti-LSF antibody . Antibodies were diluted in Dako antibody diluent with background-reducing component .2 (0.5×0.5 mm).Immunohistochemical evaluations of TSG101 and LSF expression were performed independently by two pathomorphologists. The cells were counted on an Axiophote (Opton) fluorescence microscope  with ×200 magnification on a field of 16 squares (4×4 squares), which corresponded to an area of 0.25 mmPositive staining was scored according to the percentage of cells with positive staining and staining intensity. Measurement of immunoreactive cells was performed using Cell-2 software, version 4.1 . The quantitative method is based on the analysis of the color distribution and the optical density. The software identifies cells with a greater optical density than the background and, on the basis of the color ratio, classifies cells as immunoreactive. To determine the percentage of positive cells in the sections, the counts of immunopositive cells were divided by the total cell count. For each case, a minimum of 5,000 total cells were counted in a single section.Data were analyzed with Statistica software, version 6.1. . Statistical significance was analyzed using Kruskal-Wallis with post-hoc Dunn’s test (immunohistochemistry) and one-way analysis of variance with Tukey’s post hoc test (qPCR). The difference was considered to be statistically significant when P-values were <0.05.TSG101 promoter sequence were analyzed using Cister software  as described previously  lower percentage of TSG101-immunopositive cells in cervical cancer and HSIL samples compared with that in non-tumor (HPV-negative) cells . The TSGLSF mRNA in dysplastic (LSIL and HSIL) and cervical cancer cells. LSF mRNA level was upregulated to 42.43% in HSIL samples, 30.6% in LSIL samples and 37.54% in cancer samples, compared with that in non-tumor HPV-negative samples  . LSF wasIn addition to HPV viruses, numerous factors, such as oncogenes and tumor suppressor genes are involved in cervical cancer development.Our previous results demonstrated a decreased expression of TSG101 in cervical cancer cells (TSG101 is constitutively expressed in a number of human tissues (TSG101 was found in thyroid papillary carcinomas, and breast, ovarian and gastrointestinal tumors, while downregulation of TSG101 was observed in endometrial and cervical cancers ( tissues . However cancers ,36.TSG101 promoter and regulate its expression is LSF.Our bioinformatics analysis showed that one of the factors that may bind with the qPCR and immunohistochemical analysis revealed high expression levels of LSF in cervical cancer cells compared with non-cancer samples. These results suggest that LSF is important in cervical tumorigenesis and that LSF mRNA levels generally do not fluctuate. LSF mRNA has been suggested to be a normalization control in gene expression profiling . LSF is LSF is a transcription factor involved in the regulation of a variety of viral and cellular promoters. It acts as a transcription activator and repressor . LSF stiTSG101 gene promoter and HPV-dependent cervical carcinogenesis. Fan et al identified LSF as a downstream mediator of Notch1 signaling and showed that LSF mediates, at least in part, Notch-1-induced carcinogenesis (High expression of LSF in cervical cancer HPV-positive cells suggests that this protein may be involved in downregulation of the ogenesis . Notch gogenesis , head anogenesis , lung caogenesis , colon cogenesis , acute mogenesis  and diffogenesis . LSF wasogenesis . In liveogenesis . Microarogenesis . It has ogenesis ,30,50. Oogenesis .A major cellular target of LSF is the thymidylate synthase gene, which encodes the enzyme involved in the production of dTTP, required for DNA synthesis. Deregulated LSF expression may facilitate entry into the G1/S phase of the cell cycle, promote DNA synthesis, stimulate transformation and facilitate cancer cell survival. Inhibition of LSF results in either apoptosis during S phase or cell cycle arrest at the G1/S transition ,30,50.The role of LSF as a mediator in cervical cancer development must be confirmed in future studies.