Lung cancer is a leading cause of cancer morbidity and mortality worldwide. Several studies have suggested that Human papillomavirus (HPV) infection is an important risk factor in the development of lung cancer. In this study, we aim to address the role of HPV in the development of lung cancer mechanistically by examining the induction of inflammation and epithelial-mesenchymal transition (EMT) by this virus.In this case-control study, tissue samples were collected from 102 cases with lung cancer and 48 controls. We examined the presence of HPV DNA and also the viral genotype in positive samples. We also examined the expression of viral genes , anti-carcinogenic genes ), and inflammatory cytokines in HPV positive cases.OR = 3.37, 95% C.I = 1.58–7.22, P = 0.001). The most prevalent virus genotype in the patients was type 16 (38.8%). The expression of p53 and RB were decreased while and inflammatory cytokines were increased in HPV-positive lung cancer and HPV-positive control tissues compared to HPV-negative lung cancer and HPV-negative control tissues. Also, the expression level of E-cad and PTPN-13 genes were decreased in HPV- positive samples while the expression level of SLUG, TWIST and N-cad was increased in HPV-positive samples compared to negative samples.HPV DNA was detected in 52.9% (54/102) of the case samples and in 25% (12/48) of controls. A significant association was observed between a HPV positive status and lung cancer (Our study suggests that HPV infection drives the induction of inflammation and EMT which may promote in the development of lung cancer. Lung cancer is one of the leading causes of cancer morbidity and mortality worldwide . There aE1, E2, E4–E7, and late region (L) consisting of L1 and L2 [Human papilloma virus (HPV) is one of the most important human oncogenic viruses , which h1 and L2 . E6 and 1 and L2 . E6 inte1 and L2 , 17. The1 and L2 , 19.In response to harmful stimuli and invading pathogens, the innate immune system becomes activated through a variety of receptors, leading to the generation of an acute inflammatory response. This inflammation aids in the removal and clearance of the stimulus. However, should the stimulus fail to be removed the development of chronic inflammation occurs which is strongly associated with cancer .Chronic inflammation as a result of viral infection is responsible for an estimated 25% of all human cancers , 22. In The second facet of high-risk HPV (hr-HPV) related tumour development is EMT, which plays an important role in solid cancer progression through multiple biochemical changes. EMT is well known to enhance cell migration, invasion and cancer development .There are several genes involved in EMT, including SLUG, PTPN13, E-cad, N-cad and TWIST. SLUG protein is involved in important cellular events including EMT and also has anti-apoptotic activity . PTPN13 In this study we investigated, for the first time, the role of hr-HPV in EMT and lung tumour development. We also assessed the prevalence of HPV in lung tumour samples; examining the expression level of viral and cellular genes and the associations between these expressed genes in EMT and lung tumour development.This case-control study was conducted between November 2017 and September 2018. One hundred and two lung cancer samples and forty-eight normal lung tissue samples. Control samples were age and sex matched, with the tissue samples collected from a peripheral region of the surgically removed lung cancers and non-cancer patients with fibrosis. All samples, cases and controls, were fresh tissue with a Tumor Proportion Score (TPS) > 50%. Control samples were age and sex matched. The TNM system was used to denote the stage of cancer as decided by a consultant oncologist and oncological surgeon. Gender, age, smoking status, tumour type and tumour stage were clinical parameters of patients that are shown in . We had β-globin as described before [Total DNA extraction from tissue samples was performed by QIAamp® DNA Mini Kit . Quality of extracted DNA was assessed by conducting PCR for d before . All samTotal RNA extraction was conducted by RNeasy Mini Kit .L1 and E7 genes [HPV genome detection was conducted using PCR for E7 genes . HPV genE2/E6 ratio was used. An E2/E6 ratio > 0 and < 1 indicates that the virus is in a mixed physical state, with both episomal and integrated forms of the virus [To determine the physical status of the HPV genome, the he virus .Total RNA was extracted and purified from the tissue by using RNEasy Mini kit . For cDNA synthesis, 1 μg of total RNA was reverse transcribed using the QuantiNova Reverse Transcription Kit . CDNA synthesis was performed in a thermal cycler in the following order: 27 °C for 10 min, 38 °C for 15 min, 44 °C for 40 min, 72 °C for 15 min. All the primers which were used to detect viral genes  are listed in a table in the . To deteFor viral genes we used serial dilutions of E2, E6 and E7 genes cloned in PUC57 vector . Serial dilution was containing equivalent amounts of these genes from 72 to 865 million copies per reaction, served as a standard control.GAPDH. The primers are listed in cDNA was synthesized using the PrimeScript First Strand cDNA synthesis kit . Quantitative RT-PCR analyses were performed using the Power SYBR Green PCR Master Mix . The relative expression level of each mRNA was normalized using For tissue homogenization, all fresh tissue samples were weighed and the tissue lysate was prepared according to the manufactures protocol . Approximately 50 μg of each tissue was excised and washed with ice-cold PBS.The level of p53, RB, IL-1, IL-6, IL-11, NF-kB, NF-κ PTPN13, E-cadherin, N-cadherin and TWIST was assessed using Abcam’s p53 Simple Step ELISA® Kit , Human Retinoblastoma ELISA® kit , Human Retinoblastoma ELISA® kit , Human IL-6 ELISA® Kit, Human IL-1 beta ELISA® Kit, Human IL-11 ELISA® Kit, NF-kB p65 Transcription Factor Assay® Kit , Human Tyrosine-Protein Phosphatase Non-Receptor Type 13 (PTPN13) ELISA Kit , Human E-Cadherin, N-Cadherin ELISA Kit .and TWIST ELISA Kit .The RONS level was assessed by OxiSelect™ Intracellular ROS/RNS Assay kit . For this purpose, cell lysate was used and preparation of this based on Kit instructions.P-value of less than 0.05 was considered as statistical significance.Continuous variables are presented as mean ± standard deviation and categorical variables are presented as N (%). Normality test was checked using Kolmogorov–Smirnov test for the continuous variables. For comparing the central tendency  between two groups, two-independent samples t-test or Mann-Whitney non-parametric test and between more than two groups, one-way ANOVA or kruskal-wallis test were used. Chi-square/ or Fisher exact test was performed for assessing the associations of the categorical variables. The unit of all expression RT-PCR is (2^-DCt)*1000. Internal normalization was performed using an internal housekeeping or reference gene (GAPDH) and external normalization was applied by standardized approach. In addition, correlation analysis was done by Spearman’s correlation coefficient between viral and cellular factors. All of statistical analyses were analysed using GraphPad Prism 6 and STATA software versions 11.2. False discovery rate was corrected by Benjamini-Hochberg approach for multiple comparisons. A two-sided p = 0.77). There were three types of lung tumour tissues; squamous-cell carcinoma (51.9%), adenocarcinoma (32.3%) and SCLC (15.7%). The highest and lowest stages of cancer in this study were IIIB (30.4%) and IA and IIB (1.9%) respectively. HPV DNA was detected in 52.9% of the lung cancer specimens and in 25% of control samples. There was a significant association between the presence of HPV and lung tumour . Genotype 16 was the most frequently isolated genotype in both cases (38.8%) and controls (50%). No significant association was observed between all genotypes and the occurrence of lung tumour (p = 0.651) (p = 0.434). There were no significant differences in the frequency distributions of lung tumour stages between HPV+ and HPV- groups (p = 0.163). More information is presented in In this case-control study, we examined 102 lung cancer cases and 48 controls, with the mean ± SD age; 56.36 ± 12.49 and 57.0 ± 12.24, respectively. Seventy-four (72.5%) of the cases and 31 (64.5%) of the controls were male, respectively. The cases and control groups were matched based on age (= 0.651) . HPV DNAE7 which was most highly observed in stage IV samples (mean ± SD:13.56 ± 5.13). The lowest level of viral gene expression examined was E6 in stage IB samples (mean ± SD: 3.0 ± 1.75). The gene expression level of viral factors E2 and E6 were highest in stage IIB and stage IV respectively. Stratification of the samples based on the tumour type reveals the expression level of E7 in adenocarcinoma samples (mean ± SD: 11.94 ± 4.93) and E2 in SCLC (mean ± SD: 3.67 ± 1.15) were the highest and lowest respectively (Table 2). The expression level of viral genes in control samples and tumour samples are illustrated in Fig. In the HPV+ lung carcinoma patients, the virus was present in its integrated form in 27.8% of cases. The incidences of episomal and mixed forms of HPV genome were 5.5 and 66.7% respectively. In the control HPV+ group, the incidence of HPV genome status was 25, 0 and 75% integrated, episomal and mixed forms of HPV respectively Table . The genp < 0.001). The level of inflammatory factors, were considerably higher in HPV+ cases and controls compared to the HPV- cases and controls groups. The levels of EMT involved factors found to be significantly higher in HPV infected group compare to HPV non-infected group . Among the EMT involved genes, PTPN13 and E-cad were significantly downregulated in HPV+ cases and controls compared with HPV- cases and controls (p < 0.001). SLUG, N-cad and TWIST were significantly upregulated in HPV+ cases and controls compared with HPV- cases and controls (p < 0.05). The highest expression levels were related with SLUG, N-cad and TWIST in HPV+ compared with HPV- groups . More details are presented in Fig. 2. Significant negative correlations were observed between the expression level of viral genes and the protein expression levels of regulatory host proteins, Rb and p53. Among the inflammatory factors examined, the correlations between E2 expression level with IL-1 and TNF-α were statistically significant, and the correlations between IL-6 with E6 and E7 were statistically significant (p < 0.01). The correlation between E2 expression level and PTPN13 was positive but with SLUG, E-cad, N-cad and TWIST was negative. The expression level of E6 significantly correlated with the protein level of PTPN13. The expression level of E7 has the negative correlation with E-cad and N-cad (p < 0.05). Conversely, there were positive correlations between E6 gene expression and IL-1, IL-6, IFN-α and IFN-β protein levels and RONS production (p < 0.05)  [In the current study, 52.9% of lung tumour samples were positive for HPV. Moreover, we demonstrate that increased expression of HPV genes is associated with decreased expression of regulatory cellular genes, RB and p53, and as a result increased risk of lung cancer. In an investigation Nadji et al.  studied 141 lung carcinoma samples and 92 non-cancersamples as controls. Results demonstrated that 25.6% of cases and 9.0% of controls were positive for HPV infection respectively. The reported odds ratio for HPV infection was 3.48  . Robinso= 0.002) . A meta-= 0.002) . Another= 0.002) . The mos= 0.002) –40. Prev= 0.002) . In anotPrevious investigations have noted the physical status of HPV DNA as an important marker for tumour progression in other cancers, such as breast cancer . In thisp < 0.001 for all)  the impact of HPV on the expression of genes involved in EMT, ii) the impact of HPV in the development of chronic inflammation and microenvironment alteration. EMT promotes cancer development through enhancing cellular migration and invasion , 45. Oncl) Table . This sil) Table , with vils Table . Unlike ls Table , Table 3The tumour microenvironment is a key factor in tumour development and several epidemiologic and clinical researches have proposed a strong association between inflammation related to chronic infection and lung cancer , 56–58. P < 0.001). They propose that HPV infection could be considered as a biomarker/risk in some cancers in individuals with chronic inflammation [Our results demonstrate that the expression of numerous inflammatory factors was higher in HPV+ samples than HPV-negative samples Table . Previouammation . Previouammation –64.In the current study the highest expression level of viral genes was in stage IV and the lowest level was in Stage IA and IB. In the other words, viral genes can be related to chronic inflammation and EMT Table . This isA key risk factor associated with HPV infection is smoking status. Previous studies have demonstrated the relationship between smoking and HPV infection in some cancers such as lung and cervical , 65. In Limitation of the current study were including:i) Limitations on the number of cases considered for the study and the lack of statistical representation for some tumor stages; ii) protein and RNA samples are pooled representation of the different cell types from the original tumor/control tissue; iii) the absence of medical information regarding a HPV infection before cancer diagnosis for the patients analyzed in this research.In summary, the presence of HPV was detected in 52.9% of lung cancer samples among which most were at stage III and IV (73.5%). Infection of HPV directly promotes local inflammation which in turn promotes tumorigenesis and cancer development. We demonstrate that HPV is associated with lung cancer development, although the role of hr-HPV in lung cancers requires further study. To the best of our knowledge, this is the first study reporting the role of HPV genes expression in EMT and the association between this virus and chronic inflammation in lung cancer patients.Additional file 1.


There was no significant difference in HR-HPV positivity between clinician-collected and self-collected specimens among women from the community-based clinic  or the referral clinic . HPV16, HPV35, and HPV33/52/58 group were the most frequently detected genotypes at both study sites. Self-sampling for HPV testing received a high positive response of acceptance . The overall agreement between hpVIR assay and HC-2 was 87.7% (k = 0.754). The study found good agreement between clinician-collected and self-collected genital specimens. Self-collection can have a positive impact on a cervical screening program in South Africa by increasing coverage of women in rural areas, in particular those unable to visit the clinics and women attending clinics where cytology-based programs are not functioning effectively.Human papillomavirus (HPV) testing on vaginal self-collected and cervical clinician-collected specimens shows comparable performance. Self-sampling on FTA cards is suitable for women residing in rural settings or not attending regular screening and increases participation rate in the cervical cancer screening programme. We aimed to investigate and compare high-risk (HR)-HPV prevalence in clinician-collected and self-collected genital specimens as well as two different HPV tests on the clinician collected samples. A total of 737 women were recruited from two sites, a community health clinic (n = 413) and a referral clinic (n = 324) in the Eastern Cape Province. Cervical clinician-collected (FTA cards and Digene transport medium) and vaginal self-collected specimens were tested for HR-HPV using the  Cervical cancer (CC), is the third-ranking cause of cancer disease with an estimated 569,847 cases leading to 311,365 deaths in women globally in 2018 . HR-HPV positivity between self-collected and clinician-collected samples showed an agreement of 86.9%, with a kappa value of 0.669 .p = 0.222]. The observed overall agreement of HR-HPV infection between self-collected and clinician-collected samples was 91.4% .p = 0.601, p = 0.108). The most frequently detected HR-HPV genotypes were HPV16, HPV35, and the HPV33/52/58 group, both in self-collected and clinician-collected specimens  with inconclusive results . More thThe participants from both study sites gave a similar response regarding self-collection versus clinician-collection of specimen . More thhpVIR assay. Overall, the HR-HPV prevalence was 46.2% using hpVIR compared to 48.3% using HC-2, with an agreement of 87.7%  between the two assays Click here for additional data file.S2 Data(CSV)Click here for additional data file.


A reduction of 78.5% in incident/reactivated HPV infections was demonstrated. Data results add to the current knowledge about the mechanism of post-surgical adjuvant HPV vaccination. Our prospective study is the first to document the vaccine clinical effectiveness in preventing “reactivation” of latent HPV infections. Quadrivalent HPV vaccine administered after the surgical treatment for vulvar HSIL appears to be useful in preventing recurrent disease.Data suggest that adjuvant human papillomavirus (HPV)-vaccination in women treated for cervical HPV diseases reduces recurrent disease. This study investigates adjuvant HPV-vaccination and the rate of recurrence in women undergoing surgery for vulvar high-grade squamous intraepithelial lesions (HSIL). From January 2013 to April 2020, we enrolled 149 women in a prospective case-control study. The control group (NV-group) was treated by standard surgery alone, while the study group received adjuvant vaccination soon after surgery (V-group). A follow-up was performed by vulvoscopy and HPV test. Statistical analysis was performed by Fisher’s exact test. HSIL recurrence was observed in 24/76 (32%) patients in NV-group and in 8/42 patients (19%) of the vaccinated group. By analysing the recurrence rate related to the incident and reactivated latent HPV infection, we found a significant difference between (17/76) 22.3% in NV-group and (2/42) 4.8% in V-group ( High-risk human papillomavirus (HPV) types cause approximately 5% of all cancers worldwide . In the In 2006 HPV vaccination was licensed for primary prevention of HPV related diseases in young females . AlthougPersistent HPV infection and high-grade squamous intraepithelial lesions (HSIL) are presumed to lead to HPV-related vulvar cancer, as persistent cervical HPV infection can lead to cervical cancer. The protective role of the HPV vaccine in preventing vulvar premalignant lesions . Patients are referred to the clinic by 11 local hospitals and private gynaecologists for adjuvant vaccination, and all referred women have been included in the project. The patient population of the USL Toscana nord ovest area is about 1,000,000 women. Previously we reported a reduction of subsequent cervical disease after conisation of 81.2% .clinical effectiveness of the human papillomavirus 4-valent vaccine in preventing disease relapse after surgery. The aim of this study was to investigate possible implications of receiving adjuvant HPV vaccination, in individuals surgically treated for vulvar H-SIL, evaluating the Participation in the study was offered to all women undergoing surgical treatment for vulvar HSIL, between January 2013 and April 2020, according to the inclusion/exclusion criteria . All theWomen were enrolled in this prospective case-control study, regardless of their baseline HPV DNA status. The main outcome was to evaluate the incidence of subsequent HPV-related vulvar HSIL during the follow-up period between the two groups. The study was conducted according to the ethical standards of the Declaration of Helsinki. Written informed consent was obtained from all the participants; the clinical management was in accordance with Good Clinical Practice, and was approved by the ATNO area. The following pre- and post-surgical procedures are standardised.Vulvoscopy, punch biopsy for histological assessment and HPV test were performed during the pre-surgical evaluation; patients with a vulvar HSIL diagnosis were referred to surgical treatment. HPV test was collected by vulvar scraping, and a commercial kit  was used to detect the presence of virus by real-time PCR of L1 region of HPV and the human beta-globin gene as the internal control. The reverse specific primers for HPV detection were biotinilated. The thermal profile performed on a RotorGeneTM 6000 instrument  was 95 °C for 3 min, 50 cycles at 95 °C for 0.5 min; 44 °C for 0.5 min; 72 °C for 0.5 min; followed by a final melting from 72 °C to 95 °C with 1 °C increment at each step. The obtained melting curve was analysed in order to assign the positivity of the sample.The byotinilated HPV positive PCR products were subjected to pyrosequencing using a commercial kit . After some steps of purification from the reaction mixture with the PyroMark Q96 ID system  according to themanufacturer’s instructions, the DNA was denaturated by heating and added to sequence HPV primers. The sequencing reaction was performed in the presence of substrate and enzyme by dispensation of dATPThe surgical treatment was colposcopy guided and generally performed under local anaesthesia (using 2% Carbocaine). Most cases underwent electrosurgical wide local excision and/or laser vaporisation; small lesions in periclitoral area or lesion with minimal residual vulvar disease post-biopsy underwent laser vaporisation only. A solid-state erbium in yttrium aluminium-garnet crystal (Er:YAG) laser  with a wavelength of 2940 nm, set to ablative mode was utilised. A first clinical evaluation at the vaccination clinic was scheduled for all the patients with histologic confirmed vulvar HSIL within 30 days after surgery. All women in line with inclusion and exclusion criteria  were infAfter the counselling session, participation in the study was offered to all patients treated for vulvar HSIL . VariablPatients satisfying enrolment criteria and desiring to receive the vaccine, were included in the study as “vaccinated and follow-up” arm. All “vaccinated” patients received HPV quadrivalent vaccine . Patients received vaccination with the first dose after counselling and the remaining two doses 2 and 6 months later. The vaccine was offered to a reduced price.Women fitting enrolment criteria, once filled out the informed consent and not desiring vaccination, were enrolled in the “follow-up only” arm, as the control group (NV-group). A post-surgical evaluation was performed with the vulvar HPV test and vulvoscopy; the first follow-up evaluation was scheduled every three months in the first year after the surgery, then participants were evaluated annually until the seventh year post-treatment.Any vulvar area suspected of disease persistence or recurrence was investigated by punch biopsy. Recurrences were treated with re-excision of the area and confirmed by histological diagnosis, while vulvectomy associated with inguinal-femoral lymphadenectomy was performed in case of invasive vulvar carcinoma. Persistent vulvar HSIL at first clinical evaluation was a study exit criteria Data were collected in a regional database. A vaccination certificate was issued to the patient. Adverse events were reported to offices in charge, as per clinical practice. Only women followed for at least 24 months were included in the analysis.Clinical disease relapse (CDR) was defined as a clinical disease recurrence, histologically confirmed, of HSIL during the seven years follow-up period. In order to understand the mechanism of relapse, we divided the recurrences into three categories, based on the pathway of the HPV infection status after the surgical treatment. The clinical findings of this evaluation are reported in With the term of “Persistent Recurrence” (PR), we defined a subgroup of patients, who relapsed with a histologically confirmed HSIL related to the same HPV type found in the first surgical treatment, during the follow-up period.With the term of “Incident Recurrence” (IR), we define a relapse associated with an HPV “de novo” infection (due to an HPV type that was not present at the time of the first diagnosis), while “reactivation” was called an infection caused by the same HPV type found at first surgery.While the CDR absolute number includes both persistent and incident recurrences together, the subgroup analysis (PR/IR) of our model of recurrence is able to determine the exact role of persistent and incident infections.p value of <0.05 was considered statistically significant. The Fisher’s exact test was used to assess the significance of differences between the two groups.CDR, PR, IR, variables associated with recurrence risk in both groups were calculated. Women finally lost to follow-up remained in the analysis for the period we could follow them, and then they were censored. A During the study period, a total of 149 patients that underwent surgery for vulvar HSIL were enrolled  for disep < 0.2)/correlate (p < 0.05) with the aforementioned recurrence risk factors). Therefore, a multivariate analysis was not built  women were HPV positive with an HPV infection clearance rate of 65.8%, while the vaccinated group (V-group) showed a positive HPV test in 11/42 (26.2%), with an HPV infection clearance rate of 73.8%. p = 0.41. At recurrence, the most frequent type of HPV in NV group was HPV 16 (71%) followed by HPV 33 (21%) and HPV 18 and 51 (8% each) in V group again HPV 16 was the most frequent type (50%) followed by HPV 33 (25%)  .Data of 118 women with 24-months or more of follow up were analysed for CDR. p = 0.19), with −40.6% of clinical relapses in V-group. CDR HSIL recurrence was observed in 24/76 (32%) patients in NV-group; vaccinated group developed HSIL recurrence in 8/42 patients (19%). CDR analysis shows a trend in disease reduction (p = 0.63). Analysing the different pathways of recurrent disease  we obserp = 0.01).In NV-group we observed 12/24 women with recurrent disease via “reactivation pathways”, while in V-group showed no disease relapse, due to reactivation; difference between the two groups was statistically significant  and a clinical effectiveness of −78.5% in reducing HPV relapse.Globally the pathways related to “persistent infection vs. incident/reactivated the previous infection”, the difference between the two groups was statistically significant, with a recurrence rate of 22.3% in the NV-group and 4.8% in the V-group, with a Age distribution at the time of recurrent disease was analysed in Vaccination was well-tolerated by all the patients; no major adverse events were reported. Common minor adverse events  were well-tolerated too, with complete resolution of the symptoms within two weeks. No discontinuations in the study, due to AE were observed.Vulvar HSIL is considered the precursor lesion for vulvar squamous cell carcinoma. Early diagnosis and adequate treatment are the only strategies for secondary prevention currently available. Analysing surgical treatments, a Cochrane review  confirm In order to reduce disease relapse, adjuvant vaccination in HPV related disease is an upcoming issue ,34,35,36The interest about the potential role of HPV adjuvant vaccination is confirmed by the “VIVA trial” , an ongop < 0.05) HPV DNA positivity rates for genotypes 16, 18 and 31, RR = 1.6 (95% CI: 1.1 to 2.3), RR = 1.7 (95% CI: 1.1 to 2.8), and RR = 1.8 (95% CI: 1.0 to 2.9), in women who only tested DNA-positive for HPV16, 18 and 31 genotypes, respectively, prior to vaccination, demonstrating an earlier clearance of HPV infection in comparison with the patients included in the non-vaccination cohort. HPV vaccination reduced in a statistically significant manner . Similar data were also published in a post-hoc analysis of Joura et al. [Our data are also comparable to a very recent article published by Sand et al.  who founa et al.  in whichVulvar HSIL recurrence rate after excision ranges from 20% to 40% . This wiClinical data coming from the “SPERANZA project” about viral immunology allowed us to design a model of the pathways of disease recurrence . While tp = 0.63).Analysing the pathways of recurrence in our series we observed that vaccination is able to influence the natural history of HPV infection after the surgery: We found a minor difference between the groups in recurrence, due to “de novo” infection (2/8 V-group vs. 5/24 NV-group—p = 0.01). For the first time, we demonstrated that the mechanism of recurrence reduction is not only, due to secondary prevention of “de novo infections”  of the women , butIn Some previous studies ,49,50,51Our results add to the current knowledge on the mechanism of adjuvant vaccination in women treated for HPV related diseases, however, future studies are mandatory to confirm the findings.Strengths of the study include active follow-up of participants over seven years after the surgical treatment, with a rigorous assessment of procedures and endpoints. It is important to note that all participants were intensively tested for HPV infections, before and after surgery, which gives the opportunity to draw a natural history model of post-surgical HPV infections, understanding the pathways of disease recurrence. Limitations of our study include the modest absolute number of participants, due to the fact that vulvar HSIL represents a rare condition; moreover, even if this is a non-randomised trial, the comparison of secondary risk factors for disease recurrence between the two groups was not statistically significant, mitigating this important limitation.The quadrivalent HPV-vaccine, administered after surgical treatment for vulvar HSIL, may be useful in preventing recurrence of the disease. The HPV vaccination is safe, efficacious and well-tolerated in women with vulvar HSIL. These results of our prospective, not randomised trial, adds to the current knowledge about the mechanism of adjuvant vaccination. A large multicentre randomised trial would finally confirm these findings.


Therefore, anogenital samples from the PASSYON study were retrospectively analyzed with type-specific (TS) HPV-45 and -59 real-time quantitative PCR (qPCR) assays. The SPF10 method missed 51.1% of HPV-45 and 76.1% of HPV-59 infections that were detected by the TS qPCR assays.Human papillomavirus (HPV) epidemiological and vaccine studies require highly sensitive HPV detection systems. The widely used broad-spectrum SPF 10-DEIA-LiPA25 (SPF10 method) has reduced sensitivity toward HPV-45 and -59. Therefore, anogenital samples from the PASSYON study were retrospectively analyzed with type-specific (TS) HPV-45 and -59 real-time quantitative PCR (qPCR) assays. The SPF10 method missed 51.1% of HPV-45 and 76.1% of HPV-59 infections that were detected by the TS qPCR assays. The viral copy number (VCn) of SPF10-missed HPV-45 and -59 was significantly lower than SPF10-detected HPV-45 and -59 (P < 0.0001 for both HPV types). Sanger sequencing showed no phylogenetic distinction between SPF10-missed and SPF10-detected HPV-59 variants, but variants bearing the A6562G single-nucleotide polymorphism (SNP) in the SPF10 target region were more likely to be missed (P = 0.0392). HPV cooccurrence slightly influenced the detection probability of HPV-45 and -59 with the SPF10 method. Moreover, HPV-59 detection with the SPF10 method was hampered more in nonvaccinated women than vaccinated women, likely due to a stronger masking effect by increased HPV cooccurrence in the former group. Consequently, the SPF10 method led to a strong negative vaccine effectiveness (VE) of –84.6% against HPV-59, while the VE based on TS qPCR was 3.1%. For HPV-45, the relative increase in detection in nonvaccinated women compared vaccinated women was more similar, resulting in comparable VE estimates. In conclusion, this study shows that HPV-45 and -59 detection with the SPF10 method is dependent on factors including VCn, HPV cooccurrence, and vaccination, thereby showing that knowledge of the limitations of the HPV detection method used is of great importance.Human papillomavirus (HPV) epidemiological and vaccine studies require highly sensitive HPV detection systems. The widely used broad-spectrum SPF Persistent infection with high-risk human papillomavirus (hrHPV) is associated with multiple types of cancer, including penile, anal, vaginal/vulvar, cervical, and oropharyngeal cancer. Overall, hrHPV was responsible for 690,000 new cancer cases in 2018 , here called the SPF10 method, is widely used in epidemiological HPV monitoring and vaccine effectiveness studies. This test utilizes a cocktail of primers to detect many of the essential hrHPV types, such as HPV-16, -18, -31, -33, -35, -39, -45, -51, -52, -56, -58, and -59. Furthermore, the use of generalized primers to target a broad range of HPV types reduces the total number of primers needed (The broad-spectrum L1-based SPFs needed .10 method is the various HPV detection sensitivities. The limit of detection (LOD) for the included hrHPV types, as determined by the manufacturer, is lowest for HPV-18 and -33 with one DNA copy per reaction. Conversely, the LOD is highest for HPV-59 with 44 DNA copies per reaction, followed by HPV-45 with 9 DNA copies per reaction.One limitation of the SPF10 method in Dutch sexual health center visitors reported high vaccine effectiveness (VE) against HPV-16 and -18 and significant cross-protection against HPV-31, -35, -45, and -52. Conversely, remarkably strong negative VE against HPV-59 was also estimated  quantitative PCR (qPCR) assays in anogenital swabs of Dutch sexual health center visitors, previously described in Woestenberg et al.  study. The study design is described in detail elsewhere . Briefly10 LiPA25 version 1 method according to the manufacturer’s protocol . Briefly, HPV DNA was amplified using the broad-range SPF10 primers. Subsequently, a DNA enzyme immunoassay (DEIA) was used for the detection of HPV DNA in all samples. In order to determine HPV genotypes, amplicons of all HPV DNA-positive samples were analyzed in the line probe assay (LiPA), which is able to identify 25 HPV genotypes, including hrHPV-16, -18, -31, -33, -35, -39, -45, -51, -52, -56, -58, -59, and -68 and potential hrHPV-66. The reproducibility of the SPF10 method was analyzed and met the criteria of >90% identical or compatible HPV types. Validation of the SPF10 method with DNA isolated with the MP96 compared to the MagNA Pure LC 2.0 was successfully performed with >92% identical genotyping results.Anal and genital swabs were stored at –20°C until analysis. Then, a 200-μl specimen was spiked with Phocine herpesvirus-1 (PhHV-1) prior to DNA extraction using the MagNA Pure 96 (MP96) platform . The DNA was eluted in 100 μl elution buffer. All DNA samples were tested for HPV types using the HPV SPF10 method. For HPV-45 and -59, primers and probes were designed to target a 107-bp and a 116-bp region within the L1 gene of HPV-45 and -59, respectively  of all DNA samples, previously isolated with the MP96, was quantified with separate HPV-45 and HPV-59 type-specific (TS) real-time quantitative PCR (qPCR) assays. TS qPCR assays for HPV-45 and -59 were developed in previous research , 12. Eac10 and/or TS qPCR positive samples. Prior to sequencing, the complete L1 gene was PCR amplified in three overlapping fragments using the primers listed in Table S2. PCR amplification and Sanger sequencing were performed similarly as described for the HPV16/18 L1 gene (X77858) retrieved from the Papilloma Virus Episteme (PAVE) database (https://pave.niaid.nih.gov/). Phylogenetic and single nucleotide polymorphism (SNP) analyses were performed with BioNumerics 7.6.3. Additionally, nine HPV-59-positive samples detected by the TS qPCR only, with a VCn above the SPF10 detection limit for HPV-59, were confirmed by an external lab with the SPF10 method (20 μl SPF10 product taken for LiPA instead of the standard 10 μl), an alternative highly sensitive MPTS2 PCR Luminex assay  and with a known self-reported vaccination status.In order to evaluate the potential change in HPV-45- and HPV-59-specific VE estimates, HPV-45 and -59 detection rates were analyzed with the SPF10 method and the TS qPCR assays. κ agreement levels are stated according to reference 10 method or the TS qPCR assays. The Mann-Whitney U (MWU) test was used to compare VCn measurements between SPF10-detected and SPF10-missed HPV-45 and -59 given in copies (c) per reaction (rxn). Only VCn-positive measurements were included. Fisher’s exact test was used to analyze potential differences in the presence of SNPs in SPF10-missed and SPF10-detected HPV-59 variants. Only the SNPs occurring in more than one sample were taken along in the analysis. The effect of cooccurring HPV types on the HPV-45 and -59 SPF10 detection rate within TS qPCR positive samples was analyzed with a logistic regression model and corrected for VCn. VE was estimated as 1 – adjusted odds ratio (OR) × 100% according to Woestenberg et al. .The sequences obtained during this study have been submitted to GenBank  and 3.2% (n = 190) of the specimens were HPV-45 and HPV-59 positive, respectively. Reanalysis of all samples with the TS qPCR assays identified 1.7% (n = 109) and 10.1% (n = 598) additional swab specimens as HPV-45 and -59 positive, respectively. Sporadically, the SPF10 method detected HPV-45  and HPV-59  positivity not detected by the TS qPCR assays. The agreement between the SPF10 method and the TS qPCR assay was “substantial”  for HPV-45 but only “fair”  for HPV-59 (P < 0.0001) and HPV-59 (P < 0.0001) detection rate between the TS qPCR assays and the SPF10 method on sample level  was significantly higher (P < 0.0001) than the median VCn of missed HPV-45 . Similarly, the median VCn of detected HPV-59  was significantly higher (P < 0.0001) than the median VCn of missed HPV-59 . Thus, SPF10-detected HPV-45 and -59 generally have a higher VCn than SPF10-missed HPV-45 and -59, although (non)detection did not always coincide with the LOD as determined by the manufacturer.Since the LOD of the SPF10 method was stronger for HPV-59 than HPV-45 and served as the incentive to further analyze the authenticity of the HPV-59-positive samples. L1 sequencing was performed on a subset of HPV-59-positive samples (n = 228), which confirmed the presence of HPV-59. Of this subset, 43% (n = 99) were determined HPV-59 positive with the SPF10 method and TS qPCR, and 57% (n = 129) were determined HPV-59 positive with only the TS qPCR. All sequenced samples had an HPV-59 VCn above the LOD of 44 c/rxn, ranging from 51.4 to 175 × 106 c/rxn, with the exception of one sample with a VCn of 33.7 c/rxn. Furthermore, the sequences were dichotomized into variant lineages A or B, based on Chen et al. (n = 187) was more abundant than lineage A , although no phylogenetic distinction between the SPF10-detected and SPF10-missed HPV-59 variants was found . However, HPV-59 variants harboring any SNPs within the SPF10 primer target region of L1 were significantly more likely to be missed (P = 0.0027) by the SPF10 method compared to wild-type HPV-59 variants (100% similarity to the SPF10 target region) (10 method (P = 0.0392). Overall, the results indicate that detected and missed HPV-59 variants are phylogenetically similar, but missed HPV-59 variants more often carry the A6562G SNP than do detected HPV-59 variants.The difference between the detection rates of the TS qPCR assays and the SPFn et al. . Lineage region) . Specifi10 method. After correcting for the effect of VCn, HPV-45 detection probability increased with additional HPV types, although not consistently with increasing number of cooccurring HPV types  were analyzed with the SPF10 method and the TS qPCR assays  were vaccinated and 36.6% (n = 667) were nonvaccinated. The SPF10 method and the TS qPCR detected significantly more HPV-45 in nonvaccinated women  than vaccinated women  (P < 0.0001 for both tests). The adjusted SPF10-based VE estimate against HPV-45 of 86.7%  was comparable to the TS qPCR-based VE estimate of 82.7% . Interestingly, the SPF10 method detected significantly more HPV-59 (P = 0.017) in vaccinated women  than in nonvaccinated women . The TS qPCR detected similar HPV-59 positivity (P = 0.902) in nonvaccinated women  and vaccinated women . The adjusted SPF10-based VE estimate against HPV-59 was strongly negative at –84.6% , but this changed to 3.1%  with the TS qPCR . Conversely, no big differences between the detection probability of single HPV-59 in vaccinated compared to nonvaccinated women  was observed.HPV-45 and -59 detection rates among women eligible for vaccination  and 51.1% (109/213) of TS qPCR assay positive results, respectively. In contrast, the TS qPCR assays missed only 8% (9/113) of SPF10 HPV-45-positive results and 1.1% (2/190) of SPF10 HPV-59-positive results. The lower HPV-45 and -59 detection limit of the TS qPCR compared to the SPF10 method is emphasized by the significantly higher VCn of SPF10-detected HPV-45 and -59 compared to the VCn of SPF10-missed HPV-45 and -59. Similarly, previous research reported that HPV-16, -18, and -52 with low viral load was often missed by the clinically validated GP5+/6+ HPV detection assay, while being detected by the more sensitive SPF10 method  and technical limitations .The detection sensitivity of the SPF0 method . Further0 method . Contrar10-missed HPV-59 and several HPV-45-positive samples was above the SPF10 LOD. Nonetheless, the generated HPV-59 L1 sequences (VCn above the LOD) suggest that TS qPCR-detected HPV-59 positives are genuine. Phylogenetic differences and/or the presence of SNPs might alter primer alignment efficiency, explaining the detection failure of HPV-59 with a VCn above the SPF10 LOD. Although no phylogenetic distinction was found between L1 sequences of SPF10-detected and -missed HPV-59 variants, more missed HPV-59 variants carried the A6562G SNP. A6562G is located 11 bases from the beginning of the SPF10 target region, possibly causing less efficient SPF10 primer binding. Detection failure due to primer misalignment has been observed previously for HPV-53, -59, and -82 detection with the GP5+/6+ assay . Moreover, individual factors  or sample characteristics might explain why detection of one HPV type increases the chance of detecting HPV-45 with the SPF10 method. As a result, the impact of HPV cooccurrence is different for HPV-45 and -59 detection by the SPF10 method.Detection of HPV-45 and -59 may be hampered by cooccurring HPV types competing for PCR reagents, a process called masking. The SPF primers –29. Our 10 method-based HPV-45 and -59 prevalence in vaccinated and nonvaccinated women was within the range of that observed in the Finnish and UK studies. Nevertheless, the SPF10 method underestimated HPV-45 and -59 prevalence in both vaccinated and nonvaccinated women compared to the TS qPCR assays. For HPV-45, the SPF10 method and the TS qPCR showed a similar relative increase in prevalence in vaccinated women and nonvaccinated women, resulting in comparable VE estimates. Interestingly, the TS qPCR assay showed a stronger relative increase in HPV-59 prevalence in nonvaccinated women compared to vaccinated women, alleviating the highly negative VE estimate obtained by the SPF10 method. In fact, the TS qPCR assay found similar HPV-59 prevalence among vaccinated and nonvaccinated women, leading to a negligible VE estimate. Previous studies also reported relative increases of HPV-45, -58, and -59 persistent infections among the nonvaccinated group compared to the vaccinated group with the MPTS12 RHA detection assay instead of the SPF10 method, resulting in a large increase in VE . Recent studies reported that HPV-45 prevalence among 16- to 24-year-old women in the UK ranged from 2.6 to 4.2% in 2008 (prevaccination) and decreased to 0.2 to 0.9% in 2016 (postvaccination) , 31. A Fse in VE . Therefo10 method and the TS qPCR assay suggest unmasking rather than type replacement. Type replacement is the genuine increase of nonvaccine HPV types due to a reduction in vaccine-targeted HPV types after vaccination. So far, no evidence for HPV type replacement has been found, possibly due to limited follow-up since the widescale implementation of HPV vaccination protective effects of the vaccine. Vaccinated women carry fewer coexisting HPV types with possibly lower viral load compared to nonvaccinated women . Furthermore, HPV-68 and -73 cannot be discriminated from each other. Nevertheless, these complications do not apply for HPV-45 and -59, which require single probe lines. VCn measurements are not normalized for cellular content, e.g., by quantifying β-actin with a qPCR. However, the main goal of this study was to examine the detection failures of the SPF10 method in a reaction. Furthermore, retrospective testing of HPV-45 and -59 with the TS qPCR assays might have been affected by the decrease in HPV-45 and -59 DNA concentrations over time. Nevertheless, this analysis emphasizes the high detection sensitivity of TS qPCR assays, as significantly more HPV-45 and -59 was detected in relatively old samples. Also, we did not perform VCn quantifications for the cooccurring HPV types. Although beyond the scope of this article, VCn quantifications will be necessary to perform in-depth analyses of the effect of HPV cooccurrence on HPV-45 and -59 detection with the SPF10 method.Our study has several strengths and limitations. Our study population is at high risk of HPV infection, possibly generating a higher HPV-45 and -59 prevalence compared to general populations. Vaccination status was self-reported, but previous research indicated that recall bias is likely to be limited . HPV gen10 detection performance is limited mostly for HPV-59 and to a lesser extent for HPV-45 compared with the TS qPCR assays. Suboptimal detection by the SPF10 method is influenced by low VCn, intratype genetic variation, and HPV cooccurrence. Use of the SPF10 method to monitor HPV-45 and -59 may lead to inaccuracies, especially in study populations varying in HPV prevalence, vaccination coverage, and amount of cooccurring HPV types. For HPV-59, specifically, differences in detection performance of the SPF10 method compared to the TS qPCR assay resulted in the change of a highly negative VE estimate to a negligible vaccination effect. Consequently, HPV-45 and -59 detection with the SPF10 method alone is insufficient and should be combined with a TS qPCR assay in order to improve the accuracy. Understanding the limitations of the HPV detection method used within a specific study population is crucial for correct test usage in epidemiological studies and vaccine surveillance.In summary, we demonstrate that SPF


How to best triage human papillomavirus (HPV) positive women remains controversial in an era of HPV primary screening of cervical cancer. Here, we assessed the long-term risk stratification for triaging HPV 16 positive women by standalone HPV 16 methylation and combined with E6 oncoprotein. A total of 1742 women underwent screening with HPV DNA testing, cytology, and visual inspection with acetic acid (VIA) in 2005 and were followed for 10 years. Seventy-seven women with HPV 16 positivity determined by HPV genotyping test were examined via E6 oncoprotein detection and bisulfite pyrosequencing for quantitative methylation of L1 and LCR genes of HPV 16.p < 0.05).The 10-year cumulative incidence rate (CIR) of cervical intraepithelial neoplasia grade 3 or severe (CIN3+) for HPV 16 positive women was 25.3% (95% CI 14.7–37.3%), which significantly increased in women with high methylation at six sites  and in women with positive E6 oncoprotein. A methylation panel based on the above six sites showed a competitive risk stratification compared to cytology (HR 11.5 vs. 8.1), with a higher 10-year CIR of CIN3+ in panel positives (57.2% vs 36.8%) and comparable low risk in panel negatives (5.7% vs 4.8%).The sensitivity and specificity for accumulative CIN3+ was 85.7% (95%CI 60.1–96.0%) and 78.4% (95%CI 62.8–88.6%) for a methylation panel and 57.1% (95%CI 32.6–78.6%) and 86.5% (95%CI 72.0–94.1%) for E6 oncoprotein. The AUC values of methylation standalone and the co-testing of methylation panel and E6 oncoprotein were around 0.80, comparable to 0.68 for cytology, 0.65 for viral load, and superior to 0.52 for VIA (Our findings indicated the promising use of HPV 16 methylation alone or combined with E6 oncoprotein for triaging HPV 16 positive women based on the long-term risk stratification ability. Cervical cancer is the fourth most common female malignancy worldwide both in incidence and mortality, with an estimated 569,000 new cases and 313,365 new deaths in 2018 –6. HowevThe cytology-based triaging strategy of HPV positive women, albeit ideal and effective, is a challenge for resource-limited areas , due to More recently, Clarke et al. evaluated the clinical performance of a multi-type methylation assay composed of a total of 12 types of hrHPV genotype for detection of CIN3/AIS and indicated the apparent advantages of multi-type methylation assay as a triage method in terms of higher sensitivity and lower colposcopy referral . AdditioE6™ Cervical Test, could serve as risk predictors of  high-grade cervical lesions [Continuous E6 expression, inactivating the pro-apoptotic tumor suppressor p53, was crucial for the maintenance of the malignant phenotype and oncogenic transformation into the cervical cancer , 20. Pre lesions , 22. In  lesions . HoweverThe association of HPV DNA methylation with E6 oncoprotein has never been verified in the population, albeit with molecular biology-based evidence . MoreoveA total of 1742 women were followed up in 2005 because 255 out of 1997 women recruited in 1999 were excluded due to either lost to follow-up, death, or hysterectomy during 1999–2005. Among those, 283 women were hrHPV positive including 77 HPV 16 positive women in 2005, the analytic cohort (AC), as shown in Fig. p < 0.05). The same pattern was found even after stratification by histology-confirmed disease outcomes  and those in 2014 (prospective), and the positive relation still existed when using the 10-year cumulative E6 oncoprotein positivity rates (E6 positive in 2005 or 2014) as the outcome Fig. . The 10-p < 0.001)  with high methylation, and at least three out of these six sites with high methylation for panel B. The 10-year CIRs of CIN3+ among women with positive panel A was 11.5 (95% CI 2.6–51.6) times higher compared to those with negative counterparts. Similarly, the hazard ratio (HR) for positive panel B reached 8.9 (95%CI 3.0–26.6) . Combined with E6 oncoprotein, methylation panel A improved the sensitivity to 92.9% with reduced specificity of 73.0% but with the stable AUC value. Similar performance for methylation panel B was found. Of note, two incident cases of invasive cervical cancers detected during follow-up both showed positive panel A and B result at baseline.The triage performances of different methods for the 10-year cumulative CIN3+ were described in Table This study is among the first to assess the predictive ability of HPV 16 methylation alone or in combination with E6 oncoprotein for incident CIN3+ compared to other triage methods based on a 10-year followed-up cervical cancer screening cohort. Our findings showed the 10-year cumulative risk stratification of CIN3+ in HPV 16 positive women by the use of dichotomized HPV 16 site-specific methylation and panel-specific methylation consisiting of six CpG sites . With the combination of E6 oncoprotein, methylation panels presented a competitive clinical performance in managing HPV 16 positive women with AUC values of 0.72–0.82, compared to cytology of 0.68 and viral load of 0.65, superior to VIA of 0.52. HPV16 DNA methylation was strongly associated with viral E6 oncoprotein expression and HPV infection frequency, suggesting series of active viral events both in epigenetics and gene translation in the formation of precancer and cervical cancer.Previous studies indicated that HPV16 genome hypermethylation may be useful in predicting concurrent or even future development of cervical precancer and cancer , 12, 25.With regard to specific CpG sites of HPV 16 with good predictive utility in our study, CpG 5602 in L1 gene had won the top AUC value in the detection of CIN3/AIS in Clarke et al.’s nested case-control study reaching 0.84 ; CpG 665Expression of E6 is integral to hrHPV-induced malignant transformation . Our preFrom the perspective of triaging performances for cumulative CIN3+, methylation panes alone obtained competitive AUCs values compared with that of cytology, which were in line with the results of several other methylation studies with AUCs for the detection of CIN3+ , 35. A mThe extension of triaging potentials of HPV 16 methylation to other genotypes is of interest and of practical value. Given that HPV DNA methylation of any genotype is a general phenomenon marking the transition from HPV infection to precancers for all carcinogenic types , 36–39, Our present study has advantages in using a 10-year prospective cohort to evaluate the association between HPV methylation and E6 oncoprotein and compare their triage performances for the detection of CIN3+ with other triage options such as cytology. In addition, we used CIN3+, a histologically confirmed immediate precancer, as a primary disease endpoint, which might be more reproducible and of more clinical significance for cervical screening than CIN2+ . HoweverIn conclusion, we provided the population-based data for HPV 16 viral methylation and E6 oncoprotein as two novel and promising tests that might be useful as triage tests for HPV16-positive women. We also confirmed the virtues of standalone DNA methylation and E6 oncoprotein of HPV 16 or their combination in prediction of the future risk at cervical precancers and cancer. Extension of carcinogenic type bases on these two tests is expected to have a more attractive clinical value, but the study of large-scale screening population in real-world settings in addition to technical refinement of molecular approaches would be warranted. In an era of HPV primary screening, such translation of these objective and molecular approach tools will no doubt improve the screening algorithm and accelerate the access to cervical cancer screening.A total of 1997 non-pregnant 35–45 years old women with no history of screening or hysterectomies were enrolled in Shanxi Province of Cervical Cancer Screening I (SPOCCS I) study in 1999 . All womParticipants underwent liquid-based cytology , Hybrid Capture 2 (HC2) , visual inspection with acetic acid (VIA) in 1999, 2005, 2010, and 2014 (except for VIA in 2014) , 48. WomHC2 assay was conducted to detect the presence of a total of thirteen hrHPV genotypes (type 16/18/31/33/ 35/39/45/51/52/56/58/59/68). Samples were deemed as HPV-positive if the signal strength in relative light units (RLU) compared with standard positive control (RLU/CO) was 1.0  or higher. The semi-quantitative viral loads of women positive for HPV were then categorized into two levels: low viral load (1.0–99.9 RLU/CO) and high viral load (≥ 100.0 RLU/CO), as previously described ., and 2014 using Total Nucleic Acid Isolation kit . We measured DNA purity and concentration using a NanoDrop spectrophotometer . Then PCR-based reverse hybridization line probe assay (INNO-LiPA Extra)  with a SPF10 primers set  was performed in HC2-positive specimens during 2005–2014 to discriminate twenty-eight individual genotypes [Total DNA was isolated from 200 μl ThinPrep PreservCyt Solution  with cytological exfoliated cervical cell specimens collected in 2005, 2010enotypes .Two hundred nanograms of DNA from HPV16 positive samples in 2005 were modified using the EpiTect DNA bisulfite conversion kit . With bisulfite conversion, unmethylated cytosine (C) residues are preferentially deaminated and converted into uracil (U), which is substituted for thymine (T) residues with methylated C’s remaining unmodified during PCR amplification. PCR with 1.5–2 μl bisulfite-converted methylated template DNA in a final 25 μl reaction volume was performed using the Qiagen PyroMark PCR kit with primers sets specified for each CpG site of L1 and long control region (LCR) gene of HPV 16  is an immunochromatographic test using lateral flow format to detect the E6 oncoproteins of HPV type 16, 18, and 45 (only HPV 16 was taken into consideration in this study), as previously described . BrieflyThe study in 2005 was taken as the baseline of this analytic cohort (AC) study. Histologically confirmed CIN3+ was taken as primary study endpoint. With the methylation data from HPV 16 positive specimens in 2005, a total of 30 CpG sites of HPV 16 L1 and LCR region were included in the final analysis after exclusion of CpG sites due to the invalid data (Supplementary figure The 5-year/10-year cumulative incidence rate (CIR) of CIN3+ or CIN2+ during up to ten years of follow up among HPV 16 positive women without CIN2+ stratified by baseline site-specific methylation status (high/low methylation) was estimated by Kaplan-Meier methods and compared by log-rank test. Cox proportional hazard models were used to identify significant high-methylation CpG sites. The 5-year/10-year accumulative risk of CIN3+ or CIN2+ associated with the number of high-methylation sites at baseline was evaluated as site-specific methylation status. We calculated E6 positivity rate with respect to the baseline numbers of high-methylation sites, cross-sectional in 2005, prospective in 2014, and accumulative either in 2005 or in 2014, respectively. Similarly, we calculated HPV 16 positivity frequency with respect to the baseline numbers of high-methylation sites. As three tests for genotyping were conducted during 2005–2014 among baseline HPV 16 positive women, HPV16 infection frequency could be once, twice, or three times., and VIA.Methylation panels were further explored based on the number of high-methylation CpG sites with statistical significance in Cox proportional hazard analysis of 10-year risk at CIN3+. Two panels consisted of the same CpG sites but with different number of high-methylation sites as cutoff value. Panel A was regarded as positive if two or more out of these significant CpG sites were hypermethylated, and panel B used at least three hypermethylated CpG sites as cutoff. We combined methylation panel and E6 oncoprotein with the definition of positivity for the condition if either test was positive. We compared 10-year CIRs of CIN3+ and hazard ratio by methylation status and E6 oncoprotein status standalone or their combinations with those by other triage approaches including cytology, viral loadFinally, we evaluated the clinical performance of triaging HPV 16 positive women for all triage methods in terms of colposcopy referral rate, sensitivity, specificity, positive predictive value, negative predictive value, and area under curve (AUC) for 10-year cumulative incident CIN3+. Paired Chi-square test was used for comparison of sensitivity and specificity and Z tests for AUC values. All statistical tests were two tailed with a 0.05 significance level. All analyses were performed the using SAS 9.2 and R3.1 software.Additional file 1: Figure S1. Site-specific methylation levels of HPV 16 by disease outcomesAdditional file 2: Figure S2. Five-year cumulative incidence rate and hazard ratio of CIN3+ in HPV 16 positive women with site-specific high methylation of HPV 16Additional file 3: Figure S3. Five-year cumulative incidence rate and hazard ratio of CIN2+ in HPV 16 positive women with site-specific high methylation of HPV 16Additional file 4: Figure S4. Ten-year cumulative incidence rate and hazard ratio of CIN2+ in HPV 16 positive women with site-specific high methylation of HPV 16Additional file 5: Table S1. Cumulative incident risk of cervical lesions by the numbers of positive HPV 16 methylation sitesAdditional file 6: Table S2. Methylation status of six significant CpG site of  HPV 16  in 2005 in all accumative incident CIN3+ womenAdditional file 7: Table S3. Primers for PCR and pyrosequencing of methylation at each CpG site of HPV 16 L1 and LCR genes


With the introduction of oncogenic Human Papillomavirus (HPV) testing into cervical screening there is a renewed focus on primary prevention among high-risk groups. To date, little is known about the effectiveness of this program, and the extent to which individual-level factors, such as psychosocial health and agency, may play a role. In particular,it is unclear if knowledge of one’s oncogenic HPV status impacts on adherence behaviors amongst women with screening abnormalities. The purpose of this study was to identify if clinical, demographic or psychosocial factors predict non-adherence with recommended colposcopy follow-up.This prospective pilot study included 145 women referred to a large Toronto colposcopy clinic between December, 2013 and September, 2014. Demographic, clinical and psychosocial characteristics were collected at three points in time: (1) at initial colposcopy consultation; (2) 4–6 weeks following initial consultation, and; (3) at time of follow-up appointment .p < 0.01) and those with higher-grade lesions  were less likely to be non-adherent, whereas current smokers  were more likely to be non-adherent. While not statistically significant, variation in rates of non-adherence amongst the various HPV status groups  warrants further study.Overall, 13% (n = 145) of the women were classified as non-adherent. Older women (OR = 0.73, Findings of this study indicate that younger women, those with higher-grade lesions and current smokers were more likely to be non-adherent to recommended colposcopy follow-up. While HPV status did not reach statistical significance, the direction of this finding suggests that testing for HPV may have a positive reinforcing role on adherence to follow-up. The direction of this finding warrants further study, and potentially a practical clinical goal as HPV testing for women becomes standard of care. A number of modifiable risk factors have been implicated in the progression of oncogenic HPV infection to cervical cancer . FailureA number of socio-demographic, psychosocial and clinical factors, as well as health system issues are known to influence adherence to recommended follow-up in women with screening abnormalities –8, incluroutine cervical cytology screening with greater HPV health literacy are more likely to adhere to follow-up  and self-efficacy scores [84.5 ± 15 (range 27–100)] among the study population was high. Forty-one women 28.3%) met or exceeded the clinical cut-off for the cancer-related distress for a diagnosis of PTSD (≥ 33) (Table % met or p < 0.01), those with lower grade abnormalities  and current smokers  were more likely to be non-adherent . Although this finding did not reach statistical significance, the direction of this finding provides a basis for further study as a practical clinical goal, as HPV testing for women becomes standard of care.In this study, rates of non-adherence for women attending colposcopy with cervical screening abnormalities ranged from 13.1 to 17.7% considerable lower than the existing literature 20–50%) [0% 5, 6,,6, 34–36In our initial examination of adherence behaviors in this study, a number of key predictors were identified. First, it was observed that women who had a lower-grade lesion severity and were younger, were less likely to be adherent. In unadjusted models, women returning for treatment were more likely to adhere than women returning for routine follow-up. This relationship was no longer significant once adjusted for confounders, suggesting that it may be mediated in part by lesion severity, as higher-grade lesion is associated with adherence, and women with high-grade lesions would be asked to return for treatment. Second, smoking status also reached statistical significance in that women who were current smokers were less likely to adhere. While this finding is consistent with the literature , given tWith respect to screening-related behavior, we found higher rates of non-adherence amongst those who were untested for HPV , which suggests that testing for HPV-DNA may have a positive reinforcing role on treatment adherence. This pilot work is particularly critical in light of emerging Expert Panel recommendations for the use of primary HPV testing in the cervical screening setting in Ontario , indicatintention to return for follow-up was not consistently associated with actual follow-up adherence behaviors [In contrast to the main hypothesis and previous work , 40, perehaviors , 26. Furehaviors . Higher ehaviors , 44.Finally, relatively little is currently known about how HPV burden impacts on adherence. Consistent with previous literature, we demonstrated a moderate association between HPV burden and anxiety (r = 0.44); however, neither anxiety nor HPV burden were related to adherence. Previous work has found that anxiety is a predictor of non-adherence in women who receive inadequate cytology results  or are aA number of potential limitations warrant mention. First, differences in adherence behavior and educational attainment were found between those who completed the psychosocial questionnaires at time 1) (n = 145) versus those who did not (n = 41)  and generates new hypotheses for future study (e.g.HPV testing and HPV burden and self-efficacy). As the use of primary HPV testing is set to change the cervical screening landscape, this study provides pilot data and descriptive evidence to guide further research and clinical guidelines in regards to preventative behaviors and psychosocial factors in the colposcopy setting. Given the complex health needs of this population of women, these findings provide preliminary insight into the importance of knowing one’s HPV status, and groundwork for further psychosocial and behavior study into the outcomes associated with knowing the results of a HPV-DNA test for women.Additional file 1.Supplemental Figure 1. Prospective Study Design. Supplemental Figure 2. Overview of Study Variables and Measures. Supplemental Table 1. Missing Value Comparison.


Sudden change in a pigmented lesion accompanied by the appearance of new lesions is a cause for concern for potential melanoma development. Here we present such a case, diagnosed as eruptive blue nevi in a young woman. This case highlights the importance of dermoscopy for the diagnosis of this rare phenomenon.A 24-year-old woman with no significant medical history presented with a blue lesion on her right shin. The patient reported that the lesion had been present on her leg for many years but had started growing in the preceding 2 months. In addition, she had noticed 2 additional similar, albeit smaller, blue lesions that had appeared on her right thigh and her buttocks 2 weeks before. The lesions were asymptomatic, and the patient denied any additional complaints at the time of examination.Clinical examination revealed a well-circumscribed, 6 mm in diameter, blue-gray nodule on the distal lateral aspect of the right shin . SmallerBlue nevus is a benign neoplasm composed of aberrant melanocytes located in the dermis, which can be found in 0.5%–4% of the white population. It is usually acquired during the second decade of life, presenting as a macule, papule, nodule, or plaque on the buttocks, face, scalp, hands, or feet. Several types of blue nevi exist, including common, cellular, or combined blue nevus; atypical or malignant blue nevus; and large patch/plaque lesions .When a blue nevus develops, it usually remains stable and does not change with time. Furthermore, the presence of multiple blue nevi is a rare phenomenon and usually presents as aggregated or plaque-type lesions. So far, there have been only 13 reports of eruptive blue nevi, where the number of nevi varied from 3 to hundreds . In geneAlthough the clinical and dermoscopic features of the lesions in the patient described here were typical for a blue nevus, the sudden change and the appearance of new lesions in a short period of time were suspicious, especially when considering the importance of the blue hue as part of the dermoscopic features of malignancy. This, together with the lack of any triggering factor, warranted histopathological examination of the lesion to rule out malignancy.Here we presented a rare case of eruptive blue nevi in a patient with no known triggering factors. It highlights the need for careful inspection of these lesions to rule out melanoma or malignant blue nevus, especially since melanoma has been reported to mimic benign blue nevus.


The Cervical Cancer Prevention in El Salvador (CAPE) project is a public-sector intervention introducing lower-cost human papillomavirus (HPV) testing in all four departments of the Paracentral region that screened a total of 28,015 women. After demonstrating success of an HPV screen-and-treat (S&T) algorithm over colposcopy management in the first two phases, the third phase scaled up the S&T strategy. We present results from phase III and evaluate S&T components across the entire project.During phase III, 17,965 women age 30-59 years underwent HPV testing. HPV-positive women were asked to return and, if eligible, received gas-based cryotherapy. We compare loss to follow-up and time intervals between S&T steps across the three phases.P = .173). Although most HPV-positive women completed indicated follow-up procedures within 6 months in phases I  and II , this proportion declined to 74.9%  in phase III. Mean days between testing and delivery of results to patients increased over program phases .There were no differences in HPV positivity across phases (phase I, 11.9%; phase II, 11.4%; phase III, 12.3%; A public-sector implementation of an HPV-based S&T algorithm was successfully scaled up in El Salvador, albeit with losses in efficiency. After CAPE, the Ministry of Health changed its screening guidelines and procured additional tests to expand the program. The feasibility of such screen-and-treat (S&T) paradigms has been assessed using different combinations of HPV screening and ablative treatment modalities.9Although cytology-based cervical cancer screening continues to be widely used, molecular testing of the human papillomavirus (HPV) has emerged as a more sensitive screening tool.Key ObjectiveIs it feasible to implement an effective and sustainable cervical cancer screening program in a low- or middle-income country (LMIC)?Knowledge GeneratedCombining low-cost human papillomavirus (HPV) testing with a screen-and-treat protocol, Cervical Cancer Prevention in El Salvador (CAPE) is a public sector cervical cancer screening program that was introduced and scaled up in an LMIC. Despite ongoing barriers, such as delays in result delivery and loss to follow-up of women not eligible for screen-and-treat, the program has now been expanded nationally and included in El Salvador’s national cervical cancer control guidelines.RelevanceThe program highlighted the importance of collaboration by different stakeholders to maximize the use of existing resources in the country. The CAPE model can be used as a blueprint for other nations to address the burden of cervical cancer incidence and mortality.10 but there are other tests in development.12 There is a need for evidence of best practices in HPV testing implementation in lower-resource settings. Public-sector HPV screening projects have been successful but have used cytology as a triage test or colposcopy with biopsy to confirm diagnosis before treatment.15 The feasibility of the HPV test as a screening tool has not been confirmed in regions with limited capacity for cytology and histology.Despite the potential of HPV testing, the high cost of these assays has hampered widespread adoption of S&T. Currently, one low-cost HPV test  is prequalified by the WHO,17 HPV vaccination is not yet available in the public sector. Previous research has shown that adherence to a cytology-based regimen is poor at 44%.18 To address this issue, in 2012 the El Salvador Ministry of Health (MOH) submitted a proposal to obtain careHPV tests through the QiagenCares donation program. The nonprofit Basic Health International (BHI) served as technical advisor. The application was successful, and the MOH moved forward with the Cervical Cancer Prevention in El Salvador (CAPE) project.Here we evaluate outcomes on the basis of the first public-sector S&T intervention using lower-cost HPV testing. The project took place in El Salvador, where cervical cancer mortality is 18.5 per 100,000 women (age adjusted), and the estimated screening rate is one of the lowest in Latin America at 19%.20 HPV-positive women assigned to the S&T cohort were more likely to complete treatment within 6 months compared with those assigned to the colposcopy management group .20CAPE was designed as a three-phase demonstration project to compare the feasibility of the national colposcopy management strategy against a primary HPV-based S&T paradigm and to incrementally scale up the most successful strategy. The implementation relied on the existing resources of the national public health system, with BHI providing technical support and training to MOH personnel. In CAPE phases I and II,22 In light of this evidence, the MOH made the decision to expand the S&T protocol as the approach with most potential for a national scale-up. Here we present results from CAPE phase III, compare findings from the S&T cohorts across the three phases, and discuss challenges and lessons learned throughout the project.In the S&T algorithm, women were screened with the careHPV test. Returning HPV-positive women underwent visual assessment for treatment (VAT) and, if eligible, cryotherapy treatment on the same day. Women were considered ineligible for cryotherapy if the lesion was > 75% of the cervix, penetrated the endocervical canal, or was suspicious for cancer, or if VAT was unsatisfactory (the transformation zone was not fully visible). Ineligible women were referred to colposcopy, biopsy, and treatment pending pathology findings. Cost-effectiveness analyses used CAPE and epidemiologic data from El Salvador to model long-term health and economic outcomes of S&T and conventional colposcopy management. S&T resulted in a greater reduction of lifetime cervical cancer risk (a difference of > 15%) at a lower cost per woman.23 CAPE was intended to serve to majority of women in target areas. Phase I was carried out from October 2012 to March 2013, and phase II took place between October 2013 and July 2014 (both in the San Vicente and Cuscatlán departments).20 In phase I, 2,000 women age 30-49 years were enrolled and divided equally into the S&T and colposcopy management cohorts. In phase II, 8,050 women age 30-49 years were enrolled, with 4,087 assigned to S&T and 3,963 assigned to colposcopy management. Because CAPE was designed as a demonstration project rather than a clinical trial, these were nonrandom assignments made by the MOH using the national census to obtain the number of age-eligible women living in the catchment areas of target health units. Units were then assigned to the two cohorts to match the number of women.CAPE was implemented primarily in rural areas of the Paracentral region of El Salvador . BecauseCAPE phase III (May 2015 to April 2017) enrolled 20,000 women aged 30-59 years living in communities served by 63 health units that were screening locations across all four departments of the Paracentral region . HPV-positive women were managed only by S&T. Fourteen clinics were equipped with cryotherapy units to provide treatment of eligible HPV-positive women. These were strategically distributed to minimize transportation barriers. Five regional hospitals with colposcopy facilities were designated as referral locations  for HPV-Throughout CAPE, training was provided to a total of eight laboratory technicians, 610 community health promoters (CHPs), 360 nurses, and 223 physicians. Before the beginning of phase III, BHI hired and trained five physicians to deliver group trainings to participating MOH doctors, nurses, laboratory technicians, and CHPs. In groups of 30, CHPs attended 2-day workshops that covered the female reproductive system, HPV and cervical cancer risk, and guidance on participant recruitment. Doctors and nurses were also divided into groups of 30 to take part in 1-day training workshops that included the natural history of cervical cancer, HPV testing, treatment options, and program logistics .Fourteen general practitioners were designated by the MOH to perform VAT and gas-based cryotherapy at the designated triage centers. Training included a 1-day theoretical component and a 4-day practicum with patients. To do this, three provisional clinics were set up in a local health unit. Trainees were supervised by two gynecologists. CHPs scheduled 100 women per day age 30-49 years who were either unscreened or due for a screening. For the training, women were screened using visual inspection with acetic acid, a method included in El Salvador’s guidelines at the time. Screen-positive women were assessed for cryotherapy treatment using the VAT criteria of phases I and II. In the subsequent 3 months, an experienced gynecologist conducted monitoring visits at the triage centers to ensure adequate implementation of clinical procedures. BHI personnel monitored program logistics. In phases one and two, four laboratory technicians had been trained in analysis of the HPV tests by staff from the test manufacturer; four technicians were added in phase III.Recruitment of participants was conducted by CHPs, who live in the same communities where they work. CHPs are responsible for monitoring and recording the participation of 200-500 families in primary health care programs. CHPs used their records to identify and contact, through phone calls or home visits, age-eligible women who were under-screened, never screened, or due for screening. If a woman agreed, she was scheduled to attend her local health unit within 15 days to undergo HPV testing. Appointments were organized in groups of 10-20 women for the same day. Before testing, the health unit physician or nurse delivered a cervical cancer educational session to the women and explained follow-up procedures for women with positive results. Test samples were collected by the health unit staff and transported to four MOH laboratories for refrigerated storage at 8°C, as per the manufacturer’s instructions, and eventual analyses.All women obtained an appointment to receive their results at the health unit in 30 days. Test samples can be run in 4-6 hours but must be processed in batches of 96 for the machine to run at full capacity. To maximize efficiency, tests were only run when batches were completed. The 30-day interval provided ample time to complete sample processing. During this period, unit nurses visited HPV-positive women at home to deliver their results. HPV-positive women were then scheduled to attend a triage center to undergo VAT and, if eligible, cryotherapy the same day. HPV-positive women who could not be reached were contacted at least two more times by a CHP and asked to make an appointment at a triage center to receive their results. HPV-negative women received their results at the 30-day appointment in their local health units and were recommended to be rescreened in 5 years. HPV-positive women who attended triage center appointments but were ineligible for ablation were referred to their regional hospital to undergo colposcopy and biopsy. Delivery of biopsy results and any subsequent procedures took place within the public health system but outside the context of CAPE.24 Colposcopy and biopsy information was recorded on standard MOH forms at hospitals. In phase I, BHI created and managed the only CAPE database while collaborating with the MOH to develop an online database that eventually became part of the El Salvador National Epidemiologic Surveillance System. In phases II and III, the CAPE online database was completed and management was transferred to the MOH. BHI received copies of data collection instruments and maintained an independent database that was used for all analyses presented here.Data collection instruments for screening and VAT were created by the MOH and BHI and are available online in El Salvador’s cervical cancer control guidelines.2 and t tests for sociodemographic variables and program indicators, and Kruskal-Wallis test for median number of days between procedures. The significance level was set at .05 for all tests. All statistical analyses were conducted using Stata 14.1 .For comparisons across phases, we used Wilcoxon-type trend tests for HPV positivity rates, Pearson’s χPatients and the public were not involved in the design, conduct, reporting, or dissemination of this project.20 In phase III, 17,965 women were screened using careHPV (there are no data on women who were invited to participate but refused). The mean age for these participants was 41.4 years  and the majority lived in rural areas (76.8%) versus urban areas (23.2%). Other demographic data collected in earlier phases were not collected during phase III. Available variables and differences found in the S&T cohorts across the three CAPE phases are listed in Results from CAPE phases I and II have been published previously.P = .173).25 Age-adjusted HPV positivity rates also show little difference across the three phases  in phase I, 11.4%  in phase II, and 12.3%  in phase III; these changes were not statistically significant  if they did not complete indicated management  within 6 months. In phase III, 72.8%  of HPV-positive women completed VAT and cryotherapy within 6 months of screening (adherent), 4.6%  were classified as LTFU but returned and received VAT and cryotherapy after 6 months, 11.2%  were referred to colposcopy after VAT, and records for the remaining 11.4%  were missing.P > .001; Among women referred to colposcopy, 38.7% (96 of 248) attended the initial colposcopy appointment within 6 months, but treatment records were only available for 73 of these women. Of these, 19.0% (47 of 248) completed indicated treatment within 6 months, 6.5% (16/248) after 6 months, and 4.0% (10 of 248) received treatment but had no treatment date. We were unable to confirm if the remaining 70.5% (175 of 248) were LTFU or returned for treatment but their records were missing. Thus, in phase III, a total of 75%  of women completed their indicated procedures within 6 months of screening, 5.3%  after 6 months, and the remaining 19.7%  were either LTFU or had missing data. However, when combining missing data with women who completed treatment after 6 months, did not return for their HPV test results, and did not attend indicated procedures after referral, LTFU was 25.1% , which was significantly greater than phases I (6.7%) and II , pregnancy , and unsatisfactory VAT . Data are missing for the remaining women . Among referred cases, biopsy results and treatment records were available for 84 of 248 (34.27%) and 73 of 248 (29.4%) women, respectively. Reasons for referral to colposcopy, biopsy results, and treatment types across phases are listed in v 102.8, P < .001). The median number of days between screening and colposcopy for referred patients decreased from phase I  to phase III . The median number of days between colposcopy and treatment was also shorter in phase three than in phase II  in phase I to 41 days  in phase II and 74 days  in phase III. However, HPV-positive women in phase III received their results in fewer days than HPV-negative participants . Overall, HPV-positive women were prioritized and received their results earlier than those who tested HPV negative. The limited number of colposcopists also contributed to long wait times between appointments for referred women. Although mean number of days between procedures decreased with scale-up, high rates of missing data make it difficult to interpret this change. Another challenge was the long interval between screening and delivery of results to participants. This was partly due to the loss of two shipments of reagents for HPV test processing that occurred when cold-chain requirements were not followed correctly. Although this was eventually resolved, 562 samples were frozen at −20°C for approximately 6 months until analyses could resume. However, there were no differences in HPV positivity between these and the rest of the samples  to act as project coordinator, and supervised trainings for health care providers. The MOH provided the necessary personnel and infrastructure and, as CAPE expanded, took ownership of the program, while BHI served as technical advisor.Another key element in the success of CAPE was the commitment of the national government and other stakeholders, including academic institutions, professional medical associations in El Salvador, the National Institutes of Health, and the Pan-American Health Organization. BHI and MOH representatives organized meetings and presentations with experts and local authorities and an early field visit to Nicaragua to observe a project using HPV testing. CAPE outcomes were presented at the end of each phase to government officials, which increased support for the project.24 Although data collection and management need improvement, the MOH now has a digital surveillance system for cervical cancer. Crucially, the MOH has now procured 160,000 lower-cost HPV tests to implement a national expansion of CAPE over the next 2 years. CAPE demonstrated that linking lower-cost HPV screening to a protocol that favors follow-up treatment over colposcopy and cytology can achieve high coverage levels in a low-resource setting. Similar ongoing projects in other Central American countries will provide additional data to inform the optimal deployment of this paradigm.The lean model of CAPE, based on the efficient use of the MOH’s existing resources, helped transform it into a sustainable initiative. Findings from CAPE were instrumental in changing El Salvador cervical cancer guidelines to recommend HPV testing and S&T.


Logistic regression models were performed to identify the impact of p16ink4a positivity.Retrospective analysis of disease-free (DFS) and disease-specific survival (DSS) of patients with invasive vulvar cancer at a single tertiary care center. Histology, HPV and p16ink4a expression of over 25%. Disease-free and disease-specific survival was longer in p16ink4a positive patients . In multivariate analysis, p16ink4a positivity was an independent parameter for DFS ), but not for DSS , in contrast to age and tumor stage.135 patients were included in the analysis. 32 (23.7%) showed a p16ink4a positive invasive vulvar cancer.Age and tumor stage negatively affect survival. However, disease-free survival is significantly longer in patients with p16 Transient HPV infections can be excluded and only tumors with HPV as the primary cause of the oncologic process remain [ink4a status may strongly influence the patient’s outcome, which is already established in oropharyngeal cancer [ink4a positivity and HPV status on the clinical outcome in IVC.Over the past two decades, the incidence rates of invasive vulvar cancer, which is responsible for about 5% of all gynecological cancers, increased continuously. Especially in women younger than 50 years of age, the incidence rates almost tripled over the past decades –3. Especnowadays . Similarnowadays . In a menowadays . In a rer in IVC . P16ink4s remain –12. We pl cancer . Age at ink4a was evaluated. A sensitive assay, using SPF10 broad spectrum primers PCR and DEIA (DNA enzyme immunoassay) was used for HPV–DNA detection. Positive samples were subsequently analyzed by SPF10 PCR/DEIA/LIPA25 , which is a reverse hybridization technique that detects 25 high-risk (HR) and low-risk (LR) HPV types . Furthermore, IVC cases were tested for cyclin-dependent kinase-4 inhibitor (p16ink4a), which is reported to be overexpressed in at least 90% of HPV-related VIN and IVC cases. Therefore, CINtec® PLUS Cytology Kit by Roche  was used to detect p16ink4a in the invasive vulvar cancer cases. A case was considered to be positive if more than 25% of invasive cancer cells showed a diffuse overexpression [Austria was one of 39 countries participating in the international collaborative study . Patientpression . The cliAll patients included were managed by gynecologic oncologists. Predefined uniform criteria for surgical procedure terminology, pathologic variables, and sites of recurrence were used. Based on the FIGO 2009 classification system, disease staging was performed . DependiFor the first 2 years, clinical examination was performed every 3 months, followed by 6 months intervals until the completed fifth year, then by annually consultations. At every follow up check, the patient received a vulvoscopy, vagino-rectal palpation and groin inspection. Furthermore, serum squamous cell carcinoma antigen (SCC) was evaluated. Biopsy and or computed tomography were performed when recurrent disease was suspected. Based on post-mortem results, patient’s death and causes of death were documented.ink4a could represent a transient infection with no role in carcinogenesis. Group 1 represented patients with HPV-positive and HPV-negative IVC with a p16ink4a expression in less than 25% (< 25% = negative) and group 2 consisted of HPV-positive and HPV-negative IVC with a concomitant overexpression of p16ink4a (≥ 25%). Values are shown as mean values with standard deviation (SD). To compare HPV-positivity with clinic-pathological parameters, t tests were performed. P values of < 0.05 were considered statistically significant. With respect to overall and disease-free survival, differences between groups were tested using the log-rank test and are presented as Kaplan–Meier survival curves. Multivariable analysis was performed using a Cox regression model including patient’s age , p16ink4a expression and tumor stage (FIGO III and IV vs. FIGO I and FIGO II) as independent variables. Furthermore, FIGO I and II adapted analysis was performed via t test and is presented as Kaplan–Meier survival curves as well. Statistical analyses were performed using SPSS 25.0 for MAC . Collected data: age at diagnosis, histopathological grading, initial therapy, HPV-subtype analysis, p16ink4a expression, FIGO classification, disease-free survival, disease-specific survival, nicotine abuse, coexistent lichen sclerosus or VIN, previous malignant disease, comorbidities.Patient’s data was divided in two different groups (group1/group2), assuming that cases in which HPV–DNA is detected without overexpression of p16ink4a status to exclude transient HPV infections (Table p < 0.001). In 30 (22.2%) of the evaluated histological specimen, HPV was detected. HPV 16 was the primarily diagnosed HPV-subtype and detected in 27 cases (77%). HPV 31 was detected in two cases (6.7%) and HPV 33 (3.3%) in one. In 20 (66.7%) of those HPV-positive cases, an overexpression (≥ 25%) of the tumor-suppressor-protein p16ink4a was detected. Contrary, only 12 (11.4%) of HPV-negative IVC presented an overexpression of p16ink4a . Further subgroup analysis, only including patients at FIGO stage I and II was performed.A coexistent lichen sclerosus was detected in 6 (4.4%) cases, all of them were HPV-negative. FIGO stages were well balanced between both groups ) but not disease-specific survival (DSS) ). Results on multivariate analysis are presented in Table 76 56.3%) patients suffered at least one relapse. Mean disease-free survival (DFS) in group 1 was 10 months (IQR: 5–36), whereas patients from group 2 had a relapse after 23 months (IQR: 4–65.5), % patientn = 80, 77.7%), still were significantly older (mean: 70.5 years (IQR: 57.4–83.6)), than patients of group 2 (mean: 63.1 years (IQR:46.0–79.5)), p = 0.028. Disease-free survival of group 2 still was significantly longer than in group 1; p = 0.011 ), but not concerning DSS , Table To have a closer look at patients with a favorable prognosis, FIGO stage I and II were analyzed separately Table . Patientp = 0.010 and p = 0.048, respectively).Furthermore, for sensitivity analysis, patients aged 80 years, or more were excluded in the subgroup analysis of FIGO stages I and II. Excluding the old ages as a possible bias, the difference between both groups for DFS and DSS remained statistically significant  [p = 0.501). Although patients with p16ink4a positive IVC were on average seven years younger than p16ink4a negative controls and the first recurrence occurred after an average of 2 years instead of one year, in multivariate analysis DSS depends on younger age and lower FIGO stage but not on p16ink4a status. Therefore, further research has to be done on data of relapse in IVC. Since there is no appropriate screening for VIN and IVC, elimination of HPV-related tumors is possible in countries with a good coverage of HPV vaccination [ink4a positive invasive vulvar cancer. There is a caveat which needs further investigation: In case of a relapse, the mortality of the initially prognostic favorable p16ink4a positive invasive vulvar cancer appears to be worse.In our dataset of 135 IVC, survival indicators, DFS and DSS, were significantly longer in p16with IVC –17. To bcination –22. In c