In situ hybridization (ISH) for a wide range of HPV DNA subtypes and EBV-encoded RNA was performed in the tissue samples obtained from these patients. Only two cases of primary squamous papilloma in the middle ear have been previously reported in the English literature. These papillomas developed in males of ~60-years of age and otorrhea was the most frequent complaint. Premalignant changes were observed in two of the present cases and ISH of HPV and EBV was negative in all four cases. The results of the present study indicated that chronic inflammatory stimulation, not HPV and EBV infection, is involved in the occurrence of middle ear squamous papilloma and its malignant transformation. Radical surgery and long-term postoperative follow-up are recommended due to its malignant and recurrent potential. Further genetic investigations with additional new cases are required to clarify the pathogenesis of squamous papilloma involving the middle ear.Squamous papilloma involving the middle ear as a primary lesion is an extremely rare occurrence. The aims of the present study were to investigate the presence of human papilloma virus (HPV) and Epstein-Barr virus (EBV) infections in primary middle ear squamous papilloma and to describe the clinical and pathological features of the disease along with therapeutic strategies. A retrospective review was conducted of four patients with clinical and pathological diagnoses of middle ear squamous papilloma.  Squamous papillomas are benign epithelial tumors that occur on the skin of the face and body and most often appear in the mouth or genital regions. Squamous papilloma involving the middle ear as a primary lesion is an extremely rare occurrence . Few casin situ hybridization (ISH). The pathogenesis and diagnostic, therapeutic and prognostic aspects of this tumor are also discussed in hope that the results of the present study are useful for clarifying diagnostic and therapeutic strategies for this type of papilloma and the involvement of HPV and EBV infections.Although no causal correlation has been established between viral infections, including HPV and EBV, and the development of middle ear squamous papilloma, it remains a possibility that requires important consideration. The current study reported four cases of primary middle ear squamous papilloma and the results of HPV and EBV Between 2009 and 2012, four patients were treated at the First Affiliated Hospital of Nanjing Medical University  with an initial pathological diagnosis of squamous papilloma of the middle ear. The records of these four patients were retrospectively reviewed, including the clinical history, treatment, follow-up, radiological data and pathology reports. Paraffin-embedded tissue blocks from the middle ear of these patients were recovered, sectioned and stained with hematoxylin and eosin. To avoid interobserver variations, two pathologists reviewed all pathological slides and were in agreement with the final pathological reports. All lesions in this study were associated with the middle ear and there was no evidence of prior papillomas in the external auditory meatus or nasopharynx. The current study was approved by the Institutional Review Board of the First Affiliated Hospital of Nanjing Medical University. Written informed consent was obtained from the patients.For detecting the presence of HPV, ISH was conducted with a wide-spectrum digoxigenin-labeled probe  for common HPV types according to the manufacturer’s instructions. The wide-spectrum probe targets the genomic DNA of HPV types 5, 6, 8, 11, 16, 18, 26, 27, 30, 31, 33, 35, 39, 40, 41, 42, 43, 45, 47, 48, 51, 52, 53, 54, 55, 57, 58 and 59. Sections from the tissue blocks were deparaffinized and rehydrated in graded alcohols and distilled water. Target sample pretreatment was performed in a high-power microwave oven. The hybridization reaction was detected by incubation with an anti-digoxigenin antibody tagged with horseradish peroxidase (POD), and diaminobenzidine (DAB) was applied as the chromogen. Slides were counterstained with hematoxylin and appropriate positive and negative controls were included in each assay. Positive staining was defined as the presence of dark brown granules in the nuclei of epithelial cells at the site of hybridization.EBER was detected by ISH with the EBER Detection kit  according to the manufacturer’s instructions. Next, the sections were deparaffinized, rehydrated and predigested with proteinase K and a hybridization solution containing the digoxigenin-labeled EBER nucleic acid probe was applied. Detection of the hybridized probe was performed by application of anti-digoxigenin-POD and the coloring reaction was performed with DAB. Sections were counter-stained with hematoxylin and brown nuclear staining was regarded as a positive hybridization signal.The clinical data of the four patients are summarized in Clinical management of these patients was based primarily on the preoperative imaging observations and intraoperative frozen-section examinations. Papillomas associated with premalignant changes were found in the frozen sections of cases 1 and 2 and in these cases, extended resections of the lesion  had been performed. Radical tympanomastoidectomy was used in the other two cases to guarantee radical resection of the neoplasms. All patients recovered well postoperatively and were discharged as scheduled. To date, the patients have been followed for between 6 and 36 months without evidence of recurrent disease, clinically and radiologically.in situ and high grade squamous intraepithelial neoplasia, were observed in the sections of cases 1 and 2. Representative histopathological images of the middle ear squamous papillomas and premalignant changes are shown in Histological examination of these cases confirmed the diagnosis of squamous papilloma. In addition, premalignant changes, including carcinoma HPV and EBV ISH showed no detectable HPV or EBV genomes in any of the four cases with middle ear squamous papilloma .Squamous papilloma may, in theory, occur anywhere on the epidermis and mucosa, but in the head and neck region, it is most commonly found in the mouth and throat. Primary middle ear squamous papilloma is an exceedingly rare temporal bone neoplasm and an Ovid Medline search using the search terms ‘squamous papilloma’ and ‘middle ear/temporal bone’ returned only two cases in the English literature ,8. The dThe etiology and pathogenesis of papillomas of the middle ear remain unknown. CT scans of the current patients did not detect lesions in the sinonasal cavity, nasopharynx or pharyngeal opening of the auditory tube. In addition, none of the patients had a past history of papillomas of the external ear canal or relevant surgical procedures. Thus, this infers that the tumors in the present cases did not arise in the adjacent area and become incidentally involved with the middle ear via the eustachian tube or the external acoustic meatus. Therefore, this indicates that the tumors were of multicentric primary origin. Furthermore, inverted papilloma, an additional type of papilloma, rarely occurs in the middle ear . EctopicThere is growing evidence to support the association of HPV with the pathogenesis of squamous papillomas, including genital warts and recurrent childhood respiratory papillomatosis ,12. HPV The HPV infection rate in middle ear squamous papilloma has not been previously reported and only a few studies have investigated the presence of HPV infection in middle ear inverted papilloma. Previous attempts to identify HPV in the tissue sections of seven cases of primary temporal inverted papilloma have been described in previous studies –18. In fThere were certain limitations to the examination of HPV in the present study, the most relevant of which was that the sensitivity of ISH appeared to be low compared with that of liquid-phase amplification . Thus, iEBV is a virus of the herpes family that infects the majority of the human population. This virus infects B cells of the immune system and epithelial cells. There is evidence that infection with EBV is associated with a higher risk of particular forms of cancer and may function as an oncogenic agent . Thus, tThe symptoms of middle ear squamous papilloma resemble other neoplasms involving the middle ear, including chronic otitis media with cholesteatoma or granulation tissue. Otorrhea and hearing loss are the most common complaints and the majority of cases, including the two previously reported and four present cases, have a clear history of otorrhea with antibiotic treatment that did not resolve symptoms and generally, worsened with time. This is consistent with the assumption that middle ear mucosa metaplasia, due to chronic inflammation, also induces the development of squamous papilloma . This tuNotably, middle ear squamous papilloma in the present cases occurred predominantly in males between the fifth and seventh decades of life, which is similar to the demographic features of head and neck papillary squamous cell carcinoma ,24. PapiIn the present study, four cases of middle ear squamous papilloma have been reported. Although this tumor is exceedingly rare, it is possible that its incidence is under recognized. Commonly, these papillomas develop in males of ~60-years of age and otorrhea is the most frequent complaint. Premalignant changes were observed in two of the present cases and ISH of HPV and EBV in all four cases was found to be negative. The present results indicate that chronic inflammatory stimulation, not HPV and EBV infection, is involved in the occurrence of this tumor and its malignant transformation. Radical surgery and long-term postoperative follow-up are recommended due to its malignant and recurrent potential. The current study was limited to a small number of cases and the etiology of this disease remains unclear. Further genetic investigations with newly identified cases are required to clarify the pathogenesis of this disease.


Human papillomavirus (HPV) type 16 is associated with oropharyngeal carcinomas (OPC). Antibodies (Abs) to HPV16 E6 and E7 oncoproteins have been detected in patient sera; however, Abs to other early HPV-derived proteins have not been well explored.N=40), partners of patients with HPV16+ OPC (N=11), and healthy controls (N=50).Antibodies to the HPV16 proteome were quantified using a novel multiplexed bead assay, using C-terminal GST-fusion proteins captured onto Luminex beads. Sera were obtained from untreated patients with OPC . In a validation cohort, HPV16 E1, E2, and E7 antibody levels were significantly elevated compared with healthy control samples (P⩽0.02) and partners of OPC patients (P⩽0.01).Oropharyngeal carcinomas patients with known virus-like capsid particle+ Abs had elevated serum Abs to HPV16 E1, E2, E4, E6, and E7, and L1 antibody levels, but not E5. The ratios of specific median fluorescence intensity to p21-GST compared with controls were E1: 50.7 Patients with HPV16+ OPC have detectable Abs to E1, E2, and E7 proteins, which are potential biomarkers for HPV-associated OPC. Approximately 20 million Americans are currently infected with human papillomavirus (HPV), and another 6.2 million people become newly infected each year  arrays  or HPV18 (N=10) Abs defined by the competitive Luminex immunoassay  developed by Merck  (N=20) were obtained from Brown University and were age (±5 years), residence, and gender matched to cases and processed simultaneously. Serum samples were collected using a standardised sample collection protocol and stored at −80 °C until use.Human papillomavirus serology was performed using retrospectively identified sera from 20 head and neck cancer patients (‘training set’) collected at Brown University that were known to be positive for HPV16 (N=17) and Johns Hopkins University  as well as healthy anonymous blood donors (N=30) and partners of OPC patients (N=11). Of these samples, 55 were sera and 16 were plasma (6 of these were obtained at the time of leukopheresis).To confirm the findings from the training set, serum from 30 pre-therapy OPC cases and 30 controls, collected as part of a larger research study, were evaluated. This included blood from OPC patients at Dana-Farber Cancer Institute  products were each captured onto 2000 microspheres at 40 microspheres per μl in PBS-1% BSA. Protein-bound microspheres were pooled together, then re-aliquoted to a 96-well filter plate . Microspheres were blocked with 10% each of normal sera from mouse, rabbit, goat, and rat; 0.5% polyvinyl alcohol ; 0.8% polyvinylpyrrolidone (Sigma-Aldrich); and 2.5% Chemicon (Millipore) in PBS-1% BSA. Test sera were diluted 1 : 80 in blocking buffer and incubated with the washed beads overnight at 4 °C while shaking. Biotin-conjugated goat anti-human IgG antibody  and streptavidin-R-PE  were added. To control for non-specific and GST-specific autoantibody background, the ratio of median fluorescence intensity (MFI) for individual HPV-specific Abs to the MFI for the control p21-GST antigen was measured. To determine protein expression, GST-protein tags were detected with anti-GST monoclonal Ab  and PE-conjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories).SeroMAP carboxylated microspheres  from regions #35–44 were coupled at a ratio of 5 Taq polymerase , 10 mmol l–1 Tris–HCL, pH 9.0, 50 mmol l–1 KCl,4.6 mmol l–1 MgCl2, 0.02 mmol l–1 of each deoxyribonucleotide triphosphate, 11 pmol of each primer, and 100–500 ng of DNA. The reactions were cycled at 95 °C for 2 min, followed by 20 s each of 95 °C then 55 °C and 30 s at 72 °C. The final cycle was 72 °C for 5 min and held at 15 °C until analysed. As a control for DNA quality, the DNA was separately amplified with primers to the β-globin gene . The DNA samples that were positive for HPV were genotyped by restriction endonuclease fragment analysis. The HPV PCR products were digested in three separate reactions using PstI, Rsa I, and Hae III. All PCR products were resolved on 1 mm, 5% acrylamide gels, stained with ethidium bromide, and visualised by transillumination. The pattern of restriction fragment-length polymorphism of each sample was compared with the Access Genetics database to determine the HPV genotype . Polymerase chain reaction was performed using a procedure developed by Access Genetics , which amplifies the HPV L1 domain with degenerate forward and reverse primers (N=20) and the validation control sera (N=30) were used. An MFI ratio > (the average + 3 s.d.) of all control samples was designated positive. These levels were E1: 5.4, NE2: 8.5, CE2: 6.8, E4: 2.3, E5: 4.2, E6: 9.0, E7: 6.6, L1: 9.5, and L2: 8.0. To determine the variability of serum detection for each HPV antigen, test serum from a single OPC patient (tumour HPV16+ confirmed by PCR) was assayed in duplicate on four separate days for each of the HPV16 antigens. Intra-assay and inter-assay variability was determined by calculating the coefficient of variation  × 100). Heat maps and cluster analysis were done using MultiExperiment Viewer software . For all subsequent studies, the MFI ratio of specific HPV Ab to p21-GST was used to control for non-specific protein binding.IgG Abs specific for HPV-derived proteins were detected in two representative serially diluted OPC patient sera . For botWe initially selected a retrospective training set of sera from patients known to have Abs to HPV16 VLP or HPV18 VLP by the highly sensitive Merck cLIA assay, which were predicted to have higher levels of Abs to HPV-derived early genes. The characteristics of patients with HPV-associated OPC and age- and gender-matched controls (training set) are shown in P⩽0.05)  ; Table 2P=0.914). No correlation between HPV16L1 bead array signal intensities and VLP titre were observed (R=0.638), likely related to structural differences in epitope display, which has been observed for L1-specific epitopes   ; Table 4vs 1 out of 30 controls), NE2 (25 out of 30 (83%) of cases vs 1 out of 30 controls), CE2 (24 out of 30 (80%) of cases vs 1 out of 30 controls), and HPV16E7 (19 out of 30 (63%) of cases vs 0 out of 30 controls), P<0.001. Compared with E1, E2, and E7 Abs, fewer patients had E4 Abs (13 out of 30 (43%) vs 0 out of 30 controls) or E6 Abs (15 out of 30 (50%) vs 1 out of 30 controls). All 16 patients with known HPV16+ tumours by PCR had detectable E1, E2, or E7 Abs. Four of the OPC patients had HPV16-negative tumours by PCR; of these, two had detectable Abs to both E1 and E7. This may reflect cross-reactivity with other HPV types, limitations of the HPV PCR assay, or represent HPV16 infection at other sites. As this cohort included a mix of sera and plasma, a direct comparison of the subset of serum cases and controls was performed, with the specificity of detection of HPV16 E1, E2, E6, and E7 Abs maintained (P<0.002).The HPV Abs most strongly associated with OPC were HPV16E1 (22 out of 30 (73%) of cases Given the high proportion of adults (>70%) having ever been infected with high-risk (HR) HPV in most populations , the HPV16 antibody responses in the validation set detected by bead array ELISA were grouped by unsupervised hierarchical clustering analysis . DistincIt is estimated that 75–80% of sexually active persons in the United States will be infected with HR HPV in their lifetime , 16E6 and 16E7-specific IgG were detected in 7% of cases with a relative risk (odds ratio) of 2.7, found 0.5–5 years before diagnosis (In cervical cancer, Abs to 16E6 and 16E7 develop late in the course of disease and have been shown to correlate with disease response (The previously unreported E1- and E2-specific Abs in our cohort is striking because of both the high titre and high frequency in OPC patient sera, which improves the sensitivity of detection from 70% to 100% of HPV16+ OPC when combined with E6 and/or E7 Abs. Early studies demonstrated low immunoreactivity to an E1-derived peptide in cervical cancer sera . As a reHuman papillomavirus antibody signatures may provide a rapid, convenient and inexpensive screening method for assessing the HPV status of suspected or known malignancy. Human papillomavirus+ OPC is a strong, independent factor for improved prognosis (


Recurrent respiratory papillomatosis (RRP), a chronic upper respiratory condition characterized by diffuse multiple recurring papillomas, is thought to result from human papillomavirus (HPV) type 6 or 11 infection. Although RRP is an intractable disease, malignant transformation of RRP is rare. The underlying mechanism, however, has not been elucidated. We describe the clinical course of a patient who underwent more than 130 operations for RRP associated with HPV type 6 infection and subsequently suffered spontaneous malignant transformation to squamous cell carcinoma. Immunohistochemical analysis revealed that malignant transformation might result from a genomic defect, such as p53 inactivation, leading to stimulation of uncontrolled cell proliferation by HPV type 6 for an extended period, but not directly because of HPV itself. Our results could help in the development of novel therapeutic strategies for severe RRP, although further studies are required before clinical application of molecular targeted therapies. Recurrent respiratory papillomatosis (RRP), usually a benign disease, is a chronic upper respiratory condition characterized by the occurrence of diffuse multiple recurring papillomas, which are caused by infection with human papillomavirus (HPV) type 6 and/or 11  after more than 130 operations, and which was, unusually, associated with HPV type 6 infection. We also identified protein markers of proliferation and neoplastic change in order to help elucidate the mechanism of malignant transformation in this case.The patient in this case was a 39-year-old Japanese man with a history of RRP, who had presented with progressive hoarseness when he was 1 year old and was diagnosed with laryngeal papilloma by biopsy under general anesthesia 3 years later. Surgical excision and laser ablation were performed, but the papillomas immediately recurred and resulted again in severe dyspnea. When the patient was 6 years old, a tracheotomy was performed to relieve severe dyspnea. During this procedure, a large papilloma was discovered. RRP was diagnosed, and the patient was followed-up at our hospital. Initially, various treatments were tried including interferon-α, interferon-β, interferon-γ, acyclovir, fluorouracil, retinoids, and traditional Chinese medicines, but none of these treatments resulted in a significant clinical response.The tumors originated in the larynx, gradually increased in size, and progressed to the tracheal bifurcation, although the pathological diagnosis was still one of mild dysplasia. Thus, treatment was continued in the form of repeated surgical reductions of the papilloma using an endoscopic microdebrider or potassium-titanyl-phosphate (KTP) laser to maintain the airway over the next 35 years. This involved more than 130 procedures. In 2011, granulation tissue around the tracheotomy aperture was found to have increased and a biopsy revealed indicated SCC. A larynx biopsy performed at the same time revealed additional moderate dysplasia but not malignancy. This patient received irradiation by accelerated hyperfractionation resulting in good local control, but he died from metastasis to the lung.RRP is a potentially devastating disease that can be associated with significant morbidity, and result in mortality due to airway compromise even if not change to malignant. However, malignant transformation is not commonly seen, and thus, the underlying mechanism is still unclear. In an attempt to determine the mechanism of malignant transformation in this case, HPV typing and diachronic immunohistochemistry were performed. HPV typing by multiplex-PCR detected HPV type 6 infection but not evidence of “high-risk” type HPV infection.We performed immunohistochemistry on specimens obtained from the larynx in 1989, 1994, 2000, 2005, and 2011, and on the tumor tissue obtained from around the tracheotomy aperture in 2011. All specimens from the larynx showed mild to moderate dysplasia Figure F.Figure Both the dysplastic tissue and the SCC stained strongly positive for epidermal growth factor receptor (EGFR, Figure RRP is a rare condition characterized by recurrent growth of benign papillomas in the respiratory tract, and is now known to be caused exclusively by HPV, usually type 6 and/or 11 (Go et al. Various treatments for RRP using antiviral therapies including interferon, retinoids, photodynamic therapy, zinc, anti-reflux medications, cyclooxygenase-2 inhibitors, therapeutic/preventive vaccines, and gene therapy have been trialled (Katsenos & Becker RRP is considered to be an intractable disease as it generally does not respond to therapeutic interventions, but malignant transformation is very rare, occurring in only 1–4% of patients with RRP (Jeong et al. The molecular mechanisms that underlie the malignant transformation of RRP is still unknown, but risk factors include smoking, prior irradiation, juvenile onset, and the type of infecting HPV (Katsenos & Becker To date, two mechanisms for malignant transformation of RRP associated with HPV type 11 infection have been reported. Reidy et al. showed that HPV type 11 genes can integrate into the host genome, and the three oncogenic proteins, E5, E6, and E7 produced by the integrated HPV have been implicated as malignant transforming factors (Reidy et al. The patient in the present case was diagnosed with juvenile RRP associated with infection by HPV type 6, with no history of smoking and previous irradiation, making this a very unusual case of malignant transformation. There are a number of possible explanations for the occurrence of malignant transformation in this relatively low-risk case, including re-infection by “high-risk” HPVs, an enhancement of the oncogenic potential of the existing HPV type 6 infection, the activation of oncogenic proteins in the papilloma cells because of increased cellular proliferation, or an inhibition of tumor suppressor genes.In this study, we evaluated the expression of p16, known to be a surrogate marker for “high risk” HPV infection but not yet ill-defined for “low-risk” HPV infection. The strength of p16 staining was diachronically increased in the dysplastic tissue, but p16 staining was negative in the SCC. This suggests that the multiplicity of infection or the oncogenic potential of HPV was diachronically increased if p16 staining acts as a surrogate marker, but the malignant transformation might not have been a result of HPV infection. Furthermore, EGFR, a known oncogene in head and neck cancer that is activated by the product of the HPV E5 gene (Kim et al. p53 is wild type but not expressed in cervical cancers, although some cervical cancers do express mutant p53 and not infected by HPV. Unlike cervical cancers, malignant transformation in this case might have been contributed to by a mutation in p53, leading in turn to enhanced cell proliferative stimulation by HPV type 6 over an extended period. This is distinct, however, from a direct oncogenic effect of HPV.It is noteworthy that the multi functional tumor suppressor gene, p53, was not expressed in dysplastic tissue but was strongly expressed after malignant transformation to SCC. It is well-established that the E5 gene product of HPV types 16 or 18 can inactivate p53 function leading to malignant transformation (Kim et al. The relationship between p53 expression and malignant transformation is still unclear. Some previous studies reported progressively increased expression of p53 and suggested that this was a significant event in the progression to SCC (Stern et al. Malignant transformation of RRP is almost impossible to treat. Antiviral agents and interferon have shown some promise, but unfortunately very mild curative effects. Therefore, novel treatments are needed for RRP before malignant transformation occurs, both to improve survival and to achieve a worthwhile, less toxic palliation. In this case, as well as in others previously described, EGFR signaling was activated in RRP, and is likely to have driven the growth of the papilloma. Wu et al. showed that, in papilloma-derived epithelial cells, EGFR, Rac1, and COX-2 act in a linear pathway with EGFR upstream of Rac1, which in turn results in increased expression of COX-2 (Wu et al. Although further investigation is necessary before the clinical application of molecular targeted therapies, our results can serve as a foundation for the development of novel therapeutic strategies including molecular targeted therapy of EGFR signaling for severe RRP.Written informed consent was obtained from the patient for publication of this report and any accompanying images.


We previously showed that secretory leukocyte protease inhibitor (SLPI) gene and protein expression is significantly lower in metastatic versus non-metastatic head and neck squamous cell carcinoma (HNSCC). However, we did not assess the human papillomavirus (HPV) status of these cases. Since SLPI plays a role in HIV and herpes simplex virus (HSV) infections, we hypothesized that SLPI may be involved in HPV-infected HNSCC. In HNSCC tissue (n=54), HPV DNA was determined and correlated with SLPI expression. Additionally, to investigate a possible role of smoking on SLPI expression in clinically normal mucosa, 19 patients treated for non-malignant diseases (non-HNSCC) were analyzed for SLPI expression and correlated with smoking habits. In HNSCC patients, SLPI expression showed a significant inverse correlation with HPV status. In patients with moderate/strong SLPI expression (n=19), 10.5% were HPV-positive. By contrast, patients with absent/weak SLPI expression (n=35), 45.7% were HPV-positive. Low SLPI expression was correlated with metastasis (P=0.003) independent of HPV status. HPV-positivity was clearly associated with lymph node status (81.3% N1-3 cases). In smoking non-HNSCC patients (n=7), 42.9% showed absent/weak and 57.1% moderate/strong SLPI staining. In non-smoking non-HNSCC patients (n=10) 83.3% showed absent/weak and 16.7% moderate/strong SLPI expression. For the first time, a correlation between SLPI downregulation and HPV infection was demonstrated, suggesting that high levels of SLPI, possibly induced by environmental factors such as tobacco smoking, correlate with protective effects against HPV infection. SLPI may be a potential biomarker identifying head and neck cancer patients not at risk of developing metastases (SLPI-positive), and those at risk to be infected by HPV (SLPI-negative) and likely to develop metastases. We previously demonstrated that secretory leukocyte protease inhibitor (SLPI) is significantly associated with head and neck squamous cell carcinoma (HNSCC) . AlthougThe SLPI protein, also known as antileukoprotease, is a 11.7-kDa (107 amino acids) non-glycosylated kazal-type serine protease inhibitor of neutrophil elastase, cathepsin G, chymotrypsin and trypsin –14. In pIn HNSCC, two possible types of carcinogenesis are discussed: i) an HPV-driven carcinogenesis, leading to biologically more aggressive tumors including early metastatic spread into the locoregional lymph nodes, but with comparatively better survival rates of the patients ,16; and Moreover, a recent report showed a positive correlation between cigarette smoke and SLPI-expression in a rat model . This fiFrom 2004 to 2009, tissue samples of histopathologically confirmed HNSCC were obtained from 54 patients  during surgery at the Department of Otorhinolaryngology, Head and Neck Surgery at the Christian-Albrechts-University Kiel, Germany. All samples were obtained following informed consent approved by the local Ethics Committee (D 438/10). Tissue samples were treated as follows: one section of the tumor tissue was snap-frozen in liquid nitrogen and stored at −80°C for further analysis. The remaining section of each tumor was processed for routine histopathology. DNA was extracted from 25 mg frozen tissue samples, as previously described . Total RThe anatomical location of the primary tumors was: tonsils , larynx (n=22), tongue (n=8), and soft palate (n=4). SCC of the tongue  and SCC of the soft palate  were investigated in one subgroup (n=12) since the biological behavior of soft palate SCC is more comparable to oral cavity SCC than to tonsillar SCC . Additional clinicopathological characteristics and tobacco smoking habits are presented in We also obtained tissue samples from clinically normal mucosa of the aerodigestive tract from 19 patients  who were treated for non-malignant diseases at our Department. Additional clinicopathological characteristics and tobacco smoking habits are presented in Type 3.5 LCD-Array, as previously described  assay and HPVd 20–22; .In cases with RNA available for analysis (n=37), total RNA was subjected to ultra-short type-specific RT-PCR assays which were developed for 12 HR- and 8 pHR-HPV to amplify ~65 bp of the E6*I mRNA . RNA intParaffin-embedded tissue specimens were cut into 5-μm sections, and stained for SLPI expression as previously described .The slides were reviewed by two experienced members of the Department of Otorhinolaryngology, Head and Neck Surgery, Kiel, and by an experienced pathologist. To assess SLPI protein levels, 300 cells in at least five areas were analyzed. The mean percentage of positive tumor cells was determined and cases were assigned to one of the following categories: (−) <5%; (+) 5–30%; (++) 31–75%; and (+++) >75%. Cases with a score of (−) were considered negative, cases with scores of  positive.Type 3.5 LCD-Array and/or in the presence of high viral loads by BSGP5+/6+-PCR-MPG assay. HPV infections were classified as active if HPV E6*I mRNA was detected. When RNA was not available, HPV DNA-positive cases with p16INK4A overexpression were classified as active.In the present study, specimens were classified as HPV DNA-positive only if the HPV DNA was detected by the HPVFisher’s exact test was performed to test for differences between groups, using the SAS 9.2 software. All tests were performed two-sided, and P-values <0.05 were considered to indicate statistically significant differences.The clinicopathological assessment, including the HPV detection, HPV E6*I mRNA analysis, SLPI immunohistochemistry and smoking habits are summarized in HPV DNA was detected in 33.3% of the analyzed cases. HPV16 and HPV18 were the only infecting HPV types in 88.9 and 11.1% of all cases, respectively. From the two HPV18-positive cases, 1 was HPV18 DNA alone and the other was a double infection with HPV16.Anatomical distribution of the tumor and their HPV levels was: from 20 tonsillar carcinomas, 60% tested positive for HPV DNA, including the HPV18 case and the case with double infection . From the 22 specimens derived from laryngeal carcinomas, 18.1% were infected with HPV16. From the 12 carcinomas of the tongue and the soft palate, 16.7% were HPV16-positive. The prevalence was significantly higher in the tonsillar tumors (P=0.009).Cases with available RNA (n=37) were subjected to HPV E6*I mRNA analysis. Signals for HPV RNA were detected in 17 cases, of which 14 could be assigned to HPV DNA-positive cases. The remaining 3 cases were classified as HPV-negative due to lack of HPV DNA-positivity according to the applied algorithm. However, considering HPV RNA expression as more important for an active HPV infection than existence of HPV DNA, these 3 cases would need to be classified as cases with active HPV infection. Only 1 out of the 17 HPV DNA positive cases with available RNA did not show detectable HPV mRNA. This case was classified as inactive infection, whereas the 14 HPV DNA positive cases with detectable levels of viral mRNA were classified as active HPV infections.INK4A immunohistochemistry was performed. Due to strong p16INK4A staining in 2 cases, these were classified as active HPV infections. Collectively, 16/18 cases with HPV DNA were finally classified as active HPV infections.In 3 HPV DNA-positive cases, RNA was not available for analysis. In these cases, p16In 25.9% of 54 cases, there was no SLPI staining detectable. Irrespective of the HPV status, absent, weak, moderate/strong staining was detected in 38.8, 24.1 and 11.1% of SLPI-positive cases, respectively . The casThe distribution of the N0 vs. the N1–3 cases among the group of absent/weak and moderate/strong was 20 vs. 80%, and 63.2 vs. 36.8% (P=0.003), respectively. Elevated SLPI was associated with lower metastasis whereas absent/weak abundance of SLPI was associated with increased tumor burden of the neck. The distribution of patients with (n=22) and without (n=16) metastasis among the HPV-negative cases was similar. However, out of the HPV-positive cases (n=16), 13 developed metastases. The correlation between the HPV status, SLPI expression and the N-status was highly significant (P=0.003), despite the relatively small number of cases in each group . IndepenSLPI immunohistochemistry of clinically normal mucosa obtained from 7 non-HNSCC patients with history of tobacco smoking showed absent/weak and moderate/strong staining in 42.9 and 57.1% cases, respectively. Patients without a history of tobacco smoking (n=12) showed absent/weak and moderate/strong staining patterns in 83.3 and 16.7% cases each . AlthougTo our knowledge, this is the first report showing a correlation between: i) SLPI expression and HPV infection; ii) SLPI expression, HPV infection and lymph nodal disease in head and neck cancer; and iii) SLPI expression and tobacco consumption habits in clinically normal mucosa of non-HNSCC patients.et al. In turn, A2t is associated with HPV insertion into and infection of epithelial cells (The data presented here demonstrate that high SLPI expression is correlated with reduced prevalence of HPV infection in the investigated tumor specimens of HNSCC, while the HPV-positive cases predominate in the group of absent to low SLPI expression. Therefore, even in the presence of HPV DNA, SLPI expression may prevent an HPV infection of the upper aerodigestive tract. These findings are in agreement with the current available literature regarding the role of SLPI in oral HIV infections. For example, McNeely et al suggesteal cells . Therefoet al(In addition, Wahl et al suggesteet al,28, and We previously reported that significant elevation of SLPI is associated with non-metastasized HNSCC , a phenoet al(The role of SLPI as a potent inhibitor of neutrophil elastase appears to be an important factor resulting in the protection of the mucosa and skin against proteolysis. In addition, Chan et al showed tet al, we indeet al,17. AccoIn conclusion, in the present study we identified for the first time a statistically significant inverse correlation between HPV infection and SLPI expression levels, suggesting that reduced expression of SLPI facilitates HPV infection. The previously described correlation between SLPI reduction, HPV infection, and metastasis was confirmed. In addition, we provided preliminary evidence that tobacco smoking is associated with SLPI expression.


The human papillomaviruses (HPV) are a large family of non-enveloped DNA viruses, mainly associated with cervical cancers. Recent epidemiologic evidence has suggested that HPV may be an independent risk factor for oropharyngeal cancers. Evidence now suggests HPV may modulate the malignancy process in some tobacco- and alcohol-induced oropharynx tumors, but might also be the primary oncogenic factor for inducing carcinogenesis among some non-smokers. More evidence, however, is needed regarding oral HPV prevalence among healthy adults to estimate risk. The goal of this study was to perform an HPV screening of normal healthy adults to assess oral HPV prevalence.Healthy adult patients at a US dental school were selected to participate in this pilot study. DNA was isolated from saliva samples and screened for high-risk HPV strains HPV16 and HPV18 and further processed using qPCR for quantification and to confirm analytical sensitivity and specificity.p < 0.05). Four patient samples were found to harbor HPV16 DNA, representing 2.6% of the total (n = 151). Three of the four HPV16-positive samples were from patients under 65 years of age and all four were female and Hispanic (non-White). No samples tested positive for HPV18.Chi-square analysis revealed the patient sample was representative of the general clinic population with respect to gender, race and age (The successful recruitment and screening of healthy adult patients revealed HPV16, but not HPV18, was present in a small subset. These results provide new information about oral HPV status, which may help to contextualize results from other studies that demonstrate oral cancer rates have risen in the US among both females and minorities and in some geographic areas that are not solely explained by rates of tobacco and alcohol use. The results of this study may be of significant value to further our understanding of oral health and disease risk, as well as to help design future studies exploring the role of other factors that influence oral HPV exposure, as well as the short- and long-term consequences of oral HPV infection. The human papillomavirus (HPV) has been implicated as the cause of virtually all cervical cancers worldwide -3. TheseRecent epidemiologic evidence has suggested that HPV may also be an independent risk factor for oropharyngeal cancer, revealing HPV in three times as many pre-cancerous oral lesions, and almost five times as many oropharyngeal cancers compared with normal oral mucosa -14. Of ain vitro studies have recently investigated possible mechanisms that may account for these phenotypic changes in oropharyngeal cancers  and the human β-actin housekeeping gene [GenBank: M10277]. All primers were purchased from Sigma-Aldrich  and probes purchased from Roche Applied Science DNA samples were then processed using qPCR to provide more specific and sensitive quantification. Primers and probes were designed using Roche Universal Probe library (UPL) assay design software to amplify the region overlapping E6 and E7 gene sequence of HPV16 [GenBank: HPV16 E6/E7 forward primer 5'-CAACTGATCTCTACTGTTATGAGCAA-3', HPV16 E6/E7 reverse primer 5'-CCAGCTGGACCATCTATTTCA-3', HPV16 E6/E7 hydrolysis "Taqman" probe 5'-(fam)-AGGAGGAG-(dark quencher dye)-3' (UPL probe #63) was used to amplify the 73 base pair (bp) region between the 535 nucelotide (nt) position and 607 nt position. Human β-actin forward primer 5'-GTGGGGTCCTGTGGTGTG-3', human β-actin 5'-GAAGGGGACAGGCAGTGA-3', human β-actin hydrolysis "Taqman" probe 5'-(fam)-GGGAGCTG-(dark quencher dye)-3' (UPL probe #24) amplified the 61 bp region between 2642 nt position and 2702 nt position.® 480 multiwell Plate 96 containing 1× LightCycler® 480 Probes Master (Roche Applied Sciences), 1 μM of each respective primer set (forward and reverse), 0.2 μM of respective probe, and 2 μl of DNA template; in a 20 μl final reaction volume. The probes master mix contained reaction buffer, dNTP mix (including dUTP in place of dTTP), 3.2 mM MgCl2, and Taq DNA polymerase. The real-time PCR assay was performed on a LightCycler 480 system (Roche Applied Science) with the following cycle parameters: pre-incubation for initial enzyme activation at 95°C for 10 minutes, followed by 45 cycles of 95°C for 10 seconds (ramp rate 4.4°C/second), 60°C for 30 seconds (ramp rate 2.2°C/second) and 72°C for 1 second (ramp rate 4.4°C/second). Following amplification phase, a cooling step was performed at 40°C for 30 seconds (ramp rate of 2.2°C/second). Acquisition of the fluorescence signal was performed using Mono Hydrolysis Probe setting (465-510 nm) following the 72°C extension phase of each cycle. All samples were carried out in triplicate.The real-time reaction mixture was prepared in a LightCyclerT) measured with the second derivative maximum method . Saliva samples > 0.001 copy/genome were considered HPV positive. Specificity analysis was performed on qPCR assay against HPV18 and found to be 100% specific (data not shown).The CaSki  cervical adenocarcinoma cell line was used to develop standard curves for both the HPV16 (600 copies/genome) and β-actin (2 copies/genome) genes. DNA extracted from CaSki cells was serially diluted ten-fold starting at 50 ng to 0.0005 ng . This stSensitivity and specificity were calculated as the proportion of true positives and true negatives , respectively. Following the acquisition of saliva samples and HPV screening results, demographic information from each sample was compared with the overall demographic profile of the UNLV-SDM patient pool  using a chi-square (χ2) test, to determine if any characteristic  was different than expected among the patients evaluated in this study (n = 151). A probability level of alpha (α) = 0.05 was used to determine statistical significance.p = 0.589). There were slightly more White patients in the study sample (48.3%) than in the overall UNLV-SDM population (40.8%) (p = 0.133). In addition, there were slightly fewer 18 - 64 year olds in the sample (80.8%) than in the overall clinic (85.3%) (p = 0.354).Samples of saliva were collected from 151 UNLV-SDM patients between June 2 and October 1, 2010. The patients from whom samples were collected and screened were not statistically different from the overall UNLV-SDM clinic population with respect to gender, race, or age  levels, have been largely unsuccessful due to patient apprehension and fear of blood collection . To avoiThese data demonstrated a prevalence rate of high-risk oral HPV (2.6%) virtually the same as the most recent multinational studies of healthy, cancer-free adults (3.1% to 5%) ,62. OverIn addition, although the results of this study found oral HPV infection only among four patients, who were minority and female, the vast majority of female and minority patients in this study had no evidence of oral HPV infection. As an initial pilot study, these data suggest a more comprehensive and in-depth analysis of this population may be necessary, as recent epidemiologic studies have shown that rates of oropharyngeal cancer haven risen sharply among minority females in the US, despite overall declining rates . More geThis study also revealed the presence of the high-risk strain HPV16, but not HPV18. Although some reports have suggested that oral HPV infection may involve multiple HPV strains -21,69, tThis study had several limitations that should be considered. First, although the non-invasive nature of this study was sufficient for the recruitment and screening of a significant number of patients, the overall sample size was somewhat limited in comparison to the larger multinational studies previously mentioned ,58,61. TThis study successfully recruited patients and screened saliva samples for high-risk HPV, confirming HPV16, but not HPV18, was present in a small subset of the healthy adult patients in a US dental school clinic population. These patients were female and minority. Many health disparities face females and minorities in the US, and with some evidence suggesting that smoking and oropharyngeal cancer rates may be increasing in these population subgroups, the results of this study may be of significant value to other dental, medical, and health care professionals to further an understanding of oral health and disease risk.(HPV): Human papillomavirus; (US): United States; (DNA): Deoxyribonucleic acid; (UNLV-SDM): University of Nevada, Las Vegas - School of Dental Medicine; (PCR): polymerase chain reaction; (GAPDH): glyceraldehyde-3- phosphate dehydrogenase; (qPCR): quantitative polymerase chain reaction; (bp): base pair; (nt): nucleotide; (RCF): Relative centrifugal force; (PBS): phosphate-buffered saline; (dNTP): Deoxyribonucleotide triphosphate; (dUTP): 2'-Deoxyuridine triphosphate; (dTTP): Deoxythymidine triphosphate.The authors declare that they have no competing interests.KK, SW, AB and RW conceived, monitored, and coordinated the experimental design. RB, JC, JF, DM, and JM were responsible for recruiting patients, informed consent, collecting samples, and some biomedical analysis. DT, KK, and SW carried out the DNA extractions, PCR, and qPCR analysis. KK, SW and DT were responsible for the data analysis, as well as the writing and editing of this manuscript. All authors have read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6831/11/28/prepub


Condyloma acuminatum, intraepithelial neoplasia, and squamous cell carcinoma are three relatively frequent vulvar lesions. Condyloma acuminatum is induced by low risk genotypes of human papillomavirus (HPV). Vulvar intraepithelial neoplasia (VIN) and squamous cell carcinoma have different etiopathogenic pathways and are related or not with high risk HPV types. The goal of this paper is to review the main pathological and clinical features of these lesions. A special attention has been paid also to epidemiological data, pathological classification, and clinical implications of these diseases. Vulvar human papillomavirus (HPV) infection is responsible for the development of benign tumors (condylomata acuminata), of one type of preneoplastic lesions, and of certain types of squamous cell carcinoma (SCC) . CondyloSimilarly to the cervix, most of the vulvar (pre)neoplastic lesions are induced by HPV infection (most commonly HPV 16), except for the “differentiated” (simplex) type of VIN.This allows to distinguish two types of VIN: (1) the usual VIN (uVIN)/classic VIN (WHO terminology) which is related to HPV infection; (2) the differentiated/simplex type (dVIN), non-HPV-related, but associated with vulvar dermatoses, especially the lichen sclerosus , 5.The distinction is also applicable for SCC with “HPV-related SCC,” associated with uVIN and “non-HPV-related vulvar SCC,” often associated with dVIN and lichen sclerosus , 4, 6.The incidence of HPV-associated VIN, unlike that of vulvar carcinomas, has been increasing over the past 20 years, especially in women of reproductive age, with the highest frequency reported in women of 20–35 years old –9. dVIN Approximately 95% of malignant tumors of the vulva are SCC. They represent 6.38% of all gynaecologic cancers in Belgium (Belgian Cancer Registry 2011). The American Cancer Society reports over 3,400 new cases of vulvar SCC in the USA annually .The incidence of both types (HPV and non-HPV associated) of vulvar SCC increases with age , 12. TheCondyloma acuminatum (CA) or venereal/genital warts refer to benign proliferative epidermal or mucosal lesions attributed mostly to HPV type 6 or 11, but coinfections with high-risk HPV types are frequent. More than 100 types of HPV have been identified, of which 40 can infect the genital areas. HPV are highly specific viruses showing both species and regional specificity. They represent the most common sexually-transmitted disease (STD) and are highly contagious. The prevalence of CA peaks in the early sexually active years, with two-thirds of the respective sexual partners complaining of warts. The median time between infection and development of lesions is about 5-6 months among women. Up to 20% of people with genital warts will present other STDs.The following risk factors have been described, including smoking, hormonal contraceptives, multiple sexual partners, and early coital age. Patients who develop CA complain of painless bumps and, less frequently, of pruritus, discharge, or bleeding. Lesions are commonly multiple (multicentric) and multifocal, also affecting the perianal, vaginal, and cervical regions, but oral and laryngeal regions may also be involved. Latent illness may become active, particularly with pregnancy and immunosuppression. Lesions may regress spontaneously, remain stable, or progress in size and/or number.CA are soft, raised masses, with smooth, verrucous, or lobulated aspects that may appear as pearly, filiform, fungating, or plaque-like eruptions. The surface commonly shows finger-like projections, generally nonpigmented. They mainly occur in the moist areas of the labia minora and vaginal opening, but virtually, all genital regions may be affected . Therefore, minutious colposcopic examination, using acetic acid 2–5%, is of crucial importance to detect potentially multiple involved sites.CA are perceived as disfiguring, they impact sexual lifestyle, causing anxiety, guilt, and loss of self-esteem and creating concerns about cancer risk.The most common treatments are painful and nonspecific, addressing the clinically evident lesions rather than the viral cause. Various modalities include office-based treatment  or home-based treatment (chemotoxic agents or immunomodulatory therapy). First episode patients should be STD screened. Management should include partner notification.The initial site of infection is thought to be either basal cells of the immature squamous epithelium that HPV reaches presumably through defects in the epithelium. Once HPV enters the cells, two distinct biological sequences are possible. The first form is a “nonproductive or latent infection” in which HPV DNA persists in the basal cells without virus replication. Latent infections do not show morphologic alterations and can only be identified using molecular methods.The second form of HPV infection is a “productive infection.” Viral DNA replication in the intermediate and superficial cell layers of the squamous epithelium occurs independently of host chromosomal DNA synthesis. This allows large amounts of intact virions to be formed, leading to typical morphological aspects such as “koilocytic changes.”Molecular biologic methods have identified HPV-6 as the most common HPV type in CA. HPV-11 has been found in approximately one fourth of genital warts. These two HPV types are responsible for over 90% of CA , 18.The lesions are typically exophytic and may range from discrete papillary excrescences to extensive and coalescent “cauliflower-like” masses .CA shows a striking papillary architecture. Papillae of different sizes and shapes are lined by acanthotic squamous epithelium and have a fibrovascular stroma, often containing scattered chronic inflammatory cells. Hyperkeratosis, parakeratosis, hypergranulosis, and basal cell hyperplasia are seen. Koilocytic changes , sometimes focal, are present in the most superficial layers of the squamous epidermis. Mitotic figures are observed in the lower third of the epidermis  1, 4]., 4.1, 4]The proliferation index by immunostaining (Ki67/MIB-1) in the upper third of the epithelium is considered as an adjunct test to confirm the diagnosis of CA, especially in lesions without evident koilocytic changes.The presence of Ki67/MIB-1 immunostaining has been further correlated with the detection of HPV DNA by polymerase chain reaction (PCR).In situ hybridization for HPV can also be performed on paraffin-fixed tissue sections to confirm the presence of HPV-DNA in CA. However, this test may lack sensitivity .Podophyllotoxin (0.15% cream or 0.5% solution) is an antimitotic and cytotoxic molecule that results in necrosis of genital CA. Each course of podophyllotoxin treatment comprises self-application twice daily for 3 days, followed by four rest days. However, vulvar and anal warts are more feasibly and efficiently treated with clearance rates of 45–83% after use of 0.5% podophyllotoxin solution for 3–6 weeks. Transient and acceptable burning, tenderness, erythema, and/or erosions for a few days when the warts necrotize are described. Recurrence rates of 6–100% have been reported with podophyllotoxin between 8 and 21 weeks after clearance –21. PodoImiquimod 5% topical cream (Aldara) induces interferon production and is a cell-mediated immune response modifier. It is applied to the warts three times a week at bedtime. Treatment continues until wart clearance, or for a maximum of 16 weeks. It presents minimal systemic absorption but causes erythema, irritation, ulceration, and pain at application site. In clinical studies, clearance has been reported in 35–68% of patients with treatment courses up to 16 weeks. Erythema is often seen as a side effect with imiquimod therapy and sometimes appears to precede clinical resolution. Occasionally, severe inflammation is seen necessitating discontinuation of therapy. Relatively low recurrence rates (6–26%) after successful clearance have been reported , 22, 23.5-Fluorouracil: no longer recommended for routine use, it has antimetabolic and/or antineoplastic and immunostimulative activity.Sinecatechins ointment: Sinecatechins is available as a 10% ointment in Europe and a 15% ointment in the US. It consists in a preparation of green tea catechins (sinecatechins). Evidence suggests that the mechanism of action is made through antiproliferative mechanisms. The ointment is applied three times a day until complete clearance, or for up to 4 months. It cannot be used internally or during pregnancy. Clearance rates of 47–59% over 12–16 weeks were reported. Local side effects occur as frequently as with other topical therapies. They are generally graded as mild and typically include redness, burning, itching, and pain at the site of application. In those clearing, low recurrence rates of 7–11% were observed over 12 weeks follow-up –26.Trichloroacetic acid (TCA) solution (80–90%) is used directly to the wart surface, weekly. It rapidly penetrates and cauterizes skin, keratin, and other tissues. Although caustic, this treatment causes less local irritation and systemic toxicity and has low cost. Response rates of 56–81% have been reported, with recurrence rates of 36% . TCA canCryotherapy may be performed weekly using an open spray or cotton-tipped applicator for 10–20 seconds and repeated as needed. A freeze-thaw-freeze technique is applied to each lesion at each session. Application techniques are difficult to standardize and there may be significant intraoperator differences. Cryotherapy presents the advantages of being simple, inexpensive, rarely causes scarring or depigmentation, and is safe during pregnancy. Clinical studies have reported clearance rates in the range of 44–75%, and recurrence rates of 21–42% one to three months after clearance –29.Surgical treatments have the highest primary clearance rates with initial cure rates up to 60–90%. They include electrosurgery, curettage, scissors excision, and laser therapy. Surgery may be used as primary therapy, and the majority of patients can be treated under local anaesthesia. When performed carefully, simple surgical approach leaves highly satisfactory cosmetic results. Clearance rates of 94–100% and 89–100% have been reported for electrosurgery and scissors excision, respectively, with recurrence rates of 19–29%. Formal surgery, under anaesthesia, is convenient for the removal of bulky warts, extensive warts, and anal/intra-anal warts.Many patients either fail to respond to treatment or recur after adequate response resulting in patient dissatisfaction. Global recurrence rates exceed 50% after 1 year due to repeated infection from sexual contact, persistence of virus in surrounding skin, hair follicles, sites not adequately reached by the intervention used, or immunosuppression , 30–32.A quadrivalent HPV vaccine is available for prevention of HPV-associated dysplasia and neoplasia including cervical cancer, precancerous genital lesions, and genital warts associated with HPV types 6, 11, 16, and 18. Vaccine efficacy is mediated by humoral immune responses following immunization. It is indicated for prevention of CA caused by HPV types 6 and 11 in boys, men, girls, and women aged 9–26 years. The very high efficacy of the quadrivalent HPV vaccine against HPV6/11 disease was reported in multiple randomised, controlled trials. There is now accumulating evidence that population-based quadrivalent HPV vaccination can result in dramatic declines in genital warts incidence and reduction in HPV6/11 burden –35.The term “vulvar intraepithelial neoplasia” (VIN) was endorsed by the International Society for the Study of Vulvar Disease (ISSVD) in 1986 to describe intraepithelial neoplastic proliferations of the vulvar epidermis . PreviouUsual-type VIN  occurs in young women. The incidence peaks at 45–49 years old, and has increased in recent years and has nearly doubled in the last decades, especially in young women. It is causally related to HPV infection. Other risk factors include smoking and immunosuppression. The lesions are frequently multifocal and have the potential to progress to invasive carcinoma.Differentiated VIN affects older women, tends to be unifocal, and unicentric. It is not associated with HPV infection, but it is associated with vulvar dermatosis, mainly lichen sclerosus. It also has the potential to progress to invasive carcinoma. VIN is clinically important because the rate of progression to invasive SCC is reported to be as high as 80%, if left untreated .The disease is asymptomatic in about 50% of cases. When symptomatic, the main complaints include itching, pruritus, pain, and dyspareunia. The appearance is variable from unique to multifocal lesions, flat, raised, or eroded, white, grey, red, or brown. After visual or colposcopic examination, such lesions should be biopsied for histological examination. A complete gynaecologic examination is of paramount importance to exclude any multicentric lesions that may affect the cervix, the vagina, and the anal region. The diagnosis of VIN can be subtle. To avoid delay, the physician must exercise a high degree of suspicion. Vulvar biopsy should be used liberally.A part of vulvar carcinogenesis (for uVIN and HPV-related vulvar SCC) is superposable to cervical carcinogenesis. The viral oncoproteins E6 and E7 play a key role in the (pre)malignant transformation. After viral DNA integration in human host-cells, these viral oncoproteins E6 and E7 are overexpressed. Then, E6 degrades the tumor suppressor protein p53, therefore inducing the absence of cell cycle arrest . MoreoveBased on their associations with cervical and anogenital cancers, a nonexhaustive number of anogenital HPVs  have been classified by the International Agency for Research on Cancer (IARC) as oncogenic .Among women infected with high-risk types of HPV, other factors such as smoking, immunosuppression, and long-term use of oral contraceptives can result in a doubling or tripling of risk for HSIL  and invasive cancer .The HPV-independent pathway of vulvar SCC and dVIN is not well known. Genetic mutations in TP53 , 43 or PTypical low-grade VIN appears as pale areas, whereas high-grade VIN lesions appear as white or erythematous papules or macules that frequently coalesce or show a verrucous growth. Approximately 10–15% of the lesions are hyperpigmented.Differentiated/simplex VIN can be seen as focal discoloration, ill-defined white plaques, or discrete elevated nodules but they are typically less bulky than uVIN lesions . Approxi(1) Usual/Classic VIN (uVIN). uVIN shows morphological characteristics similar to all HPV-associated intraepithelial lesions such as cervical intraepithelial neoplasia (CIN), anal intraepithelial neoplasia (AIN), vaginal intraepithelial neoplasia (VaIN), and penile intraepithelial neoplasia ..4].The International Society for the Study of Vulvovaginal disease (ISSVD) has proposed that VIN should not be graded but described as high-grade VIN lesions only (VIN 2 or VIN 3). The ISSVD has also recommended that the term low-grade VIN  should not be used anymore and that such lesions should be classified as flat condyloma acuminatum, or given an appropriate descriptive term .(2) Differentiated VIN (dVIN). Differentiated (simplex) VIN is classified as high-grade VIN (thus VIN 3) due to its associated high risk to progress into invasive SCC ., 4.1, 4]This lesion is frequently associated with lichen sclerosus and other cutaneous inflammation such as lichen simplex chronicus , 47.p16, a surrogate marker of HPV, can be used to detect HPV infection. Diffuse and intense nuclear and cytoplasmic staining typically correlates with high-risk HPV infection. Focal and weak positivity is nonspecific. p16 immunostaining is characteristically negative in the epidermis of dVIN , 51.Detection of increased proliferative activity in the upper layers of the epithelium using Ki67/MIB-1 staining has shown to be well correlated with the presence of HPV DNA by molecular analysis. Ki67/MIB-1 can help for distinction between dVIN and normal vulvar epidermis . HPV in PCR analysis of VIN 1 lesions has shown a mixture of low- and high-risk HPV (geno)types, whereas VIN 2 and VIN 3 are generally associated with high-risk HPV, most commonly HPV 16 and 18. Molecular studies have failed to demonstrate HPV DNA in dVIN. To identify dVIN, p53 staining can be used. 90% of dVIN show a high p53 positivity in the basal layer with suprabasal extension , 47.The mainstay of management for VIN has been the surgical excision. One important advantage of surgical excision is that a complete histologic assessment is performed to exclude or define the presence of occult invasive carcinoma. The goal of the surgery is to obtain a 5-mm disease-free margin to control symptoms and to avoid malignant transformation. Results are initially good, but the recurrence rate is in between 30% to 50% . Large aLaser ablative therapy is an alternative to excision. The disadvantage of ablative therapy is that a necrotic ulcer on the vulva may result and wound healing may be slow. Complete healing may take several weeks. Pain, which is severe in some patients, is the main complication with laser therapy. Bleeding and infection have also been reported. The cosmetic results appear to be excellent. Laser therapy is an acceptable treatment modality, if invasive carcinoma has been ruled out. Many consider laser therapy the treatment of choice in the management of VIN, particularly for those who have multifocal disease. In the review article of the 253 patients treated with laser, 23% recurred . Cli. Cli4]. The current World Health Organization (WHO 2003) classification of vulvar tumors describes several variants of invasive squamous carcinoma :(a) Keratinizing Squamous Cell Carcinoma NOS (Not Otherwise Specified). This is the most common histologic subtype of SCC. Neoplastic cells are mature with abundant eosinophilic cytoplasm and show keratin pearl. The nuclei are enlarged with prominent nucleoli and features readily identified in most cases include considerable nuclear atypia and mitotic activity  Nonkeratinizing Squamous Cell Carcinoma. The cells in this subtype of invasive SCC show minimal evidence of keratinization with scattered keratinized cells but lacks keratin pearl. Keratin pearl formation is not observed [observed , 4, 5. Pobserved .(c) Basaloid Carcinoma. This tumor subtype arises in association with high-grade uVIN and comprises irregular nests and cords of “basaloid” cells with scant cytoplasm. The cells are ovoid, relatively uniform in size, and the nuclei show evenly distributed granular chromatin with no evident nucleoli. No keratin pearls are observed [observed , 4, 5. Pobserved . HPV-16 observed . (d) Warty (Condylomatous) Carcinoma. This histologic subtype is architecturally characterized by the presence of multiple papillary projections with fibrovascular cores. The papillae are lined by keratinized squamous epithelium showing koilocytic changes (the most characteristic features). Keratin pearl formation in the invasive nests is often seen [ten seen , 59, 60.ten seen . HPV 16 (e) Verrucous Carcinoma. This highly differentiated variant of SCC is characterized by bulbous pegs of neoplastic cells that appear to push into the underlying stroma. The neoplastic squamous epithelium is hyperplastic and associated with prominent hyper- and parakeratosis. The tumor cells have abundant cytoplasm. Nuclear atypia is minimal. Mitotic figures are rare and koilocytosis is usually absent. Verrucous carcinoma presents no or very little metastatic potential. HPV type 6 has been identified in a number of verrucous carcinoma [arcinoma , 61–64, arcinoma . (f) Squamous Carcinoma with Tumor Giant Cells. This is a rare and aggressive variant of invasive SCC characterized by the presence of numerous multinucleated tumor giant cells. Large atypical nuclei with prominent nucleoli and brisk mitotic activity are frequent [frequent , 5.(g) Keratoacanthoma-Like Carcinoma. This variant has been included in the last WHO classification (2003) of vulvar SCC. It presents an appearance of keratoacanthoma and occurs on hair-bearing skin. Histologically, it is characterized by the presence of a central crater filled with proliferating squamous epithelium and anucleated masses of keratin. Invading nests and cords of squamous epithelium are observed in the dermis. These tumors may regress spontaneously by a poorly understood immune mechanism [echanism , 5.There is no grading system unanimously accepted for vulvar SCC. The American Joint Committee on Cancer (AJCC 2010) recommends a four-grade system: well-differentiated (G1), moderately differentiated (G2), poorly differentiated (G3), and undifferentiated (G4)  .The grading system recommended by the Gynecologic Oncology Group (GOG) is based on the percentage of undifferentiated cells .Grade 1 tumors have no undifferentiated cells, Grade 2 tumors contain less than 50% undifferentiated cells, Grade 3 tumors have greater than 50% but less than 100%, and grade 4 is essentially entirely composed of undifferentiated cells. The risk of recurrence has been reported to be higher with increasing grade –69.Immunohistochemical expression of p16 is commonly expressed in vulvar squamous tumors and VIN associated with oncogenic HPV , 71. IndVulvar carcinoma benefits from a surgical staging system based on criteria established by the International Federation of Gynecology and Obstetrics (FIGO) . The latPretreatment assessment includes confirmation biopsy and careful perineal, vulvar, and vaginal examination. MRI and PET CT are not part of a routine work up and must be prescribed on individual basis.Early stage carcinoma is best treated by a radical local excision with macroscopic tumor-free margins of 1 cm . The “trLocally advanced vulvar carcinoma based on vaginal, urethral, or anal involvement is treated by concomitant chemoradiation associating external beam, brachytherapy implant, and radiosensitizing platinum chemotherapy. In this context, nodal staging may precede the initiation of the radiotherapy . ExternaRecommended follow-up includes clinical vulvar, vaginal, and nodal examination on a three to six monthly basis. Indication of routine morphological or metabolic imaging exams must be individualized.The understanding of all the aspects of these three relatively frequent diseases that are “vulvar condyloma acuminatum, vulvar intraepithelial neoplasia, and vulvar SCC” guarantees an optimal care of the patients. A close cooperation between clinicians and pathologists is also essential concerning their accurate diagnosis.


Cervical cancer ranks as the first most frequent cancer among women in Benin. The major cause of cervical cancer now recognized is persistent infection of Human Papillomavirus (HPV). In Benin there is a lack of screening programs for prevention of cervical cancer and little information exists regarding HPV genotype distribution.Cervical cells from 725 women were examined for the presence of viral DNA by means of a polymerase chain reaction (PCR) multiplex-based assay with the amplification of a fragment of L1 region and of E6/E7 region of the HPV genome, and of abnormal cytology by Papanicolaou method. The association between HPV status and Pap test reports was evaluated. Socio-demographic and reproductive characteristics were also related.A total of 18 different HPV types were identified, with a prevalence of 33.2% overall, and 52% and 26.7% among women with and without cervical lesions, respectively. Multiple HPV infections were observed in 40.2% of HPV-infected women. In the HPV-testing group, the odds ratio for the detection of abnormal cytology was 2.98  for HPV positive in comparison to HPV negative women. High risk types were involved in 88% of infections, most notably HPV-59, HPV-35, HPV-16, HPV-18, HPV-58 and HPV-45. In multiple infections of women with cytological abnormalities HPV-45 predominated.This study provides the first estimates of the prevalence of HPV and type-specific distribution among women from Benin and demonstrates that the epidemiology of HPV infection in Benin is different from that of other world regions. Specific area vaccinations may be needed to prevent cervical cancer and the other HPV-related diseases. Benin has a population of 2.22 million women aged 15 years and older who are at risk of developing cervical cancer. Cervical cancer ranks as the first most frequent cancer among women in Benin, and the second most frequent cancer among women between 15 and 44 years of age. Current estimates indicate that every year 925 women are diagnosed with cervical cancer and 616 die from the disease [There is an annual global incidence of nearly 500 000 new cervical cancer cases, with 274 000 deaths per year .In developing countries, such as Benin, there is a lack of effective screening programs for prevention of cervical cancer. In these countries, in fact, no clinically significant reduction in the incidence of cervical cancer has occurred during the past three decades ,4. In deThe major cause of cervical cancer now recognized is persistent infection of HPV . HPV exiThe distribution of HPV types varies greatly across populations and HPV surveys have been performed in different geographical regions in order to apply appropriate vaccine strategies . HoweverIn light of these considerations, the aim of this study was to examine, through an observational study, the HPV genotype distribution and the cervical cell cytology among women from Benin by evaluating genital swabs collected from 725 women undergoing voluntary screening. In addition, socio-demographic and reproductive characteristics were related with HPV and cytological results.From January to September 2009 a total of 725 genital swabs were collected from women aged from 15 to 70 years undergoing voluntary screening at Saint Luc Hospital of Cotonou and Porto-Novo (south Benin), at the Hospital of Abomey, at the St. John Catholic Hospital of Parakou and of Lokossa , at the Hôpital de L'Ordre de Malte of Djougou, at Saint Jean de Dieu Fatebenefratelli of Tanguetà, and Natitingou in Atakora Department (north-west Benin), and at the main maternity hospital . The study protocol was approved by the local research ethic committee, and informed consent was obtained from all subjects according to the World Medical Association Declaration of Helsinki. All participants were interviewed using a structured questionnaire with respect to sociodemographic and reproductive characteristics such as age, age at first sexual activity, number of pregnancies, age at first menstruation, and number of abortions. They were also instructed about the causes of cervical cancer, signs and symptoms, prevention, early detection, and treatment. Results of HPV and cytological testing were delivered to the health centres after testing, and those with positive tests were suggested for colposcopy, biopsy, and adequate treatments.Cervical smears, suspended in ThinPrep preservative solution, were processed for HPV types at Research Laboratories, bcs Biotech S.p.A., Cagliari, Italy, on the basis of the manufacturer's instructions. In the cytological testing, cervical cells were collected with the use of the Ayre's spatula (esocervix) and cytobrushes (endocervix). The smears were processed in the Cytomorphology Department laboratories of Cagliari University, Italy, and independently evaluated by two anatomopathologists, according to the 2001 Bethesda system ,12.Screening was performed by doctors and auxiliary nurse-midwives who were trained in a preventive course using International Agency for Research on Cancer (IARC) manuals for the collection of cervical cells for HPV and cytological testing.in situ and "AGC favour neoplastic". Any abnormalities detected were dealt with in compliance with guidelines updated by the Italian Association of Colposcopy and Cervical/Vaginal Pathology [All the women were screened for the presence of abnormal cervical cytology with conventional Pap smears. Interpretation of the cytological specimens was reported according to the Bethesda System 2001, adopted in May 2001 and including revision statements of specimen adequacy, general categorization, interpretation and result of epithelial cell abnormalities as follows: "atypical squamous cells" (ASC) divided into qualifiers of 1) ASC of "undetermined significance" (ASC-US), and 2) "cannot exclude high-grade squamous intraepithelial lesion" (ASC-H); low-grade squamous intraepithelial lesion (LSIL); high-grade squamous intraepithelial lesion (HSIL); squamous cell carcinoma; atypical glandular cells (AGC), with attempts to identify whether the origin of the cells was endometrial, endocervical or unqualified; endocervical adenocarcinoma athology -17.® HPV Extraction kit (bcs BIOTECH), multiplex PCR amplification of target DNA, hybridization of amplified products to oligonucleotide probes, and finally detection of the amplified products by colourimetric determination, using the ProDect® Chip HPV Typing kit (bcs BIOTECH). The test system uses L1 viral region (143 bp) as target to detect and type 14 viruses with a high-medium oncogenic risk , and 5 viruses with a low-oncogenic risk . Moreover, to increase the rate and complete the HPV detection, this method amplifies the E6 and E7 ORF (233-268 bp) when the L1 system gives negative results [HPV DNA typing was performed by the Biochip array genotyping test based on four major processes that include specimen preparation, using the ProDect results .® HPV Extraction kit  as follows: 3 ml of ThinPrep cytological suspension were centrifuged at 2000 rpm for 20'; cell pellets were resuspended in a lysis buffer and digested with Proteinase K. The total DNA extracted was concentrated with an alcoholic precipitation and resuspended with 30 μl of Tris-EDTA buffer.DNA extraction was carried out with the ProDect® Chip HPV Typing kit : each 50 μl reaction consisted of working mix containing MgCl2, KCl, TRIS-HCl, Taq DNA polymerase, dNTPs, and biotinylated GP5+/GP6+, pU-1M/pU-2R primers and β-globin primers together with 5 μl of DNA sample. The PCR was programmed as follows: 94°C for 2 min, and 5 cycles of 94°C for 30 sec, 50°C for 1 min, 72°C for 30 sec and 40 cycles of 94°C for 30 sec, 45°C for 1 min, 72°C for 30 sec and finally, at 72°C for 10 min before cooling it indefinitely at 4°C.DNA amplification was performed by multiplex PCR with the ProDectHybridization to the oligonucleotide probe: denaturing solution was added to the PCR product; all washes and hybridization steps were undertaken in a 96-microplate wells in which the specific probes for 19 HPV genotypes, E6/E7 sequences, and β-globin reference were pre-spotted . The sysColourimetric determination: the colour change reaction was from Streptavidin-phosphatase mediated precipitation of working substrate.® Bcs Biochip Reader - bcs BIOTECH, Cagliari, Italy). Based on the microbiological profile of the sample, different patterns appeared on the chip, each corresponding to the type of pathogen potentially present in the starting sample.The Biochip reading and pattern interpretation were performed through an automated reader and specifically designated software .Statistical analysis was performed by Statistical Package for the Social Sciences 15.0 software. All variables were expressed as absolute and relative frequencies.2 test or Fisher's exact test . Mann-Whitney and Kruskal-Wallis tests were used to analyze differences between age at diagnoses, age at first sexual activity, age at menarche, age at first childbirth and HPV or Pap test status for all women.The association between HPV status and Pap test reports was evaluated by calculating crude odds ratio (OR) by logistic regression analysis with a 95% confidence interval (CI) and by Fisher's exact test. The association between HPV or Pap test status and clinical-gynaecological variables, such as number of pregnancies, number of abortions, menopausal status, and pregnancy status, was assessed through the χTo deal with missing data, a multiple imputation model with fully conditional specification was used.Of the 725 women who came to the clinic study 427 had valid HPV tests, whereas 298 have no adequate sample to HPV testing.Viral DNA was identified in 142 of the 427 samples (33.2%); 85 (59.8%) samples had single infection and 57 (40.2%) had multiple infection. Among HPV-positive samples, 125 (88%) had high-risk HPV infection , HPV-18 (14.8%), HPV-58 (13.4%), HPV-45 (9.9%), HPV-56 (8.4%), and low risk genotypes such as HPV-42 (7.7%), HPV-6 and -11 (7%) in women with both normal and abnormal cytology. HPV-59 was detected both in single and multiple infections, whereas other HR-HPV types  were more frequently identified in multiple infections (Table P < 0.0001).Among the 427 women who had a HPV testing, 354 had both Pap test and HPV testing . Mann-Whitney and Kruskal-Wallis tests showed a significant correlation between age at diagnoses and abnormal cytology (P = 0.03). No correlation was found between age at diagnoses and HPV status (P = 0.28). These tests have not shown significant correlation between HPV or Pap test status with age at first sexual activity , age at menarche , and age at first childbirth .The correlation between the HPV positivity or abnormal cytology and most of clinical-gynaecological characteristics of screened Benin women, such as number of pregnancies, number of abortions, menopausal status, and pregnancy status, assessed by Fisher's exact test, revealed no statistically significant association. Fisher's exact test showed that the abnormal Pap smear appears in 27.9% of women under 36  and 18.6% of older ones .Most of HPV-DNA positive women analysed in this study were infected with HPV-59, HPV-35, HPV-16, and -18, in decreasing order. The HPV types had different distributions in single and multiple infections. HPV-59 was present with the same proportion in single and multiple infections, while 36.84% of multiple infections presented HPV-35, followed in decreasing order by HPV-16, HPV-18 and HPV-45 (Z-test and Fisher's exact test).These data on HPV type distribution in women from Benin indicate that the epidemiology of HPV infection in this area is different from other regions of the world and also from those close to Benin such as Nigeria and Burkina Faso. The prevalence of HPV genotypes in cervical cytological samples varies greatly in different geographical regions -27. In fThe frequency of HPV in cervical cytological samples shows a strong correlation with cervical cancer incidence, but HPV-positivity may be detected in a high rate in women with negative histology or cytology. In this study, HPV DNA was detected in 26.7% of women from Benin without cytological abnormalities and many of whom harboured HPV types with very high progressive potential. These are alarming data since they result very high in comparison to those of a recent meta-analysis that estimated that the worldwide HPV prevalence in the cervix of women with normal cytology is about 10.4% . The datThe HPV type 16, although with different prevalence rates, is the most common viral type, being present in 12.3%, 18.4%, 21.4% and 25.5% of HPV-positive women without cytological lesions from Sub-Saharan Africa (Nigeria), Asia, South America and Europe, respectively . These dAs expected, in women from Benin with abnormal Pap tests HPV DNA was detected with a high rate of 52%. The odds ratio in fact shows that HPV-infected women run a double risk of having a cervical lesion compared to those uninfected. HPV-59 and HPV-35 are the most common HPV types identified in these women with cytological lesions, followed by HPV-16, HPV-18. In multiple infections, HPV-45 was the one mostly detected. These findings are disturbing since previous surveys from nearby geographically regions showed that HPV35 and HPV45 were common in sub-Saharan Africa women with high-grade squamous intraepithelial lesions and squamous cell carcinoma -26.Population-based data for HPV-type distribution is a prerequisite to development of new HPV-screening tests to predict the potential benefits of HPV vaccination and to monitor the impact of vaccination on HPV type replacement, but these data are limited or missing for many world regions . This stThe results of the association between the gynaecological and socio-demographic characteristics and HPV infection or Pap test results are inconsistent except for the relation of abnormal Pap smears to young age, age at first childbirth, and number of spontaneous abortions. Individual sexual habits, difficult to detect in populations, are probably responsible for these data.These data can be considered as nationally representative of Benin because the study analysed women from a population living there. A major strength of this study is the fact that it provides the first estimates of the prevalence of HPV among women from the general population in Benin, showing an elevated frequency of genital HPV infection in Benin women. Moreover, the high prevalence of viral types other than vaccine types should be taken into account in developing area-specific vaccines. These data will contribute to elucidating the epidemiology of HPV infection across subpopulations, and it will also be helpful in the implementation of future prevention strategies.AGC: Atypical glandular cells; ASC: Atypical squamous cells; ASC-H: ASC cannot exclude high-grade squamous intraepithelial lesion; ASC-US: ASC of undetermined significance; CI: Confidence interval; HSIL: High-grade squamous intraepithelial lesion; HPV: Human Papillomavirus; HPV LR: Human Papillomavirus of Low-Risk for cancer; HPV HR: Human Papillomavirus of High-Risk for cancer; IARC: International Agency for Research on Cancer; LSIL: Low-grade squamous intraepithelial lesion; OR: Odds ratio; PCR: Polymerase Chain Reaction.The authors declare that they have no competing interests.FP contributed to conception and design, acquisition, analysis and interpretation of data, and drafted the manuscript. MP coordinated the acquisition of the samples and performed the cytological analysis. ADM, MA, and MP performed the molecular procedures and help to draft the manuscript. ARFZ participated in study design and acquisition of the samples. LM performed the statistical analysis. MTP revised the data and contributed with important intellectual content. DM and CM contributed to acquisition and elaboration of data. PS conceived and participated in study design and coordination, and contributed to acquisition of the samples, to draft the manuscript and supervised the work of the research group. All authors read and approved the final manuscript.