E6/E7 carcinogenic process, and are characterized by effects on the host genome, which result in the inactivation of tumor suppressors  and the activation of oncogenes , thus activating oncogenic signaling pathways . This study could enhance our understanding of HPV integration and pave the way for subsequent HPV studies in PSCC.Penile squamous cell carcinoma (PSCC) has been regarded as an HPV-related cancer for a long time. However, the integration pattern and carcinogenic pathways of HPV in PSCC remain unclear. The results of this study provide insights into the HPV-related carcinogenic mechanism in PSCC, which may be less prone to involvement in the traditional E2 oncogene was not prone to involvement in integration. Eleven of the 35 (31.4%) HPV-integrated PSCCs harbored intact HPV E2; these tumors had lower HPV E6 and E7 expression and higher expression of p53 and pRb proteins than those with disrupted E2 did (p < 0.001 and p = 0.024). Integration breakpoints are preferentially distributed in or near host genes, including previously reported hotspots  and newly identified hotspots , which are mainly involved in oncogenic signaling pathways . Regarding the phosphorylation levels of JNK, p38 was higher in HPV-positive tumors with MAPK-associated integration than those in HPV-positive tumors with other integration and those in HPV-negative tumors. In vitro, KLF5 knockdown inhibited proliferation and invasion of PSCC cells, while silencing CADM2 promoted migration and invasion. In conclusion, this study enhances our understanding of HPV-induced carcinogenesis in PSCC, which may not only rely on the E6/E7 oncogenes, but mat also affect the expression of critical genes and thus activate oncogenic pathways.Human papillomavirus (HPV) is a significant etiologic driver of penile squamous cell carcinoma (PSCC). The integration pattern of HPV and its carcinogenic mechanism in PSCC remain largely unclear. We retrospectively reviewed 108 PSCC cases who received surgery between 2008 and 2017. Using high-throughput viral integration detection, we identified 35 HPV-integrated PSCCs. Unlike cervical cancer, the HPV  E6/E7-induced carcinogenesis triggered by disruption of the HPV E2 oncogene: The disruption of E2 results in a loss of negative feedback control of the expression of the E6 and E7 viral oncoproteins that are involved in the inactivation of p53 and pRb proteins [E6/E7-induced carcinogenesis, in some cases, HPV integration also induces carcinogenesis through directly activating oncogenes or inactivating tumor suppressors in the human genome [Penile squamous cell carcinoma (PSCC) is a rare malignancy worldwide, but its incidence is much higher in developing areas such as Africa, South America, and Southeast Asia. Human papillomavirus (HPV) infection is a significant etiologic driver of PSCC . In mostproteins ,5. Besidn genome ,9. HowevAPOBEC mutation [The integration pattern and characteristics of HPV in the PSCC genome are also unclear. Several studies have indicated that HPV-positive PSCC harbors a different genomic profile ,11,12,13mutation ; HPV-posE6 and E7 expression); 35 of the 41 PSCCs were verified to harbor HPV integration using HIVID analysis and further analysis of HPV integration was performed. Moreover, 67 HPV-negative cases were used to compare the clinicopathological characteristics and the expression of key genes with 35 HPV-integrated cases. The study design is shown in In this study, we used tumor specimens from 108 PSCC patients who underwent surgical intervention at the Sun Yat-sen University Cancer Center between 2008 and 2017. The inclusion criteria were as follows: (I) Histologically confirmed PSCC; (II) no anti-cancer treatments received before surgery; (III) no history or concurrence of other malignant tumors; (IV) complete clinicopathological data. Histologic subtypes of PSCC were reviewed by two pathologists according to the fourth edition of the World Health Organization’s classification . Tumor s+ PSCC samples were subjected to library preparation as previously reported  covering the full-length HPV genomes of 18 subtypes , namely,Adaptor, low-quality, and short sequence (less than 40 bp) data were first removed to obtain clean data, which were then mapped to 314 genomes of 32 HPV subtypes  using BuNext, the initial integration sites were obtained by SVdetect analysis , high-coq-value was <0.05.Subsequently, the genomic regions/elements and genes involved in HPV integration were analyzed. High-confidence results were annotated using ANNOVAR , referriAfter routine deparaffinization, rehydration, antigen retrieval, and endogenous peroxidase inactivation, FFPE specimen slides (4.0 μm-thick) were subjected to immunohistochemistry (IHC) staining using primary antibodies against phospho-(p-)JNK , Danvers, MA, USA), p-ERK , p-p38 , p-STAT3 , β-catenin , CEP19 , NRROS , CADM2  or KLF5 , p53 , pRb , and the Dako REAL™ EnVision™ Detection System  according to the manufacturer’s instructions. To score the IHC staining, 3–5 (depending on the size of the tumor area) independent microscopic fields (100×) of each tumor specimen were examined in terms of the integrated optical density (IOD) via Image Pro Plus 6.0  . The sta450nm) [Two PSCC cell lines previously established by our group, Penl2 (RRID: CVCL_VF95) and 149RCa (RRID: CVCL_VF96) , were cu450nm) .The migration ability of PSCC cells was assessed using a wound healing assay ,25. Brie5 149RCa cells or 1 × 105 Penl2 cells were seeded into Boyden chambers (8 µm pore) with Matrigel coating . After 24 or 36 h of incubation, the cells on the lower surface of the filter were fixed, stained, and counted.The invasion ability of PSCC cells was evaluated using a Transwell assay as described previously ,26. Brie® Premix Ex TaqTM (TaKaRa) with specific primers  after being normalized to the control .Total RNA was extracted from PSCC cells or RNA later-preserved tissues using Trizol reagent , and then reverse-transcribed into cDNA using HiScript II . qPCR assay was performed using SYBR primers . The relχ2 test, Fisher’s exact test, and Pearson’s correlation analysis were employed for correlation analysis, the Mann–Whitney and Kruskal–Wallis tests were used for rank-sum testing, and the Kaplan–Meier method with the log-rank test was used for survival analysis. Disease-specific survival was defined as the time from surgery to either death caused by disease or last follow-up. The difference between two or more groups was analyzed by Student’s t-test or analysis of variance (ANOVA). Statistical significance was set at p < 0.05.All statistical analyses were performed using SPSS 22.0 . The We performed an HIVID assay in 41 HPV-positive PSCC previously identified by qPCR . HIVID dp < 0.001)  were observed HPV integration, while the rest three tumors only harbored episomal HPV DNA. In total, 2252 HPV integration breakpoints were identified in 35 PSCC specimens  . Among t< 0.001) .p = 6.6 × 10−62), common fragile sites (p = 8.5 × 10−25), SINE-Alu (p = 3.9 × 10−43), and satellites (p = 6.9 × 10−9), but were relatively scarce in areas without those elements (p = 5.1 × 10−217).We deciphered the pattern of HPV integration in the human genome and found that although the integration event distributed in all chromosomes in the human genome a, they wp = 3.1 × 10−10), but not in intergenic regions (p = 6.7 × 10−10) (p = 3.2 × 10−5) and 3′ untranslated regions (p = 7.8 × 10−3), but not in 5′ untranslated regions (p = 1.7 × 10−4); in intergenic regions, breakpoints were clustered in regions that were <100 kb from genes , but were scarcer in regions >700 kb from genes (p = 2.2 × 10−16)  d. In int× 10−16) e.E6/E7 carcinogenic process often triggered by HPV E2 disruption. The integration of E2 causes the disruption of E2, thus increasing the level of HPV E6/E7 protein and decreasing the level of p53 and pRb [E2 was significantly less prone to be involved in HPV integration than expected (p = 1.25 × 10−5)  of them harbored <3 HPV E2 integration events. Although the number of cases is relatively small, patients with intact E2 had higher disease-specific survival rates than those with disrupted E2 (p = 0.094)   (E1 (p = 1.36 × 10−32) was more prone to involve in integration, while several other regions, such as E6 (p = 3.42 × 10−4), E7 (p = 7.06 × 10−6), and the LCR region (p = 7.39 × 10−4) were less prone to involvement in integration  b, which = 0.094) c. In tum< 0.001) d. Moreovctively) e. Other egration b. The whng of L2 .3) than in those with grade G1–2 .As HPV integration could also induce carcinogenesis by activating oncogenes or inactivating tumor suppressors in the human genome, to investigate which gene and signaling pathway play a key role in HPV-induced PSCC, we identified 2277 genes that were likely affected by HPV integration . These gp = 6.44 × 10−9; p = 0.042, 0.030, and 0.323, respectively) and HPV-negative tumors  (p = 0.018) and HPV-negative tumors (p < 0.001)   b–d. Next< 0.001) e. Howevectively) . Taken tKLF5, LRP1B, and KLF12) have previously been described in cervical cancer [CADM2, CEP19, CSMD1, and NRROS) are reported for the first time in the present study. Moreover, some hotspot genes were located in closer proximity, such as KLF5–LINC00392–KLF12, CADM2–LINC02070, TACR3–CXXC4, NRROS–CEP19, and LINC02170–CMC2, which may have been affected by shared integrations  to examine their expression in PSCC by IHC staining    a, KLF5 lctively) a. Howevectively) .CADM2, and another occurred in the downstream region. We report CADM2 as an HPV-integration hotspot gene in this study. We preliminarily investigated the in vitro function of CADM2, and found that knock down of CADM2  , p-JNK , and p-p38   than that in HPV-negative tumors ( <0.001) a, we sup< 0.001) d. These In the present study, we determined the pattern of HPV integration in PSCC and investigated the impact of HPV integration on penile carcinogenesis. Several findings may contribute a lot to our current knowledge of HPV-induced PSCCs.E2 were observed than expected in PSCC, whereas no such tendency exists in cervical cancer [E2, indicating that these tumors are not triggered by HPV E2 gene disruption. Even in 24 tumors with disrupted E2, half (12/24) of them only harbored less than three E2 integration sites, suggesting that E2 disruption may not play a critical role in those tumors. The similar pattern was also observed in other HPV-related squamous cell carcinomas, such as oropharynx squamous cell carcinoma [E2 harbor lower expression of HPV E6 and E7, and higher expression of p53 and pRb than tumors integrating HPV E2 DNA. These results remind us that many HPV-induced PSCCs are highly possibly generated by directly affecting critical host genes. Meanwhile, patients with intact E2 had marginally better survival than those with disrupted E2 (p = 0.094). A similar result also exists in oropharynx squamous cell carcinoma [First, we identified a carcinogenic mechanism in PSCC that is uncommon in cervical cancer. Significantly fewer integration breakpoints in HPV l cancer . Eleven arcinoma ,29,30. MKLF5 and KLF12 (13q22.1), MYC (8q24.21), and TP63 and TPRG1 (3q28) [+ tumors, including in chromosomal regions 2q33.2-q33.3 and 2q35. Macedo et al. [Second, we first depicted the integration pattern of HPV in PSCC in a direct way. Previous studies in other HPV-related cancers have revealed that HPV integration events are more likely to occur inside or near gene regions and in within genome instability elements. Loci at 13q22.1, 8q24.21, 3q28, 17q23.1, and 17q23.2 are the top five hotspot cytoband regions. Specifically, integration-affected hotspot genes include 1 (3q28)  found thCADM2, which has never been reported to be hotspot gene in other HPV-related tumors. All of the integrations in the CADM2 gene were caused by the HPV16 subtype in our study. CADM2 has been reported to be a tumor suppressor in hepatocellular carcinoma and in glioma [Third, we identified many hotspot genes and pathways that were associated with HPV-induced carcinogenesis in PSCC. One of the hotspot genes was n glioma ,32, and n glioma . In thisn glioma ,35,36,37n glioma . Thus, Hn glioma .CADM2 gene and the activation of the oncogene KLF5, as well as the MAPK and JAK/STAT signaling pathways, other effects induced by HPV integration may also exist. As several new HPV integration clustered regions  were observed in PSCC, we plan to investigate hotspot genes in these regions to further clarify the additional carcinogenic mechanisms involved in PSCC.The major limitation of this study is the relatively small sample size (35 integrated tumors), which limits making a definite conclusion. As PSCC is a rare tumor, a large-scale study would be difficult to carry out. In addition to the inactivation of the tumor-suppressor E6/E7 carcinogenic process, but also characterized by affecting the host genome, which resulted in the inactivation of tumor suppressors  and the activation of oncogenes  and oncogenic signaling pathways . Our findings enhance our understanding of HPV integration and pave the way for subsequent HPV studies in PSCC. Further large-scale multicenter studies and mechanical studies of KLF5 and CADM2 are warranted.We described a whole genome-wide study of HPV integration in PSCC, and identified an HPV-related carcinogenic mechanism not only through the traditional


The aim of this study was to evaluate the diagnostic accuracy and efficiency of p16/ki-67 dual stain in the identification of CIN2+ lesions, in Greek women with ASCUS or LSIL cytology.A total of 200 women, 20 to 60 years old, were enrolled in the study. All samples were cytologically evaluated and performed for p16/ki-67 and high-risk HPV (HR-HPV) test. All patients were referred to colposcopy for biopsy and histological evaluation. Three cervical cancer (CC) screening strategies were designed and the total direct medical costs of the procedures during our clinical trial were evaluated, from a healthcare perspective.HPV 16 as expected was the most common HR-HPV type followed by HPV 31 and HPV 51. The risk for CIN2+ was significantly higher in HPV 16/18 positive cases. p16/ki-67 demonstrated a high sensitivity for CIN2+ identification in both ASCUS and LSIL groups . HR-HPV test with sensitivity 52.3% and 65.5%, as well as colposcopy with sensitivity 14.3% and 36% respectively in ASCUS and LSIL group, showed inferior results compared to p16/ki-67. The specificity of p16/ki-67 for ASCUS and LSIL was 97.2% and 95.2% respectively, inferior only to colposcopy: 100% and 100%, lacking however statistical significance. HR-HPV test instead, presented the lowest specificity: 76.4% and 71.4% respectively in comparison to the other two methods. From a healthcare perspective, the costs and benefits of the tests implementation for the annual screening and triaging, in three CC screening strategies, were also calculated and discussed.The results of the study indicate that p16/ki-67 is a safe and rapid assay that could be used to detect CIN2+ among women with mild cervical lesions, presenting both high sensitivity and specificity and could minimize the psychological and economic burden of HPV screening. Cervical cancer (CC) is the fourth most common malignancy in women globally. It has been established as a great disease model for prolepsis, as it has been significantly reduced during the past decades due to the population-based screening programs and vaccination . The maiFurthermore, besides the screening approach, triage tests are needed to identify among HPV positive women, those that are at a higher risk and should be referred to colposcopy and further diagnostic evaluation. HPV testing is been proven to be a useful triage tool for minor cytological abnormalities, such as atypical squamous cells of undetermined significance (ASCUS) when we screen women over 30 years old , 10.INK4a protein [On the other hand, HPV testing as primary screening presents high sensitivity but low specificity, resulting in an increased number of HPV positive patients who should be referred to colposcopy. That type of management increases the burden in colposcopy clinics and the risk of overtreatment. Thus, an optimal triage strategy which combines both high sensitivity and specificity, is needed to distinct the majority of women at low risk of developing cervical cancer and identify those at a higher risk, who should be referred to colposcopy for further management or treatment , 12. The protein , 13–16.The aim of this study was to evaluate the diagnostic ability of p16/ki-67 dual stain in a homogenous Greek population, in order to identify the optimal management strategy of HR-HPV positive women with ASCUS or low-grade squamous intraepithelial lesion (LSIL) cytology as part of an organized cervical cancer screening setting.st Department of Obstetrics and Gynecology, "Alexandra" Hospital, National and Kapodistrian University of Athens, in Greece. A total of 200 Greek women aged from 20 to 60 years old with ASCUS/ LSIL cytology, HR-HPV positive and with histological findings CIN≥ 1 , were selected as a subpopulation of a larger study. These women attended the clinic from March 2018 through April 2019 for their routine cytology-based cervical cancer screening and after being thoroughly informed participated in the study. Liquid-based cytology (LBC) samples were collected and after HPV testing and p16/ki-67 dual stain were performed, women were referred to colposcopy for evaluation and cervical biopsies. Three cases were excluded as invalid cytology and one as invalid p16/ki-67 due to low cell count.The present study was conducted in the 1The exclusion criteria of the current study were HR-HPV negativity or biopsy result of CIN0, treatment for CIN, prior hysterectomy and pregnancy. Written informed consent, medical history and demographic information were obtained from all the 196 enrolled subjects . Medical® HPV test and CLART® HPV3 genotyping assay.Liquid-based cytology samples were stored in PreservCyt solution  vials according to the manufacturer’s instructions. A first slide was prepared using Thinprep T2000 processor system  for cytological evaluation in accordance to the 2014 Bethesda system . For thi® HPV test  which provides individual results for HPV 16 and HPV 18 genotypes and pooled results for the rest 12 HR-HPV types: 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68 in a single analysis. Briefly, target DNA primers amplify a sequence of approximately 200 nucleotides within the L1 region of the HPV genome of the sample with the use of polymerase chain reaction (PCR) to detect the 14 high-risk HPV types [® 4800 fully automated System.Qualitative in vitro HPV genotyping analysis was performed using Cobas® HPV3 kit , an in vitro diagnostic kit that detects 49 HPV genotypes including High Risk , Prob. High Risk , Low Risk  and genotypes of undetermined risk (for research only): 34, 64, 67, 69, 74, 86, 87, 97, 101, 102, 103, 106, 150, 151. Detection is based on CLART® technology which includes PCR amplification of a 450bp fragment within the L1 viral region using type-specific probes. The visualization of the amplified products is performed in a low-density microarray platform and interpreted in an automatic Genomica’s reader  [HPV genotyping of the 12 HR-HPV types, that were not evaluated separately with Cobas technology, was performed with the CLART  reader) . For the® PLUS Cytology Kit is an immunocytochemistry assay for the qualitative detection of p16INK4a and ki-67 proteins in cervical cytology preparations. The proteins are detected using a ready-to-use cocktail of primary monoclonal antibodies: human p16INK4a protein (clone E6H4™) and primary recombinant rabbit antibody directed against human ki-67 protein (clone 274–11 AC3) [® PLUS kit  on an automated slide-stainer platform: Ventana BenchMark XT Slide Stainer.The CINtec–11 AC3) . The sliINK4a (brown cytoplasmic stain) and ki-67 (red nuclear stain) regardless of their morphologic appearance. Negativity was established when no synchronous p16/ki-67 dual staining in the same cell was observed, in accordance to the manufacturer’s criteria and instructions [INK4a and/or ki-67staining was visible or if there were less than 5,000 squamous cells in the slide [All slides were evaluated by two experienced cytopathologists unaware of histology and HPV testing results. A case was interpreted as positive if one or more cervical epithelial cells were dual stained for both p16ructions . In Fig he slide .INK4a overexpression and a dark red nuclear stain for ki-67 expression within the same cell (red arrow); unaffected cervical epithelial cells without p16/ki-67 staining (black arrow).p16/ki-67 dual-stained examples (×40 magnification) in cases of (A) atypical squamous cells of undetermined significance (ASCUS), (B) low-grade squamous intraepithelial lesion (LSIL) cytology. Positive p16/ki-67 dual-stained cells with a brown cytoplasmic stain for p16The population enrolled in our study, underwent colposcopic evaluation of the cervix and the colposcopic findings were reported according to the ASCCP Terminology for Colposcopic Practice . If no vWe designed and evaluated three different CC screening strategies based on the current algorithms used: Cytology (LBC), HPV test with 16/18 genotyping, Co-testing (Cytology & HPV test with 16/18 genotyping) with the addition of p16/ki-67 dual stain as a triage strategy.The costs of the lab-tests for screening  and the diagnostic methods  presented in In each CC screening strategy we included the direct medical costs of the procedures per screened woman from a healthcare perspective. Specifically, direct medical costs consist of screening tests used  and diagnostic methods  plus office visit . The costs for the annual follow-up screening tests as well as any treatment needed for CIN were not included.Women with Normal cytology return to Routine Screening.a) Triage with HPV 16/18 genotyping.Women with HR-HPV test (-) return to Routine Screening, those with HPV16/18(+) are referred to colposcopy, whereas women with HR-HPV test (+) & HPV16/18(-) repeat cytology in one year.b) Triage with p16/ki-67 dual stain.Women with p16/ki-67(+) results are referred to colposcopy while those with p16/ki-67(-) repeat cytology in one year.Women with Cytology ≥ ASCUS:Women with HR-HPV test (-) return to Routine Screening.a) Triage with cytology.Women with HR-HPV test (+) & HPV16/18(-) with cytology < ASCUS, repeat cytology in one year.Women with an HR-HPV test (+) and cytology ≥ ASCUS are referred to colposcopy.b) Triage with p16/ki-67.Women with p16/ki-67(+) are referred to colposcopy, while those with p16/ki-67(-) are re-tested with HPV 16/18 genotyping in one year.Women with HR-HPV test (+):Women with Cytology <ASCUS and HR-HPV test (-) return to Routine Screening.Women with Cytology ≥ ASCUS and HR-HPV test (+) are triaged with p16/ki-67 dual stain. When p16/ki-67(+) the patients are referred to colposcopy and those with p16/ki-67(-) repeat co-testing in one year.2) and Odds Ratio (O.R.) with 95% Confidence Interval (CI) were calculated. Diagnostic accuracies of HR-HPV test, Colposcopy and p16/ki-67 method for the detection of CIN2+ were evaluated using Sensitivity, Specificity, Positive Predictive Value (PPV) and Negative Predictive Value (NPV). Comparison between them was performed using the Paired Samples t-test. A Receiver Operating Curve (ROC) analysis was conducted to compare the prognostic abilities of HR-HPV test and p16/ki-67 for the detection of CIN2+ calculating the respective areas under the curve (AUC) with 95% CI, using the maximum likelihood estimation method, which has the advantage of being free of assumption about the Gaussian distribution of underlying variables. All tests were two-sided and statistical significance was set at p < 0.05. All statistical analyses were conducted using SPSS vr 21.00 .Data were expressed as mean ± SD for continuous variables and as percentages for categorical data. The Kolmogorov—Smirnov test was utilized for normality analysis of the parameters. To evaluate the risk of CIN2+ lesions, according to HPV genotype, the Chi-square test  were analyzed. Among those, 93 cases had ASCUS cytology, 103 had LSIL and 4 cases were excluded as invalid due to low cellularity. The mean age of all 196 finally enrolled women was 37.5 years old, with participants < 30 years old (27%) and ≥ 30 years old (73%). Only a small percent (12.8%) of the women were HPV vaccinated, the majority (90.8%) had a pap test performed during the previous years and 26.0% had other vaginal infections. Most women (91.8%) had over 2 sexual partners during their lifetime and approximately half the population was nulliparous. Smoking habits of our population were equally distributed .All patients were selected to be HR-HPV positive and the HR-HPV test results were classified as HPV 16/18 (+) or HPV 16/18 (-) when no HPV 16 or HPV 18 was detected. In our population, 80/196 (40.8%) cases were HPV 16/18 positive and 116/196 (59.2%) had some other of the 12 HR-HPV types. In cases of multiple infections, patients were classified according to the highest risk type for its constituents. For example, if a case distributed both HPV 16 (+) and HPV 31 (+), it was then classified into the HPV 16/18 (+) group. The percentages of HR-HPV genotypes in our study are presented in HPV 16 was the most frequent high-risk type in 66 out of 196 cases (33.7%) followed by HPV 31 (17.3%) and HPV 51 (13.8%). The HPV 56, HPV 58 and HPV 66 distributed similar percentages (11.2%–11.7%), followed by HPV 18 (8.7%). The HPV 52 and HPV 59 presented a percentage of 7.7% and the rest HR-HPV types: 33, 35, 39, 45, 68 presented lower frequencies.In order to evaluate the risk of CIN2+ according to HPV genotype, patients were sorted into fourteen groups depending on HR-HPV genotype’s positivity as shown in Among the 93 ASCUS samples presented in The performance of the combination of the two assays in identifying CIN2+ cases in ASCUS group was 2/55 (3.6%) when HPV16/18(-)/ p16/ki-67(-), 8/10 (80%) when HPV 16/18(-)/p16/ki-67(+), 0/17 (0%) when HPV 16/18(+)/p16/ki-67(-) and 11/11 (100%) when HPV 16/18(+)/p16/ki-67(+) as shown in Regarding the LSIL cytology group as shown in The diagnostic accuracy of each test for the detection of CIN2+ is presented in The p16/ki-67 Positive Predictive value (PPV) and Negative Predictive Value (NPV), in the ASCUS group were: 90.4% (95% CI 68–98) and 97.2% (95% CI 89–99) as indicated in The ROC curve analysis of the two methods, HR-HPV test and p16/ki-67, is shown in In order to evaluate the costs of the three different proposed CC screening strategies Figs –4 we estst Cytology screening strategy (nd HPV test screening strategy approach (rd Co-testing (Cytology & HPV test with 16/18 genotyping) screening strategy and triaging with p16/ki-67 dual stain  inhibitor that plays an important role in cell cycle regulation. The over expression of E7 oncoprotein, in persistent HR-HPV infections, leads to p16INK4a upregulation which might serve as a biomarker for the detection of cervical precancerous lesions [INK4a and ki-67 in the same cell, does not physically exist in normal cells and this co-expression is considered a valuable marker for cell-cycle deregulation and cell transformation due to HPV infection [The p16 lesions , 25. ki- lesions . The co-nfection . Therefonfection , 29.In our study, besides p16/ki-67, we classified HPV positive patients into two categories, as HPV 16/18 (+) and HPV 16/18 (-) according to algorithms for primary HPV screening . In concWe also identified the distribution of the 14 HR- HPV types in our Greek population. Briefly, our results are fully consistent with previous studies in Greece, where HPV 16 is the most prevalent type followed by HPV 31 and HPV 51 –36. ThisIn order to evaluate the accuracy of the methods, we compared the p16/ki-67 dual stain with the HPV testing and colposcopy, which are currently proposed by the screening guidelines. In our study, the results showed that p16/ki-67 method had both high sensitivity and specificity as triage test, both in the two cytology groups (ASCUS/LSIL), improving the diagnosis of CIN2+ lesions.Among women with ASCUS and LSIL, sensitivities of p16/ki-67 dual stain were 90.4% and 95.0% respectively, higher than that of the HR-HPV test (52.3% and 65.5%). Previous studies have shown that p16/ki-67 can be a reliable tool for risk stratification of HPV positive women with mild cervical lesions, reporting high sensitivity values similar to that of HPV test , 37–40.In both ASCUS and LSIL cases, p16/ki-67 showed higher specificity for CIN2+ diagnosis, 97.2% and 95.2% respectively, while no statistical significance was identified when compared to the HPV test (76.4% and 71.4%). Previous studies have shown that p16/ki-67 had higher specificity compared to HPV testing , 37–40. In our National Health Care System, colposcopy is widely used to detect cervical lesions and biopsy under colposcopy guidance, confirms or excludes CIN2+ lesions. We retrospectively collected colposcopy reports of the population we studied in our Department of Obstetrics & Gynecology which is a referral Department for gynecological cancer. We compared these reports to the histological findings, in order to evaluate the prognostic accuracy of colposcopy.We observed that in ASCUS and LSIL cases, 38.1% and 65.7% respectively of the histologically confirmed CIN2+ were underestimated by colposcopy and defined as “Negative” or “LSIL”. Whereas, the p16/ki-67 depicted more CIN2+ cases among the two groups  and showed clearly higher sensitivity  than Colposcopy  (p> 0.005). On the other hand, there was no statistical significance in specificity between p16/ki-67 and colposcopy impression (95–100%).There are many factors that could explain this observation such as, training and experience of the clinicians involved, inadequate colposcopy due to lack of objective criteria to classify the lesions macroscopically and co-existing pathologies which tamper with the appearance of the normal mucosa.Furthermore, we presented the ROC curve to show the diagnostic performance of p16/ki-67 and HPV test. The AUC of p16/ki-67 was greater in both cytology groups (0.938/0.952) compared to the HPV test (0.644/0.685). These results are comparable to those of a recent study were AUC of p16/ki-67 dual stain was (0.778), also significantly higher than that of HPV test (0.503) .Additionally, in both cytology groups, we evaluated the combination of the two methods, HR-HPV test with 16/18 genotyping and p16/ki-67, in order to identify how many cases of CIN2+ will be missed. In the LSIL cases, when HPV16/18 and p16/ki-67 were both positive or negative, no women were misdiagnosed. In case of conflicting results between HPV16/18 and p16/ki-67, taking into consideration the p16/ki-67 positivity alone, we were able to diagnose 91.3% of women with CIN2+ that would otherwise escape diagnosis and only 8.7% were missed. On the other hand, we observed that if only HPV 16/18 was positive we could detect only 20% of CIN2+ and the rest were missed.Looking into the ASCUS cases, when both HPV16/18 and p16/ki-67 were negative, 3.6% of women with CIN2+ were missed, whereas when HPV 16/18 (+)/p16/ki-67 (+) all CIN2+ were diagnosed. In case of conflicting results between the two tests, when only p16/ki-67 was positive, 80% of women with CIN2+ were accurately diagnosed. Finally, in HPV 16/18 (+)/p16/ki-67(-) cases, no CIN2+ were missed.Our study showed that p16/ki-67 presented both high sensitivity and specificity values, with only a few CIN2+ being missed. Thus, it appears to be an excellent triage tool for accurate stratification of women with mild cervical lesions. Our results are in line with other articles where the effectiveness of the screening strategies may be improved by the addition of p16/ki-67 dual stain in the triage , 43.st strategy, Cytology primary screening, the utilization of p16/ki-67 as an optional triage tool has reduced the costs by €3.881,26 in the Public Healthcare System and by €14.997,50 in Private Practice compared to HPV test triage. Additionally, a cost reduction of €21.647,50 in Private Practice was reported in the HPV test-based screening scenario by adapting dual stain in the triage, whereas an elevation of €4.694,18 was shown in the Public Healthcare System in comparison to cytology triage. Finally, the costs for the Co-testing screening strategy were proved to be the highest among all screening scenarios.Regarding the analysis of the total direct medical costs of the three different CC screening scenarios, from a healthcare perspective, in our clinical trial, in the 1It seems that the model Cytology as primary screening strategy and triage with p16/ki-67 dual stain may not only have high clinical and diagnostic impact, avoiding over-diagnosis and over-treatment, but also budgetary impact lowering annual costs for screening and seems to be the most realistic and safe scenario for CC screening.As for the limitations of our study, the number of cases was small, as well as the fact that the follow-up of the women enrolled was not evaluated and is yet to be accomplished. Further evaluation of the follow-up is necessary to examine the application of the p16/ki-67 biomarker in triaging women with mild cervical lesions, as shown in many previous scientific studies. The role of p16/ki-67 negativity in combination with HR-HPV positivity remains to be fully interpreted and understood.Summarizing our results, p16/ki-67 dual stain presented both higher sensitivity and specificity, providing good clinical outcomes, while lowering the annual direct medical costs of CC screening. Cytology in combination with p16/ki-67 dual stain is a powerful, safe and affordable tool to triage women with mild cervical lesions, especially in low- and middle-income countries where accurate but also budgetary adaptations of health care services are required.


Most cervical cancers were closely associated with human papillomavirus (HPV) infections. Therefore, understanding the ecological diversity of HPV prevalence and genotype distribution among various populations in different geographical regions was essential for optimizing HPV vaccination and maximizing the vaccination effects. A total of 12,053 patient data from the three-level hospitals in Hengyang city were retrospectively analyzed. In this study, the HPV prevalence was 10.16% overall, and the multiple-type infection rate was 1.83%. The HR-HPV infection rate was 8.52%. The top six HPV genotypes were as follows in descending order: HPV16, HPV58, HPV52, HPV39, HPV51, and HPV53. The HPV prevalence in the group above 60 years old was the most, and their HR-HPV infection rate corresponded to the most too. The infection rates of HPV and HR-HPV among outpatients were both lower than those among the hospitalized-patients, respectively. Among the hospitalized-patients, the infection rates of HPV and HR-HPV among the 50–60 years group were the most in both. The HR-HPV ratio-in-positive among HPV-positive patients with the histopathologic examination was higher than that among those patients without. Among 52 HPV-positive patients with cervical squamous carcinoma, the ratio-in-positive of HPV16 was 61.54%. This study demonstrated that the HPV prevalence varied with age among women from Hengyang district of Hunan province in China and showed that HPV16, HPV58, HPV52, HPV39, HPV51, and HPV53 genotypes were more popularly distributed in this region, which could provide the experimental basis for Chinese public health measures on cervical cancer prevention. Human papillomavirus (HPV) is a spherically non-enveloped and double-stranded DNA virus and prefers to infect the epithelial cells of the skin and mucous membrane. HPV has more than 200 genotypes, and its infection is widespread in the crowd . AccordiThe cervical cancer susceptibility can be affected mainly by HPV infection and varied with some other factors, such as HPV awareness and toll-like receptor gene polymorphisms –9. It waHengyang city has 7.337 million permanent residents from over 40 nationalities and is the deputy center of Hunan province in China, which also is the second-largest city in this province. Therefore, the HPV prevalence and HPV genotype distribution of this city are significant for Chinese epidemiological data, and it can provide important advice for Chinese public health measures on cervical cancer prevention.In this study, the data of 12,053 women from the tertiary hospitals in Hengyang city between July 2018 and July 2019 were retrospectively analyzed. HPV testing was used as a routine examination for those suspected gynecologic patients from better-off families. HPV positive was identified by PCR, and HPV PCR-Flow fluorescence assay was used for HPV genotyping. 27 HPV genotypes were tested, including 17 HR-HPV genotypes  and 10 LR-HPV genotypes .p-value of 0.05 was considered statistically significant. The statistical data analysis was performed by SPSS version 13.0 .The prevalence of HPV infection, presence of single or multiple HPV infections, and HPV genotypes, as well as, their corresponding 95% CIs were estimated with binomial distribution analysis. The Chi-square test was performed to compare the differences in the infection rates (the positive amount occupied among its corresponding whole population) of HPV or HR-HPV and the ratio-in-positive (the positive amount occupied among its corresponding positive population) of HR-HPV or LR-HPV among various patients. A A total of 1,224 women were positive for HPV infection in 12,053 women, among which 1,003 were positive for a single genotype infection and 221 for multiple infections . It was In the current study, 221 women were positive for multiple infections, among which a total of 163 patients were with double infections, 36 patients with triple infections, and 12 patients with quadruple infections, and there were patients even with septuple or sextuple infections . As showFrom 12,053 patients, 27 different HPV genotypes were identified. As shown in The distribution of the remaining top five genotypes was as follows in descending order: HPV58, HPV52, HPV39, HPV51, and HPV53, which differed from HPV prevalence in Guatemala, where the top six genotypes were HPV16, HPV18, HPV39, HPV58, HPV52, and HPV45 . FurtherAmong 12,053 women, HPV16 was the most frequent genotype with an infection rate of 2.19% . Moreover, the HPV16 positive patients occupied 21.57% among 1,224 HPV positive patients, that is, the ratio-in-positive of HPV16 was 21.57% , which was near to that (22%) in Guatemala , indicatThe amount of all clinical HPV strains found from 1,224 positive women was 1,538, among which 535 clinical HPV strains were from 221 women with multiple infections. As shown in p < 0.01)  more than that  of hospitalized-patients, their HPV infection rate  was lower than that  of hospitalized-patients ( < 0.01) , the sam < 0.01) . It may p > 0.05)  .p < 0.01), respectively, followed by that of 50–60 years group . The HPV infection rates of the most and the second prevalent group were slightly higher than those in Guatemala  (As shown in  to 15%) . Therefo to 15%) . The HPV to 15%) , but in  to 15%) .p < 0.01).Among all HPV infections, HR-HPV infection was identified as the infection with at least one HR-HPV genotype, and its infection rate was 8.52% . The group above 60 years old with the highest HPV infection rate  had the highest HR-HPV infection rate , which was obviously higher than that (7.4%) in Beijing from 2014 to 2015. It may be due to that the investigated population in Hengyang was from gynecologic patients and was different from the population that received free screening in Beijing . On the p > 0.05) or among those of all age groups (p > 0.05) had no statistical significance  was higher than that of LR-HPV  among 1,224 HPV-positive patients. Although HR-HPV ratio-in-positive of the group above 60 years old  and below 20 years group  were the most or least, respectively, the difference between them (ificance .p > 0.05). However, the HR-HPV infection rate  of the hospitalized-patients was higher than that of the outpatients  (p < 0.01) and even the average level , showing that the diseases related with HR-HPV infection would be more severe than those infected by LR-HPV only, thereby indicating the importance of the therapeutic vaccine.As seen from p < 0.01, p < 0.01), followed by those of above 60 years patients . The high prevalence of HPV and HR-HPV among above 50 patients was in accordance with the situation that the cervical cancers are likely to happen in 50–70 years old patients, and might be attributable to their decreased immunity for clearing HPV infections, suggesting that the older people were the high-risk population for HPV.The investigated population focused was 20–50 years old , so the same as the hospitalized-patients . Moreover, the patients of this age bracket were the most  among the 507 HPV-infected hospitalized-patients. Among 4,048 hospitalized-patients, the HPV infection rate  and HR-HPV infection rate  of 50–60 years patients were both the most (p > 0.05), and that difference among all age groups had no statistical significance (p > 0.05)  and those below 21 years had the least , they had an obvious difference in HR-HPV ratio-in-positive ( > 0.05) .p < 0.01) or even the average level  (p < 0.05), which confirmed that HR-HPV can make the disease severe and even result in organ damage.Among 507 hospitalized-patients with HPV infection, 284 patients had been performed with histopathologic examination according to their illness, but the other 223 patients did not need examination. Among the 284 HPV-infected hospitalized-patients with the histopathologic examination, 70 patients were with cervical carcinoma or its precancerous lesions, 157 with uterine fibroid or its related lesions, and 57 with the pelvic neoplasm or its related lesions. Among 507 HPV-infected hospitalized-patients, the HR-HPV ratio-in-positive of 284 patients with the histopathologic examination was 90.49% (257/284) and higher than that  of 223 patients without (p < 0.05)  ( < 0.05) , suggestAs shown in On the basis of the study on the awareness and knowledge levels on HPV , this stThe original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.The human data in present study were collected in line with the Helsinki Declaration and was approved by the Ethical Review Committees of University of South China.SYT analyzed and interpreted the patient data and was a major contributor in writing the manuscript. YQL, YH, and HYS also analyzed and interpreted the patient data collaboratively. YPW and YMW performed the collection of data. All authors read and approved the final manuscript.The authors acknowledge the grants of the Provincial Natural Science Foundation of Hunan Province (2021JJ50014), Scientific Research Project of Hunan Provincial Health Commission (202105012137), Municipal Science project of Hengyang City in Hunan Province (202002042122), National Natural Science Foundation of China (81402169), Educational Committee Foundation of Hunan Province (19C1616), Hunan Provincial Key Laboratory for Special Pathogens Prevention and Control (2014-5) and Construct Program of the Double First-Class discipline of University of South China (2020SYL).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Persistent human papillomavirus (HPV) infection will eventually lead to clinical problems, varying from verrucous lesions to malignancies like cervical cancer, oral cancer, anus cancer, and so on. To address the aforementioned problems, nanotechnology-based strategies have been applied to detect the virus, prevent the interaction between virus and mammalian cells, and treat the virus-infected cells, due mainly to the unique physicochemical properties of nanoparticles. In this regard, many nanotechnology-based chemotherapies, gene therapy, vaccination, or combination therapy have been developed. In this Minireview, we outline the pathogenesis of HPV infection and the recent advances in nanotechnology-based weapons that can be applied in combating HPV-associated diseases. Human papillomavirus (HPV) is an envelope-free double-stranded circular DNA virus composed of DNA core and protein capsid. HPV parasitizes on the surface of the genitals and leads to the proliferation of the squamous epithelial cells on human skin and mucous membrane . Most in2 laser therapy, photodynamic therapy, and surgery. They are used for both mild diseases and precancerous lesions. Although these strategies have been widely used in treating HPV infections, they often suffer from high recurrence rates and have a result in scar hyperplasia or increased infection rate  have been widely developed and applied in biomedicine and other fields since the late 1960s . Nanomatet al. described the pathogenic role of exosomes and micro ribonucleic acids (miRNAs) in HPV-mediated inflammation.Several studies have elaborated on the role of inflammation in the initiation and development of cancers, as illustrated in et al. confirmed that extracellular survivin was associated with HPV infection in HPV-18 positive Hela cells  in the late 1990s . In whicThe application of gold nanoparticles in HPV detection can be presented in many forms, including photoelectric analysis, fluorescence analysis, electrical and colorimetric analysis, etc . Photoeln = 52) are exciting, as high as 85.7%/100% and 85.7%/91.7%, respectively  available for HPV infection are comprised of virus-like particles (VLP) made of the type-specific major capsid protein L1, have little protection capacity against viral types which have no epitopes on the vaccine scaffold . Further vaccine . Further vaccine .2 laser therapy, mainly target benign, proliferative lesions. Besides, these therapies have no elicit effect and can be used only after the lesions have formed. In response to these shortcomings, HPV-based E6 and E7 oncoproteins, two key factors that can promote the abnormal proliferation of epithelial cells in the cervix, eventually arouse more and more attention in gene therapy   with poly (β-amino ester) (PBAE) together to afford the hyperbranched copolymer PAMAM-PBAE (hPPCs). The resulting hPPCs were used to form a polyionic complex with CRISPR/Cas9 plasmid that targets the HPV16 E7. hPPCs significantly enhanced the cellular uptake of plasmid, escape of plasmid from endolysosome entrapment, and the subsequent gene editing efficiency . SimilarMoreover, the combination of chemotherapy and gene therapy is another promising strategy to combat HPV-induced malignancies. For example, Di et al. constructed a biomimetic dual-drug delivery system (Si/PNPs@HeLa) to deliver both paclitaxel and siRNA targeting E7. With the help of HeLa cell membranes camouflage, the system could avoid immune surveillance and accumulate on the tumor region, hindering the growth of tumor tissue successfully (inhibiting rates of 83.6% by tumor volume). Besides, the researchers also concluded that HeLa cells resistant to paclitaxel could be resensitized by knocking down the E7 gene .et al showed that the engineered aluminum hydroxyphosphate nanoparticles (AAHPs) could be used to enhance antigen-specific antibody responses induced by HPV18 L1-VLPs by adjusting their surface charge. After a series of experiments, the researchers concluded that the positively charged AAHPs provoked much stronger and long-lasting antigen-specific humoral immune responses compared to their negatively charged or neutral counterparts (co-hydroxymethyl glycolic acid)-based delivery systems were developed for delivering the synthetic long peptide and a toll-like receptor 3 ligand. Similar therapeutic efficacy was observed in polymeric NPs and mineral oil formulations. The most important is that there is no adverse effect in the application of polymeric NPs containing poly IC (Apart from the preventative L1-VLP or possible L2-VLP vaccines mentioned above, therapeutic vaccines that promote antigen-specific humoral immune response are under development and will gradually be put into practice. In the development of therapeutic vaccines, it is important to choose an appropriate adjuvant. Recently, Liang terparts . As an a poly IC .As an emerging technology, the application of nanotechnology in HPV infection is receiving increasingly more attention. Nowadays, most patients come to clinics or hospitals only after lesions have been discovered, such as verrucous lesions, cervical intraepithelial neoplasia, or even cancers. Therefore, it is very important to strengthen the publicity and education of relevant groups. As for HPV detection, the testing work in most developed countries is much better than that in underdeveloped countries and regions. Therefore, there is an urgent need to develop methods of high efficacy, low cost, ease to store, and transport. Our review has listed several nanomaterials, like Sso7d-modified Au NPs. Although the preclinical experimental results were exciting, a larger sample and further study in clinical forms are needed. For the treatment of HPV infection, the HPV vaccine neutralizing antibody is indeed a good choice. The latest method is to exploit the cross-antibody-neutralization effect to amplify the range of HPV types that vaccines work on, such as by the way of constructing a multiepitope single-molecule formulation. Apart from antibody neutralization of HPV vaccines by redesigning epitopes or changing types of adjuvants, gene therapy alone or in combination with chemotherapy are also efficient strategies to treat HPV-induced diseases. Of note, it is of great importance to perform repeated experimental verifications and consider safety, cost-effectiveness, and technical difficulties in choosing proper treatment for HPV-associated diseases.


U.S. HPV vaccine uptake remains below the Healthy People 2030 goal of 80% series completion. Parental concerns and misinformation about the efficacy and safety of the Human Papillomavirus (HPV) vaccine remain, and may be addressed by digital interventions tailored to their concerns. Reported here are results from a small scale randomized trial testing a mobile web app for parents and their adolescent daughters (ages 11–14 years) encouraging HPV vaccination in New Mexico, an ethnically-diverse U.S. state.Methods: A clinic-cluster randomized trial where pediatric clinics (n = 9) were recruited and randomized, and parent-adolescent pairs (n = 82) within clinics received either the Vacteens.org/Vacunadolescente.org mobile web app or Usual and Customary (UC) HPV Vaccination information. Parents completed online surveys at baseline and 3-months. Daughters' HPV vaccine data were collected from the New Mexico State Immunization Information System 1 year post baseline.Results: Three month survey results found Vacteens.org/Vacunadolescente.org parents to have higher positive HPV vaccine beliefs, informed decision making, intent to vaccinate and vaccine confidence outcomes than UC parents. HPV vaccine data found higher first dose HPV vaccination , and higher HPV vaccination series completion .Conclusions: The small trial results showed the Vacteens.org/Vacunadolescente.org web app prompted positive vaccine-related attitudes and beliefs, and more HPV vaccination initiation and series completion. Mobile web apps can make decision-making tools for HPV vaccination widely available on digital platforms, reducing vaccine hesitancy, and confusion and increase HPV vaccine uptake. In the U.S. uptake of Human Papillomavirus (HPV) vaccine remains far below the Healthy People 2030 goal of 80% series completion . Nationaand health beliefs   and the Reported here are findings from a randomized trial on a smartphone web app for parents and adolescent girls (ages 11–14) that was intended to encourage HPV vaccination in New Mexico, an ethnically-diverse U.S. state. The trial tested the following hypotheses:Vacteen/Vacunadolescente mobile web app will express more favorable HPV vaccine beliefs, informed decision making, intent to vaccinate for HPV, self-efficacy for HPV vaccination, and benefits and risks of HPV vaccination for their daughters than parents assigned to the Usual and Customary (UC) Information control group.H1: Parents assigned to the Vacteen/Vacunadolescente mobile web app will initiate and complete the HPV vaccination series than daughters of parents assigned to the UC Information control group.H2: More daughters of parents assigned to the GoHealthyGirls website) to a mobile app platform, Vacunadolescente.orgVacteens/, and provided both English and Spanish versions. Mobile web apps are mobile device sensitive web sites designed to function like native operating system (Android or iOS) apps, but avoid the problems of operating system exclusivity. They maintain app functionality across mobile platforms, and are more easily updatable should that be required. The web app developed in this project was an informed decision-making website for parents and adolescent daughters (ages 11–14) that employed both Informed Decision Making , HPV vaVaccination records were acquired by matching parent identification information to the NM-SIIS database. Record acquisition was performed by an honest broker, who was blind to clinic and parent randomization status. Participants' parents last name, adolescents last name, first name and birthdate were used as matching variables in vaccination record acquisition. First and second shot completion data were recorded from participant daughters' records.t-tests were used. We chose to use one-tailed tests because our hypotheses were directional and a less conservative approach to analysis was believed to be appropriate for this relatively small sample evaluation ; Informed Decision Making ; parents in the Vacteens.org/Vacunadolescente were also more likely to intend to vaccinate their daughters right away than later or not at all . In addition, parents in the Vacteens.org/Vacunadolescente group were significantly more confident about their vaccination choices , and a trend toward being more aware of the benefits and risks of vaccination .Three-month follow-up surveys were completed by 38% (n = 82). A review of first shot date and date of entry into the study determined that some daughters (n = 13) had received their initial HPV vaccinations prior to study, and thus were not qualified to participate. Data for these cases were excluded from the analysis; exclusion occurred equally from the Vacteens.org/Vacunadolescente (n = 7) and UC Information (n = 6) groups, leaving a final N = 69 for analysis. Analyses of first dose data revealed a significant treatment group difference , such that rate of HPV vaccination initiation in the Vacteens.org/Vacunadolescente condition (59.4%) was 18.8% higher than the UC Information condition (40.6%). Further, HPV vaccination series completion in the Vacteens.org/Vacunadolescente group was statistically-significantly higher  (68.4%) compared to the UC group (31.6%), an absolute increase of 36.8%.HPV vaccine uptake data from the NM-SIIS database was obtained for all daughters of parents enrolled in the trial  and series completion . The small sample of parent-daughter pairs may limit confidence in the outcome, but the effect sizes and odds ratios are in the moderate range, suggesting a substantial effect of the Vacteens.org/Vacunadolescente web app that would potentially make large in-roads into vaccine uptake when distributed widely.The results of this trial indicated that the Vacteens.org/ Vacunadolescente mobile web app. First, it could be used in conjunction with a pediatric clinic practice, where physicians recommend use of the mobile web app prior to well-child visits, sports physicals, or vaccination appointments. Parents who browse the app may make informed decisions about vaccination before the visit and be ready for vaccine initiation, saving valuable time in the doctor-patient interaction, time that is already at a premium. It also may make it more comfortable for providers to talk with parents about HPV vaccination, knowing that the topic was already presented and many of parents' concerns were covered in the mobile web app. Further, tools provided in the Vacteens.org/Vacunadolescente mobile web app, like the text and email follow-up reminders, could make vaccine dose completion more likely, as our data show. Thus, in combination with presumptive recommendations  to seek the HPV vaccine for them. The Vacteens.org/Vacunadolescente web app focused its messaging on this younger 11–14 years age range, and parents of older teens  may need somewhat different messaging that is sensitive to older teens having more agency in vaccination decisions.The present investigation carries some limitations. The small sample size is a limitation, and further research will be needed to confirm the impact of the web app on vaccine uptake and related variables. The findings are also limited to young adolescent girls ages 11–14, even though HPV vaccination is recommended for boys. We are currently conducting a trial with a version of the web app tailored to parents of young male adolescents in the same 11–14 years of age. The loss of some parents due to already having had their daughter vaccinated, is of some concern; however, the results remained statistically significant with moderate effect size. It seems that some parents were simply not sure as to whether they had their daughter vaccinated for HPV. Paper-based methods for tracking vaccination, especially in adolescence, are now rarely used, and parents may lose track of vaccination instances. Currently, the New Mexico Department of Health provides an online portal where parents can search for their child's vaccination record (a number of states have begun to adopt this technology), but parents may be unaware of this resource. The loss of a substantial amount of 3-month follow-up survey data due to trial termination by the funding agency is a limitation to the study. The loss of these data certainly limits conclusions available from the study survey analyses. In retrospect, the trial termination (due to insufficient progress in clinic recruitment) is regrettable given the promising data that the investigation was able to obtain. The location of the trial in New Mexico may limit its generalizability due to its ethnic mix, containing predominately Hispanic and Native American minority participants. Whether the mobile web app would be just as effective with African American parents or other minority group parents is unknown. A final limitation is the young age of the sample (11–14 years). HPV vaccination is recommended for individuals up to age 26 and we cannot be certain that the The results of this investigation suggest that mobile web app technology, systematically developed for ease and convenience of use on mobile and other computing devices and guided by DOI and IDM theories of health behavior, that communicates about parents' concerns, lack of information, and misinformation parents hold regarding the HPV vaccination, can substantially improve HPV vaccine uptake. A recent review of social media and mobile technology interventions to improve HPV vaccine uptake  indicateThe raw de-identified data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by University of New Mexico Main Campus Institutional Review Board. Written informed consent to participate in this study was provided by adult participants, and adolescent assent was provided by adolescent participants' legal guardian/next of kin.WW oversaw research project in all aspects, conducted data analysis, and major amount of manuscript writing. GZ was involved in message construction, measurement, and manuscript writing. AK was involved in message construction, clinic recruitment, and manuscript writing. DB was involved in message construction, web app development, testing, and manuscript writing. JR was message language translation and web app development. LC was involved in clinic recruitment, message construction, and vaccine data. VM was involved in message development and measure assessment. RS was involved in clinic recruitment. All authors contributed to the article and approved the submitted version.WW, DB, JR, and VM were employed by company Klein Buendel Inc. Outside of the present work GZ has served as an external advisory board member for Merck and Moderna and as a consultant to Merck. In addition, he has received investigator-initiated research funding from Merck administered through Indiana University. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


In the Netherlands, the HPV-vaccine uptake was 52% during the 2009 catch-up campaign (birth cohorts 1993–1996). This increased to 61% in the regular immunization program (birth cohorts 2000–2001). However for birth cohorts 2003–2004 the uptake declined to 45.5%. With this study we aimed to gain insight into social, economic and cultural determinants that are associated with HPV-vaccination uptake and which subgroups with a lower HPV-vaccination uptake can be identified. In addition, we investigated whether the influence of these factors changed over time.To study the determinants of HPV-vaccine uptake we performed a database study using different aggregation levels, i.e. individual level, postal code level and municipality level. All Dutch girls who were invited for HPV-vaccination through the National Immunization Program in the years 2012, 2014 and 2017  were included in the study population. We conducted multilevel logistic regression analyses to analyze the influence of the determinants on HPV-vaccination uptake, taking into account that the delivery of HPV-vaccine was nested within municipalities.Results showed that in particular having not received a MMR-vaccination, having one or two parents born in Morocco or Turkey, living in an area with lower socioeconomic status and higher municipal voting proportions for Christian political parties or populist parties with liberal-conservative views were associated with a lower HPV-vaccination uptake. Besides some changes in political preferences of the population and changes in the association between HPV uptake and urbanization level we found no clear determinants which could possibly explain the decrease in the HPV-vaccination uptake.In this study we identified current social, economic and cultural determinants that are associated with HPV-vaccination uptake and which low-vaccination subgroups can be identified. However, no clear determinants were found which could explain the decrease in the HPV-vaccination uptake. Tailored information and/or consultation for groups that are associated with a lower HPV-vaccination uptake might help to increase the HPV-vaccination uptake in the future.The online version contains supplementary material available at 10.1186/s12889-021-11897-0. Vaccination against human papillomavirus (HPV) targeting girls 12 years of age is part of the Dutch national immunization program (NIP) since 2010. Prior to this, a catch-up campaign for 13–16-year-old girls was initiated in 2009. The bivalent HPV16/18-vaccine was used starting with a three-dose schedule up to 2013 and a two-dose schedule from 2014 onwards. HPV16 and − 18 together are estimated to account for 70% of all cases of cervical cancer [The HPV-vaccination uptake is low compared to the coverage for other vaccines in the Dutch NIP. During the catch-up campaign in 2009, the vaccine coverage was 52% for birth cohorts 1993–1996 . This inResearch among girls who were targeted for the initial catch-up campaign and their mothers showed that socio-demographic determinants, such as socioeconomic status (SES) and country of birth were associated with HPV-vaccination uptake , 8. In aIt is unknown whether the influence of the various social, economic and cultural determinants on HPV-vaccination uptake changed over time in the Netherlands. In addition, it is unknown which determinants could explain the recent decrease in the HPV-vaccination uptake. With this study, we aim to gain insight into the determinants that are associated with HPV-vaccination uptake and which low-vaccination subgroups can be identified, and to investigate whether target groups can be identified that are associated with the decline in HPV-vaccination uptake.We performed a database study to investigate various determinants of HPV-vaccination uptake on different aggregation levels: individual, postal code and municipality. The sample included all girls invited for HPV-vaccination through the NIP in the years 2012, 2014 and 2017, respectively from birth cohorts 1999, 2001 and 2004. For 2017 was the latest complete dataset available; in 2014 the vaccination schedule was changed and this was the last year before the decline in vaccination uptake; in 2012 and 2017 the Dutch National Elections for seats in the House of Representatives were held.Anonymous individual-level data were obtained retrospectively in 2018 from the national vaccination register (Praeventis), using the 2018 municipality division . The individual level variable Ethnicity was defined as country of birth of both parents, for which most common country of birth combinations were used.Additional data, on postal code and municipality level, were extracted from the publicly available data of Statistics Netherlands (CBS), The Netherlands Institute for Social Research (SCP), and the Electoral Council (Kiesraad), or were provided by the Municipal Health Services (MHS). If data was not available for a certain invitation year, data of the most recent year was used  unless multicollinearity (> 0.70) was found between two or more predictor variables. To calculate the correlation between all predictor variables in order to detect multicollinearity Spearman’s correlation coefficient and the phi coefficient (2 × 2) were used [Multilevel logistic regression analyses were used to determine the association between the dependent variable HPV-vaccination uptake of a completed series (2 or 3 doses depending on invitation year) and predictor variables. The multilevel models included two hierarchical levels where girls who were invited for HPV-vaccinations level 1) were nested in municipalities (level 2). First, the associations between HPV-vaccination uptake and independent variables , adjusted odds ratios (AOR) and 95% confidence intervals (95%CI).The study was approved by the research ethics committee of the Radboud University Nijmegen Medical Centre, Nijmegen, the Netherlands; CMO number 2018/4744.In the following paragraphs, main results of the multilevel univariate and multivariable logistic analysis of the invitation year 2017 are presented per predictor variable. Additionally, these results are compared to the associations between HPV-vaccination uptake and the predictor variables in the invitation years 2012 and 2014. Tables of the multilevel univariate analysis (Table As the correlation between MMR-vaccination status and DT (aP)-IPV-vaccination status was > 0.80 in the multicollinearity analysis, only MMR-vaccination status was included in the multilevel multivariable logistic regression models. In the multilevel univariate and multivariable models MMR-vaccinations status was significant and positively associated with HPV-vaccination uptake  had a significantly lower HPV-vaccination uptake compared to girls whose parents both were born in the Netherlands , Socialist Party (SP), Democrats 66 (D66) and Green Left (GL) Tables . This inGirls who lived in a municipality with a higher voting proportion for the populist parties with liberal-conservative views PVV & FvD had a significantly lower HPV-vaccination uptake in 2017, yet, in invitation years 2012 and 2014 either a positive or no statistically significant association between HPV-vaccination uptake and PVV & FvD voting proportion was found , ethnicity, socioeconomic status of the postal code area, urbanization level of the municipality, road distance to vaccination location and municipal voting proportions in national elections were predictors for the HPV-vaccination uptake. Subgroups with a lower HPV-vaccination uptake in 2017 were in particular girls who have not received a MMR-vaccination (HPV-vaccine uptake 6.0% versus 48.9% when having received two MMR-vaccinations), who have one or two parents born in Morocco or Turkey (HPV-vaccine uptake 16.5–26.1% versus 49.4% when having two parents born in the Netherlands), who live in an area with a lower socioeconomic status (HPV-vaccine uptake 36.9% versus 54.8% when socioeconomic status is high) and higher voting proportions in municipalities for Christian political parties (CU&SGP)  and populist parties with liberal-conservative views . Besides some changes in political preferences of the population  of 0.86 (95% CI: 0.83–0.90) in 2017 versus 2012) and changes in the association between HPV-vaccination uptake and urbanization level  we found no clear determinants which could possibly explain the decrease in the HPV-vaccination uptake.Several groups in the Netherlands are known to have objections against vaccination in general. Among the orthodox Protestants, who live geographically clustered in the so-called Dutch Bible Belt, approximately 40% has not received childhood vaccinations . In addiRegarding ethnicity, highest HPV-vaccination uptake was found among girls with both parents born in the Netherlands. Lowest uptake was in particular observed for girls with one or two parents born in Morocco or Turkey. This was also found in a study following the catch-up campaign in the Netherlands . In a syGirls living in areas with lower SES appeared to have lower HPV-vaccination uptake than girls living in areas with higher SES. This relation between SES and HPV-vaccination uptake was also shown in a previous study in the Netherlands . UnderlyIn the most recent invitation year, 2017, a road distance to the vaccination location of more than five kilometers showed in the multilevel multivariable logistic regression model a very small but statistically significant association with a lower HPV-vaccination uptake. In contrast, no significant association was found between road distance to vaccination location and HPV-vaccination uptake in 2012 and 2014. Another Dutch study showed that the average road distance was 5.7 km and was comparable between 2014 and 2017 . People In 2012 and 2014, girls living in areas with higher urbanization levels had a lower HPV-vaccination uptake than girls living in areas with lower urbanization levels. However, in 2017, this association was not found. The Dutch study performed among girls eligible for the catch-up campaign in 2009 showed that unvaccinated girls lived in more urbanized areas .Regarding voting proportions in national elections, we found a lower HPV-vaccination uptake in girls living in municipalities with a higher voting proportions for the Christian political parties (CU&SGP), compared to the national mean. The association between high political preference for Protestant Christian parties and low HPV-vaccination uptake was shown before in the Netherlands . Apart fAlso in 2017, a higher municipal voting proportion for populist parties with liberal-conservative views was found to be associated with a lower HPV-vaccination uptake. Previous database studies found that voters for Party for Freedom (PVV) and Forum for Democracy (FvD) may have less confidence in the government, media, and social institutions , 16. AlsIn birth cohorts 2002 and 2003, i.e. who were vaccinated in 2015 and 2016, a sharp decrease in vaccination uptake was observed [Tailored strategies are critical in reaching groups with suboptimal vaccination uptake . We wereBesides the strength that individual data was used on vaccination status to determine the HPV-vaccination uptake, this study has also some limitations. Data on social, economic, cultural and political determinants were not collected for the purpose of this study and only available on postal code level or municipality level. Therefore, associations on these aggregation levels represent the group of individuals within a given area and might not directly apply to an individual. For example, it concerns the voting behavior of adults in the municipality, while these girls were not yet allowed to vote themselves. On the other hand, the decision about vaccination is also mostly made by the parents of the girls. Furthermore, for some determinants data was not available for the specific years included in this study. In this case the most recent data was used. Proportions for the political parties in national elections were only available for 2012 and 2017. For road distance, only data was available for 2014 and 2017. Therefore, the results for 2012 and 2014 should be interpreted with caution. Also, we used home addresses obtained in 2018. Girls might have been moved in the years before, but we think that these movements outweigh each other and therefore had a very small effect on the analyses. Besides that, some variables contained a large number of missings. Especially for ethnicity, which counted low numbers for some categories in all cohorts, especially in 2012 and 2014. This limits the comparability of these variables over time. Besides the investigated determinants, there are other determinants that are possibly associated with the HPV-vaccination uptake. For example, school-education or being the oldest girls in the family (i.e. the first who is eligible for HPV-vaccination). Unfortunately, no information on these or other potential determinants was available in the databases.In this study we identified current social, economic and cultural determinants that are associated with HPV-vaccination uptake for public health relevance. Customized information and/or consultation should be prepared for identified target groups that are associated with a lower HPV-vaccination rate. We found no clear determinants which explain the decrease in the HPV-vaccination uptake. The vaccination coverage recently increased again in the Netherlands , probablAdditional file 1.Additional file 2.


In multivariable logistic analysis, third-grade students had the most increased awareness of CC , HPV  and HPV vaccine  when compared to first-grade. Awareness and knowledge regarding HPV infection were insufficient among students in SOHS. As the future healthcare providers, these results highlight the need to supplement targeted education to improve their awareness and knowledge of HPV and vaccination.Cervical cancer (CC) is one of the most common causes of cancer-related deaths worldwide. CC is mainly caused by human papilloma virus (HPV), which can be prevented by vaccination. We conducted a cross-sectional study in secondary occupational health school (SOHS) through a questionnaire aimed to assess the awareness and knowledge regarding HPV infection of students. A total of 2248 students participated in the survey, 45.3% of them had heard about CC, while only 21.9% of them had heard about HPV; and 74.2% had no idea of the causal link between HPV infection and CC. Most participants displayed poor awareness and knowledge about HPV infection. The results suggested that age, grade, major, academic performance, etc. were correlated with higher awareness of CC, HPV and HPV infection ( The cervix is the organ connecting the uterus and the vagina, in which cervical cancer (CC) develops. CC is the fourth most common cancer for women worldwide, of which annual absolute estimates of new cases and deaths were 569,800 and 311,400 in 2018 [®, quadrivalent Gardasil® and nonavalent Gardasil®) have been approved in succession for use in mainland China [Persistent HPV infection is the most important and necessary cause of almost all cases of CC . HPV infnd China .® there was very low among adolescent girls [Nevertheless, there is no sufficient data about the coverage rate of HPV vaccination due to the lack of national population-based plan on HPV vaccination in mainland China. It is Hong Kong the first place to license an HPV vaccine around China, and yet the uptake rate (7.2%) of Cervarixnt girls . A revient girls . Lackingnt girls ,13. Accont girls . StudentHence, the aim of this study was to assess the awareness and knowledge about HPV infection among students at SOHS and identify the influencing factors to help design pertinent intervention measures for this population.This study was a cross-sectional survey conducted between September and December 2019, targeting Chinese SOHS students. The sample size of this study was calculated using a rate of 16.8% of students aware of HPV which was acquired from pilot studies, a marginal error of 3% and 95% confidence interval. Considering a low response rate and design effect, there was a 30% increase in the sample size which was eventually determined as 776. A two-stage sampling was performed to recruit the participants. First, stratified sampling was conducted for medical and non-medical classes. Then, cluster sampling was employed to the selected classes. Finally, the participants consisted of 2248 students comprising 428 boys and 1820 girls responded to the survey carried out in the classrooms based on the WeChat platform Application (App).The questionnaire of HPV infection was developed by the research team with the advice of experts. It was consisted of two domains: socio-demographic characteristics of participants were assessed with sixteen items; ten questions assessed awareness and knowledge regarding HPV infection among respondents. For Q1–7, Q9, Q10, responses of “Not Sure” were recoded as “No” in the statistical analysis. For Q8, it was with multiple choices and every option was correct. In this study, awareness in the questionnaire meant that the participants had heard about cervical cancer, HPV and HPV vaccine. Knowledge meant more details about HPV infection in the rest of the second domain of the questionnaire. These data were used to explore factors associated with HPV awareness and knowledge.This study was approved by Ethical Review Committee of Shantou University Medical College, Shantou, China (SUMC-2019-62). Informed consents were collected from participants in our study and all responses would be confidential.p < 0.05 was considered as the significance level.Data were analyzed using SPSS 26.0  and STATA 14.0 . To evaluate the data, descriptive statistics, percentage, frequency, mean and standard deviation were used, as appropriate. The chi-square test was used to analyze the differences. Multivariate logistic regression analysis was undertaken to assess the relationship between awareness, knowledge and characteristics among the respondents, and odds ratios (ORs) along with 95% confidence interval (CI) were also calculated. Only significant variables remained in the models. For all the analyses above, Declaration: We used ‘AIDS’ incorrectly in order to be able to communicate the commonly used term to the subjects of the study.In this study, data of 2248 students were collected and analyzed with a 100% response rate. Most of the participants were female (81.0%), younger than 16 years old (60.5%) and lived in rural (73.0%). The majority of students (86.8%) were majored in the medical specialty. More than half of students were in their first-year (52.8%), followed by second-year (42.9%) and third-year (4.3%). Most students had relatively lower personal, paternal and maternal educational status, family income and academic performance. About 4.9% of the study populations had sexual experience. Among them 30 respondents (1.3%) had the history of CC in the family and 287 participants (12.8%) had the family history of other cancers. For all of them, 754 respondents (33.5%) had received at least one dose of the HPV vaccine .Generally, awareness and knowledge on HPV infection were insufficient. In this study, it was found that 1019 respondents (45.3%) had heard about CC; 492 respondents (21.9%) had heard about HPV and 555 respondents (24.7%) had heard about HPV vaccine. Mostly, 1768 respondents (78.6%) didn’t know that men could be infected by HPV. Similarly, more than seventy percent of the participants didn’t know that HPV infection could cause CC and some other cancers. Their ignorance regarding the link of genital warts and liquid-based cytology of HPV infection should be taken seriously. Only 192 students (8.5%) realized that it was asymptomatic after HPV infection. Almost all the respondents (96.8) knew that AIDS was a STI, while only about half (51.6%) of the participants considered that HPV infection as STI, followed by syphilis (45.5%), gonorrhea (43.4%), fungal diseases (26.3%), herpes (24.2%) and hepatitis B (19.5%) .n = 492) who had heard about HPV, the majority (72.8%) had access to HPV-related information from the Internet, followed by their education (58.9%), television or radio (38.0%), health professional (30.9%), family or friends (26.4%), brochures (20.7%) and magazine or newspaper (13.2%) (Among those ( (13.2%) .p < 0.001). As for the respondents who were in higher ages, grades, majored in medical specialty and (or) had better academic performances, they knew more about Q1–7, Q9 and Q10 (p < 0.05). However, there was no difference in the awareness and knowledge of Q1–7, Q9 and Q10 between rural and urban residents. Among students themselves with higher education level, awareness and knowledge concerning Q1–7 and Q10 were more than their counterparts, while there was no difference in knowledge about that HPV infection was often asymptomatic. Paternal educational status reached significance with Q2, Q7, Q9 and Q10, whereby students whose father had higher education level were more likely to be aware of HPV and knew that HPV could cause genital warts, HPV infection was often asymptomatic and could be detected by liquid-based cytology. Maternal higher educational status was associated with the students’ increased awareness and knowledge of HPV, HPV vaccine, availability for HPV of men and methods for preventing HPV infection compared to their counterparts. The students with higher family income more endorsed knowledge of HPV vaccine and HPV’s relation to men and CC. Significant differences in characteristics of sexual experience and family history of CC were found in the responses to Q1, Q2 and Q9. It was showed that there was statistically significant association between the characteristic of family history of other cancers and the responses to Q1–7. HPV-vaccinated participants demonstrated significantly better awareness and knowledge of Q2–7, Q9 and Q10 . The strongest association between characteristics and the awareness of CC, HPV and HPV vaccine was grade. The three models revealed that those students with higher grades and (or) majored in medical specialty were more likely to be aware of CC, HPV and HPV vaccine (p < 0.05). Urban dwellers were 1.34 times more likely to be aware of CC than rural residents . The participants with higher education before enrollment were more likely to know HPV vaccine . The respondents whose father with higher education were more likely to realize HPV. Students with higher family income were 1.65 times more likely to have heard of HPV vaccine. Compared to those whose academic performances were fair or below, participants with better academic performances were more likely to be aware of CC  and HPV . The first model also revealed that subjects having family history of CC  and other cancers  were associated with higher odds of awareness of CC than counterparts. Students with family history of other cancers were 1.45 times more likely to know HPV compared to their counterparts. HPV-vaccinated participants were more likely to be aware of HPV  and HPV vaccine   and HPV vaccine  than males. Increased awareness of CC, HPV and HPV vaccine was significantly associated with older students  .To the best of our knowledge, this is the first study implemented in southern China to measure the awareness and knowledge of students in SOHS regarding HPV infection. It is hard to compare our results with previous studies due to the lack of surveys in the same group. We acquired a good response of 100%, which suggested that the results could represent this population. Most of students were female, majored in medical specialty, lived in rural area and had no sexual experience. We found the vaccination rate of HPV vaccine in our participants was lower than that found in another study of Germany conducted in 2011, where 67% of female students aged 18–20 years were vaccinated  and loweThe recruited students had relatively low level of awareness and knowledge regarding HPV infection 21.9%), which was far below than studies conducted in Spain (54.34%)  and Ital.9%, whicFurther analysis revealed that the Internet was their first source of information about HPV infection among participants aware of HPV, while previous studies reported the reverse results ,30,31. IIn our study, female students knew more about CC and HPV-related cancers compared to males, while there was no sex difference in awareness of HPV and HPV vaccine and knowledge of other detailed information about HPV, which was similar to a study among the junior middle school students . HoweverThe limitations of this study are as followed. Firstly, the study found a number of correlations between the level of HPV knowledge with the characteristics of respondents, but explaining causality needs to be further explored based on research features. Secondly, as the participants filled in the questionnaire themselves, there was partial recall bias. Thirdly, the participation of the third-grade students was obviously lower than the other two grades, which was because the majority of them were on an internship outside school. Finally, this survey was only aimed at students in health schools. Multiple schools and groups are planned to be included in the survey to elucidate the cognitive level and possible influencing factors in the later stage.In conclusion, the results underscore that the overall awareness and knowledge regarding HPV infection among students in SOHS is insufficient and superficial. As most of the respondents will be the future healthcare providers, it is crucial to equip them with professional and adequate knowledge through targeted education campaigns and curriculums for the implementation of HPV vaccine and prevention of CC among them and more general population.