U) cervical cancer has been debated. Here we report the results of detailed molecular classification of HPVU cervical cancer, and validate HPVU as a biomarker of poor outcomes. We identify that HPVU cervical cancer tumors harbor mutations affecting cell cycle progression, and in vitro experiments reveal HPVU, but not HPV+, cells are sensitive to palbociclib monotherapy. HPVU status can be translated into the clinic as a predictive biomarker of poor patient response to standard of care treatments and these patients may benefit from personalized treatment plans. Our results identify palbociclib as a lead candidate as an alternative treatment strategy for HPVU cervical cancer patients. We also suggest that primary cervix tumors be routinely tested for HPV prior to treatment to identify patients who will benefit from more aggressive precision-driven therapy.Persistent HPV infection is a known driver of cervical carcinogenesis, but the existence and biological relevance of HPV undetected (HPVU) have been underappreciated in clinical decision making. In this study, two independent CC datasets were used to characterize the largest cohort of HPVU tumors to date . Genomic and transcriptome tumor profiles and patient survival outcomes were compared between HPV+ and HPVU tumors. In vitro analyses were done to determine efficacy of the selective CDK4/6 inhibitor palbociclib on HPVU cancer cell lines. Patients with HPVU CC tumors had worse progression-free and overall survival outcomes compared to HPV+ patients. TP53, ARID1A, PTEN, ARID5B, CTNNB1, CTCF, and CCND1 were identified as significantly mutated genes (SMGs) enriched in HPVU tumors, with converging functional roles in cell cycle progression. In vitro HPVU, but not HPV+, cancer cell lines with wild type RB1 were sensitive to palbociclib monotherapy. These results indicate that HPVU status can be translated into the clinic as a predictive biomarker of poor patient response to standard of care treatments. We suggest primary cervix tumors be routinely tested for HPV prior to treatment to identify patients who will benefit from more aggressive precision-driven therapy. Our results identify palbociclib as a lead candidate as an alternative treatment strategy for HPVU CC patients.Cervical cancer tumors with undetectable HPV (HPV Cervical cancer (CC) is the fourth leading cause of cancer amongst women worldwide . PersistU) tumors [U [In the context of cervical cancer, HPV screening is recommended for women aged 30–65 in addition to pap smears every 5 years . However) tumors ,10,13,14umors [U .U is an indicator for poor progression-free and overall survival. Overall failure rates for standard of care chemoradiation are as high as 67% in some series for patients with HPVU tumors [U CC before initiating treatment has the potential to improve outcomes by allowing for personalized treatment planning.For patients receiving the standard of care concurrent administration of cisplatin chemotherapy with pelvic irradiation ,8,9,16, U associated genomic alterations. To date there have been no comprehensive studies integrating the investigation of genomic characteristics and therapeutic options for HPVU CC compared to HPV+ tumors. In this study we aimed to (1) characterize the genomic landscape and transcriptome of HPVU compared to HPV+ CC tumors; and (2) investigate clinically actionable targets that are unique to HPVU CC tumors and can be exploited for therapeutic intervention.In our previous work, we reported on the landscape of genomic alterations in cervical cancer , but theGenomic analyses were performed on 2 sets of data A. For th+ positive and HPVU tumors [U (n = 11). These HPVU samples with corresponding RNAseq data (n = 10) were then validated by aligning to reference genomes for high-risk HPV 16, 18, 31, 33, 45, 52, 56, 58, and 59. HPVU was defined as any sample with less than 1 HPV read in 1 million human reads. All 10 WUSM samples called as HPVU by DNA probes were also negative using the RNAseq cutoffs.The CESC TCGA standard of HPV detection of at least 1 HPV read per million human reads was employed to identify patients with HPVU tumors . In the U tumors . The WUSU and 355 HPV+ tumors in the NOR-TCGA set and significantly mutated genes (SMGs) were identified using a false discovery rate (FDR) of 0.1. SMGs were also analyzed for either co-occurrence or mutual exclusivity of mutations with other SMGs. GISTIC2.0 [U tumors using FDR ≤ 0.25. Targeted gene exome sequencing for 237 pan cancer associated genes was performed on 86 tumor–blood pairs from the WUSM cohort . Additionally, differential expression (DE) analysis was performed using R version 3.5.2 and DESeq2 package on the 10 HPVU and 58 HPV+ tumors from the WUSM cohort. DE genes used for subsequent GSEA pathway analysis had a log2 fold change > 1 or < −1 and p-adj < 0.01. Pathway analysis was restricted to hallmark gene sets that had at least 10% of the pathway contributed from DE genes and an FDR < 0.01.RNAseq analyses were performed for 354 NOR-TCGA  and 68 WUSM cohort tumors independently and used as test and validation datasets, respectively, to assess transcript expression differences between HPVU cervical cancer cell lines, HT3 and C33A harbor mutant RB1 , SCC-47 from Millipore, and SCC-154 from University of Pittsburgh and maintained in IMDM media  with 10% heat inactivated FBS and incubated at 37 °C in 5% CO2. Mycoplasma testing was performed periodically to verify no infection. Experiments were performed on cell lines under passage 30. Palbociclib was dissolved in 0.01% of dimethyl sulfoxide (DMSO). All three HNSCC cell lines had WT RB1 genotypes, while Fadu also had overexpression of cyclin D1 as assessed by Western blot using anti-cyclin D1 (92G2) .Wild-type (WT) RB1 is necessary for palbociclib to be effective . Howevertant RB1 . TherefoCells were treated with increasing doses of single agent palbociclib (PZ0383) . Treated cell viability was normalized to untreated control cells. Combination palbociclib and radiation treatment was also evaluated, cells were treated with palbociclib 1hr prior to single-fraction 2 or 4 Gy radiation treatment. Five days after treatment cell viability was quantified using alamarBlue cell viability reagent according to the manufacturer’s protocol .G1 cell cycle arrest was evaluated by treating cells with vehicle (0.01% DMSO) or 0.25 µM palbociclib and harvesting them 24 and 48 h after treatment. Cells were stained with propidium iodide and run for flow cytometry on the MACSQuant analyzer . Attenuation in proliferation was evaluated by treating cells with either vehicle or 0.25 µM palbociclib and counting cells at 6-, 9-, and 12-days post-treatment. A representative day 12 plate was crystal violet stained and visualized.P less than 0.05 was set as the threshold for significance of all study outcomes.The Kaplan–Meier method and log rank test were used to determine differences in progression-free and overall survival, using the R version 3.5.2 packages survminer and survival. Fisher exact test was used to identify SMGs in the NOR-TCGA and WUSM cohorts. Mann–Whitney/Wilcoxon tests were used to identify SMGs with differential transcript expression. The Wilcoxon signed-rank test and student t-tests were used for in vitro experiments. U patients from this cohort had worse disease-specific survival outcomes compared to HPV+ patients (p < 0.001)    B. The pa = 0.06) C. Surviv= 0.001) D. Patien+ tumors E,F. Furt+ tumors .U tumors in the NOR-TCGA set revealed a total of 10,182 somatic mutations, including 6314 missense, 586 nonsense, 2232 silent, 266 splice site mutations, as well as 480 deletions, 90 insertions, and 214 miscellaneous mutations. The aggregate non-silent mutation rate was 3.1 per Mb. MutSig2CV analyses revealed TP53, PTEN, KRAS, and ARID1A as significantly mutated genes (SMGs) at the false discovery rate of q < 0.1. Recurrent mutations in these genes occurred in 50%, 29%, 21%, and 33% respectively . For the first set, the comparison of relative mutational frequency was focused on the 1569 genes which met at least one of the following criteria: (i) MutSig2CV p value < 0.1 in either HPVU or HPV+ tumors; and (ii) any gene with >1 mutation in HPVU tumors and zero mutations in HPV+ tumors. These criteria allowed for the investigation of genes which would otherwise be deemed non-significant in MutSig2CV analyses due to the small sample size, with a stringent false discovery rate <0.05 following Fisher’s exact test and Benjamini–Hochberg correction. The genes with higher frequencies of somatic mutations in HPVU compared with HPV+ cervical tumors include TP53, RICTOR, ARID1A, ARHGEF2, ZNF331, CTNNB1, KIAA3012, and MC5R , were TP53, ARID5B, ARID1A, PTEN, CTNNB1, CTCF, and CCND1  revealed 6 significant focal amplifications in the following cytobands (listed in genomic order with some genes in the peaks in parentheses): 3q26.31 , 7p15.3 , 8p11.22 , 11q13.3 , 11q22.1 (TRPC6), 17q12 (ERBB2), 19q12 (CCNE1), and Xq21.33 (no gene in the peak but close to FAM133A) (S4). In addition, six significant focal deletion peaks were identified in cytobands 3p14.2 (FHIT), 4q35.2 , 5q12.3 , 15q22 , 19p12 (RPSAP58), and 22q13.32   .U tumors into two unique subsets: an SMG-rich subset with tumors harboring most of the SMGs except for TP53, and an SCNA-rich subset which harbored most of the SCNAs and displayed strong overlap with TP53 mutation (ZNF331 mutations co-occurred with CTCF (p = 0.007623) and ARID1A (p = 0.00659) mutations, and KIAA1012 mutations co-occurred with MC5R mutations (p = 0.000198) , ZNF331 (p = 0.0466), and NCAPH2 (p = 0.0466) mutations  . TP53 muutations .ARHGEF2, CCND1, and CTNNB1 all had higher transcript expression in HPVU tumors from both the NOR-TCGA and WUSM patient cohorts  and G2M checkpoint pathways (FDR = 0.002347) (The SMGs  cohorts . NCAPH2 TCGA set . Further.002347) .U tumors had higher CCND1 transcript expression in both the NOR-TCGA and WUSM cohorts, and CCND1 was present in a significant 11q13.3 amplification peak in the NOR-TCGA set. In HPV+ cervical cancer, expression of HPV E7 drives bypass of the G1 cell cycle checkpoint by targeting Rb for proteasomal degradation. Since HPVU cervical cancers may bypass the G1 checkpoint via a different mechanism than HPV+ tumors, we hypothesize that HPVU tumors may be more sensitive than HPV+ tumors to the FDA approved CDK 4/6 inhibitor palbociclib .All seven SMGs identified in the NOR-TCGA and WUSM combined genomic analysis have converging biological roles in coordinating G1 to S phase cell cycle progression, shown in U cervical cancer cell lines C33A and HT3 were tested for sensitivity to palbociclib monotherapy alongside the HPV+ SiHa and Caski cervical cancer cell lines. We did not observe any sensitivity of either the HPVU or HPV+ cell lines to palbociclib  cell lines. The HPVU Fadu and HPV 16 positive SCC-47 and SCC-154 HNSCC cell lines all are RB1 WT, and the Fadu cell line overexpressed cyclin D1  (+ cell line’s viability (HPV< 0.001) A. Palbociability A. We treiability .p < 0.05; D12, p < 0.01), while HPV+ SCC-47 and SCC-154 showed no delays in growth rate after treatment with palbociclib  or 0.25 µM palbociclib and harvesting them 24 and 48 h after treatment. G1 cell cycle arrest was maximal 24 h after palbociclib treatment, with an average increase in G1 cells, across three independent replicates, of 13.6%, 2.2%, and 2.0% for Fadu, SCC-47 and SCC-154 cells respectively B,C. Fadubociclib D,E.U CC. The most prevalent clinical test for HPV is the use of immunohistochemical (IHC) staining of primary tumor biopsies for p16. Although p16 IHC is reproducible and easy to perform in the clinical laboratory setting, p16 expression can occur independently of HPV infection, and may overestimate the number of tumors assumed to be HPV+ [U tumors. In the research setting the current gold standard of HPV testing is RNAseq based detection of HPV viral transcripts [+ tumor varies between research groups. For HNSCC, the TCGA definition of HPV negativity was less than 1000 HPV transcript reads per tumor sample [Various methodologies are used to detect HPV in patient tumors, however there is currently no universal consensus on defining HPV be HPV+ ,26. Sincnscripts ,17. Usinr sample . The TCGr sample . Future U tumors may result from the loss of HPV nucleic acids as the tumors become larger and more advanced [U status, suggesting there may be other potential explanations. While the association between HPV infection and cervical cancer is incontestable, there is no evidence to suggest that the cervix is uniquely protected from the non-HPV-related carcinogenic processes that occur in other tissues of the body. In addition, one would intuitively expect that HPV-driven cervical cancer will more likely be diagnosed earlier rather than later in life if HPV detection and treatment are not done at the precancerous stage. Therefore, our confirmation of an association between HPVU tumors and older age of diagnosis supports the possibility that at least some HPVU tumors may indeed not be driven by HPV infection [Apart from the limits of detection, it has also been postulated that HPVadvanced . Althougnfection .TP53, PTEN, ARID1A, ARID5B, and CTNNB1 as SMGs in HPVU tumors [CCND1 and CTCF as SMGs put the converging role of cell cycle progression in HPVU cervical cancer into clear perspective. In particular, the contribution of CCND1 was highlighted both by activating P287T mutations and copy number amplifications [U tumors harbor TP53 mutations given the role of the HPV E6 oncogene in abrogating p53 function. It is also intriguing that several novel HPVU associated SMGs have direct and indirect links to HPV biology, suggesting that the effect of the somatic mutations in the absence of HPV may be similar to the synergy between the wild type forms and HPV in cancer. For example, KIAA1012 encodes TRAPPC8, a transport protein essential for successful HPV cellular entry [CTCF in the absence of HPV may mimic HPV-CTCF interactions and influence tumorigenesis.Similar to previous studies, we identified U tumors . AlthougU tumors , multiplU tumors ,17. Notaications . It is nar entry . The E2 ar entry . It is tRICTOR as an SMG mutation suggests that it may be an oncogene in the context of HPVU tumors with the possibility for therapy with mTORC2 inhibitors. The relatively low expression of RICTOR in HPVU tumors is therefore puzzling but may be explained in part by an indirect effect of HPV E6 on RICTOR expression. RICTOR is a target of mir-218 [RICTOR expression may be downregulated. Interestingly, a tumor suppressive function has recently been demonstrated for RICTOR in p53-mutant medulloblastoma [RNF111 encodes the Arkadia protein which has been shown to enhance tumor suppression in colorectal, renal cell, and lung carcinoma by enhancing TGF-beta signaling [PPWD1 encodes a peptidylprolyl isomerase which contains WD40 domains and is thought to play a role in protein folding and pre-mRNA splicing [The identification of  mir-218 , which i mir-218 . Therefoblastoma . Some geignaling ,36,37, wsplicing ,39.U compared to HPV+ CC that contribute to treatment resistance, there is also increasing evidence that micro- and long non-coding RNAs also contribute to disease resistance. One such study found that mir-21 and mir-155 were more highly expressed in CC tumors as compared to normal cervix tissue [+ and HPV− CC tumors, however regardless of HPV status both mir-21 and mir-155 were shown to be a significant predictive indicator of cervical cancer. The authors postulated that the expression of these micro-RNAs may be of high diagnostic value as they have the ability to help predict and identify both HPV+ and HPV− CC development.Although our study’s primary focus was in the identification of coding genes differentially mutated and/or expressed in HPVx tissue . AdditioU CC tumors harbor mutations and alterations in transcriptional regulation to overcome G1 cell cycle arrest. We identified mutations and/or overexpression of β-catenin and cyclin D1 which binds to CDK4/6 and inhibits Rb and allows cell cycle progression. The FDA approved CDK4/6 inhibitor, palbociclib was evaluated for efficacy as a single agent therapeutic in HPVU and HPV+ HNSCC cell lines. In response to single agent palbociclib treatment the HPVU Fadu cell line exhibited increased G1 cell cycle arrest 24 h after treatment, prolonged proliferation attenuation up to 12 days after treatment and decreased cell viability compared to untreated controls. The HPV+ SCC-47 and SCC-154 cells showed no significant effects on G1 cell cycle arrest, proliferation rate or cell viability after palbociclib treatment.In this study, we are the first to show that HPVU cervical cancer cell lines due to the C33A and HT3 cell lines harboring RB1 mutations [U cell line C33A is preferentially sensitive to cyclin D1 inhibitors [RB1 is not frequently mutated in either HPVU or HPV+ CC patients from either NOR-TCGA or WUSM cohorts. Additionally, RB1 mutations have not been identified as frequently mutated in cervix cancer cohorts from other independent studies [U CC with wild type RB1. Lastly, a recent publication found palbociclib was able to sensitize HPVU but not HPV+ HNSCC cell lines to radiation treatment [U cancer cells and attenuating their ability to actively engage their cell cycle. Therefore, although combination treatment does not synergize to decrease cell viability, this strategy may be effective to attenuate HPVU cancer cells ability to proliferate. Altogether the results from our study indicate that HPVU cell lines are uniquely responsive to single agent palbociclib treatment and set the stage for clinical testing to determine if patients with HPVU cervical carcinomas could benefit from the addition of palbociclib into their treatment plan.A caveat of our present study was that we were unable to assess palbociclib sensitivity using HPVutations , and palhibitors ,42. Of n studies ,17. Therreatment . HoweverU as a poor prognostic marker in cervical cancer. Patients with HPVU cervical tumors had higher recurrence and mortality rates after surgery and standard of care CRT treatment compared to patients with HPV+ tumors (U tumors prior to the initiation of treatment. These patients are likely to fail their standard of care treatment and may benefit from alternative treatment plans, which could include radiation or chemotherapy dose escalation, including the addition of biologically targeted agents. In this study, using the largest cohort of HPVU cervical cancers reported to date, we have performed detailed biological characterization which has identified cell cycle regulation as a rational target for HPVU tumors, and, using a panel HPV+ and HPVU cell lines, we have shown that the CDK4/6 inhibitor palbociclib is a potential alternative treatment strategy.HPV status has the potential to be a powerful prognostic biomarker of CC patient progression-free and overall survival. In this multi-institutional study, we have identified and validated HPV+ tumors . In curr


Small cell neuroendocrine carcinoma of the uterine cervix (SCNEC) is a rare cancer involving the human papilloma virus (HPV), and has few available treatments. The present work aimed to assess the feasibility of SOX2 and HPV statuses as predictive indicators of SCNEC prognosis.The associations of SOX2 and/or high-risk (HR)-HPV RNA in situ hybridization (RISH) levels with clinicopathological characteristics and prognostic outcomes for 88 neuroendocrine carcinoma (NEC) cases were analyzed.INK4A and HR-HPV RISH expression and SOX2/HR-HPV RISH co-expression were detected in 68(77.3%), 76(86.4%), 73(83.0%), and 48(54.5%), respectively. SOX2-positive and HR-HPV RISH-positive SCNEC cases were associated with poorer overall survival  and disease-free survival  compared with those expressing low SOX2 and negative HR-HPV RISH. Alternatively, univariate analysis revealed that SOX2 and HR-HPV RISH expression, either separately or in combination, predicted the poor prognosis of SCNEC patients. Multivariate analysis revealed that the co-expression of SOX2 with HR-HPV RISH may be an independent factor of OS  and DFS  prediction in SCNEC.Among these patients with SCNEC, SOX2, P16Overall, the results of the present study suggest that the co-expression of SOX2 with HR-HPV RISH in SCNEC may represent a specific subgroup exhibiting remarkably poorer prognostic outcomes compared with the expression of any one marker alone.The online version contains supplementary material available at 10.1186/s12885-021-08059-1. Small cell neuroendocrine carcinoma of the uterine cervix (SCNEC) is a highly aggressive and rare malignant cervical cancer < 3%). The incidence of lymph node and distant metastases is high in the early stage of SCNEC, although SCNEC is usually only detected at the advanced stage –3. SCNEC%. The inSOX2) gene, located on chromosome 3q26.3-q27, belongs to the SOX family [The sex-determining region Y-box 2  human papilloma virus (HPV) infection . The occpression . The detpression . FurtherThe present study retrospectively examined the expression levels of SOX2 and HPV mRNA in SCNEC and investigated the relationships between the expression levels and clinicopathological characteristics in SCNEC cases.www.researchdata.org.cn; RDD approval number: RDDA2020001710).In the present retrospective study, we enrolled 88 patients with histologically confirmed SCNEC, who had under surgical resection at Sun Yat-sen University Cancer Center between January 2010 and December 2014. Patients were enrolled when they were diagnosed with primary SCNEC, with available clinical information. The last follow-up was conducted in June 2020. The study protocol was approved by the Institutional Ethical Board of Sun Yat-sen University Cancer Center. The raw data relevant to the study were imported into the Research Data Deposit public platform , anti-Chromogranin A , anti-CD56 , anti-MSH6 (SP93) , anti-MLH1 (Roche (M1), Germany), anti-PMS2 (Dako (EP51), Germany), anti-Ki-67 (ZA0502), and anti-MSH2  antibodies. Phosphate buffer was used to wash the sections three times before incubation with biotinylated goat anti-mouse antibodies. Next, DAKO liquid 3, 3′-diaminobenzidine tetrahydro-chloride (DAB) was used for staining, and Mayer’s hematoxylin was used for counter-staining. The block-like and diffuse staining at every core was positive for P16INK4A, whereas the patchy or non-stained sections indicated a negative result. Additionally, ≥1% positively-stained cancer cell nuclei indicated a positive result for MSH2/MSH6/PMS2/MLH1. The SOX2 positive samples were scored as follows: 0, < 5% cells with positive staining; 1, 5–24% cells with positive staining; 2, 25–49% cells with positive staining; 3, 50–74% cells with positive staining; and 4, 75–100% cells with positive staining. Positive staining intensity was graded as follows: 0, 1, 2, and 3 suggested negative, weak, moderate, and strong staining, respectively. Then, the percentage score was multiplied by the intensity score to obtain the eventual score. Thereafter, 6.5 was used as the median IHC score to classify high or low SOX2 expression levels.SCNEC tumor tissues and lymph node metastatic tissues, were obtained to construct a TMA. Thereafter, the constructed TMA block was sectioned  and stained for immunohistochemical analyses. Sections were de-waxed in xylene and ethanol and treated with 3% hydrogen peroxide diluted with methanol. Thereafter, avidin-biotin was used to block all sections overnight at 4 °C. The sections were then incubated with anti-SOX2 , anti-P16According to a previous study, the tumor tissues used in PCR should not be the same as those used in TMA . HPV wasThe RNAscope scoring system was used to examine each specimen, as described in previous studies , 24. HPVINK4A with the HPV mRNA expression levels and clinicopathological parameters in patients with SCNEC were analyzed using the chi-square test. Kaplan-Meier analysis was used to analyze overall survival (OS) and disease-free survival (DFS), while the log-rank test was used for comparison. The correlation of prognosis was analyzed using univariate and multivariate Cox regression analyses. P < 0.05 (two-sided) indicated statistical significance. To construct a nomogram, the predictive power of each variable for OS and DFS was evaluated using univariate Cox regression. Thereafter, the significant variables were used in multivariate Cox analysis. To determine independent prognostic variables, the Akaike information criterion (AIC) score was used for backward selection for suitable variables. Finally, the variables were enrolled for the nomogram construction, with 1-, 3-, and 5-year OS and DFS selected as the primary endpoints. To evaluate the nomogram predicting power, a concordance index (C-index) with receiver operating characteristic (ROC) curve analysis was applied [The SPSS 19.0 software was used for data analyses. The expression of SOX2 in different SCNEC subgroups was compared using an unpaired T-test. Correlations of SOX2 and P16 applied . The dis applied .The characteristics of 88 patients with SCNEC are summarized in Table SINK4A expression profiles in SCNEC, a total of 88 paraffin-embedded SCNEC tissue samples, with the corresponding clinicopathological data, were harvested to construct the TMA cohort. SOX2 was primarily located in the nucleus, while P16INK4A was observed in the cytoplasm , 76(86.4%), and 73(83.0%), respectively   no significant difference between the groups   and relapse (P = 0.023), whereas SOX2 expression showed no significant correlation with other clinicopathological features of patients with SCNEC. However, P16INK4A expression was only significantly related to Ki67 (P = 0.005). Notably, HPV mRNA levels were significantly correlated with FIGO staging (P = 0.021), pre-operative chemotherapy (P = 0.007), relapse (P = 0.027), neuroendocrine markers (P = 0.021), and pathological classification (P = 0.014). These data suggest that the overexpression of SOX2 and HPV mRNA potentially facilitates tumorigenesis and development of SCNEC.The association of SOX2, P16INK4A, and SOX2/HR-HPV RISH were related to patient OS (P < 0.05). Moreover, multivariate Cox regression showed that parametrium invasion , stromal invasion , nerve invasion , SOX2 , HR-HPV RISH , and SOX2 /HR-HPV RISH  may independently predict prognosis in SCNEC cases. Univariate regression analysis revealed that nerve invasion, SOX2, HR-HPV RISH, SOX2/P16INK4A, and SOX2/HR-HPV RISH were related to DFS (P < 0.05). Upon multivariate regression, it was observed that nerve invasion , SOX2 , HR-HPV RISH , and SOX2/HR-HPV RISH  were evidently related to DFS. Additionally, in this study, we created a forest plot to display hazard ratio together with the corresponding 95% CIs of OS and DFS, based on Cox proportional hazards regression (Fig. INK4A, HR-HPV RISH, SOX2/P16INK4A, and SOX2 /HR-HPV RISH expression showed significant differences, which were verified through log-rank tests (Fig. Tables S P < 0.0. MoreoveVariables obtained based on Cox proportional analysis were then applied to build the respective OS and DFS prognostic nomograms Fig. . The facThe present work examined the significance of SOX2, HR-HPV RISH, and clinicopathological features in SCNEC cases. The respective nomograms were constructed according to Cox hazards analysis to predict OS and DFS for SCNEC cases. Thereafter, each point was assessed for prognostic risk, and individualized post-treatment was provided. To our knowledge, the present work is the first retrospective analysis of the value of SOX2 and HR-HPV RISH in predicting the prognosis for SCNEC.The dysregulated OCT4/SOX2 complex has been detected in various human malignant tumors , and thuINK4a/Ki-67 can serve as a candidate marker for HR-HPV infection in HPV-associated endocervical neoplasia [INK4a is currently controversial, often leading to a misinterpretation of the staining results [INK4a and Ki-67 IHC. Multivariate analysis demonstrated that SOX2/HR-HPV RISH co-expression served as an independent factor in predicting the OS and DFS in SCNEC cases. Further studies, using larger cohorts, should be conducted to validate our findings.The role of HPV in the etiology of SCNEC is well established, and HR-HPV can be detected in the majority of the patients . The preeoplasia , 35. How results , and the results , 38 and  results . Recent  results , 40. TheConsistent with our results, SOX2 was proven to be a potential marker to predict overall survival and recurrence in p16+ oropharyngeal cancer . Recent There were several limitations to this study. First, its retrospective nature may lead to inevitable selection bias. Second, this study was conducted with a small sample size from a single center. Third, the present work only focused on the significance of SOX2 and HR-HPV RISH in predicting prognosis, while other prognostic factors, such as molecular biomarkers or inflammatory prognostic markers, were not included. Therefore, the results of this work should be further validated in multi-center studies with a larger sample size.Additional file 1.


Human papilloma virus (HPV) is involved in the development of cancer of the cervix, mouth and throat, anus, penis, vulva, or vagina, but it has not been much considered as a cause of breast cancer. Recently, a number of investigations have linked breast cancer to viral infections. High-risk HPV types, predominantly HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59, are established as carcinogens in humans. In this study we aimed to detect 19 high-risk and 9 low-risk HPVs from archived breast tumor tissue among Ethiopian women.  In this study, 75 breast cancer patients from Tikur Anbassa Specialized Hospital in Addis Ababa (Ethiopia) were included. HPV detection and genotyping were done using the novel Anyplex™ II HPV28 Detection Assay at the Orebro University Hospital, Sweden. The Anyplex™ II PCR System detects 19 high-risk HPV types  and 9 low-risk HPV types . IHC for p16 was done using an automated system, the Dako Autostainer Link.  Out of the 75 valid tests, two were found to be positive (2.7%) for HPV. One of the cases was positive for the high-risk HPV16 genotype while the other was positive both for the high-risk HPV39 and the low-risk HPV6. The cell cycle protein p16 was highly expressed in the case positive for the high-risk HPV16, but it was not expressed in the case positive for HPV39.  The prevalence of HPV is low in Ethiopian breast cancer patients, but the role played by HPV in breast carcinogenesis among Ethiopian breast cancer patients cannot be commented based on these observations. P = 0.0002). Ren et al. also showed three high-risk HPV types  are positively correlated to breast cancer [Human papilloma virus (HPV) has been implicated in the development of cancer of the cervix, mouth and throat, anus, penis, vulva, or vagina, but it has not been much considered as a cause of breast cancer. However, a growing number of investigations have linked breast cancer to viral infections, including human papilloma virus (HPV), Epstein–Barr virus (EBV), mouse mammary tumor virus (MMTV), and human cytomegalovirus (HCMV) . Human pt cancer .In another study, it has been shown that women with HPV-associated cervical pathology are at increased risk from the same HPV type positive breast cancer which implies a possible link between HPV and breast carcinogenesis . There iIn Ethiopia, according to one study, more than 6,000 new cervical cancer cases are diagnosed annually and HPV is the cause of most of the cases . A studyThe participants involved in this study were previously investigated in our published work to characterize the molecular classification and determine distribution of the androgen receptor in breast cancer among Ethiopian women , 14. TheDNA was isolated from tissue core samples of each tumor specimen. The presence of malignant cells was assessed in all samples by evaluation of slides stained with hematoxylin and eosin (H&E). Genomic DNA (gDNA) was isolated from the tumor cores using the QIAamp DNA Mini Kit (Qiagen), including proteinase K treatment (Qiagen) followed by purification using the QIAcube automated system (Qiagen). The DNA concentration was measured using a Nanodrop ND-1000 (Nanodrop Technologies). The gDNA samples were stored at 4°C.The multiplex quantitative real-time PCR was done at the Orebro University Hospital. Real-time PCR amplification was performed using the Anyplex™ II HPV28 Detection Assay , in accordance with the manufacturer's protocol, in a CFX96 Real-Time Thermocycler . The Anyplex™ II HPV28 Detection Assay is a novel multiplex real-time PCR assay that permits the simultaneous amplification, detection, and differentiation of target nucleic acids of 28 HPV types and internal control (IC). The Anyplex™ II PCR System detects 19 high-risk HPV types  and 9 low-risk HPV types .Immunohistochemistry (IHC) for p16 was done using an automated system, the Dako Autostainer Link. Formalin-fixed paraffin sections were cut at 4 microns and rehydrated with water. Heat-induced epitope retrieval was performed with the FLEX TRS High-pH Retrieval Buffer for 20 minutes. After peroxidase blocking, the specific monoclonal antibody  was applied at room temperature for 20 minutes. The FLEX + Rabbit EnVision System was used for detection. DAB chromogen was then applied for 10 minutes. Slides were counterstained with Mayers hematoxylin for 5 seconds and then dehydrated and coverslipped. Slides were scanned on a Pannoramic 250 digital scanner  and images scored using the software program “CaseViewer” . Negative controls were included in the run.Statistical analysis was done using SPSS for windows version 21. Continuous data are reported as mean ± SD or number (proportions). Skew distributions are reported as the median value with minimum and maximum.There were 114 participants with FFPE available for this study. Only 75 cases had valid results in the genotyping experiment for HPV. Mean age at diagnosis of the 75 cases was 42 years (SD12), and median age was 40 (range 22–75). Most of the participants (40%) were <40 years old. About 31% of the participants were ≥50 years and 28% were 40-49 years old.Of the 75 tumors, 7% were grade 1, 29% were grade 2, and 33% were grade 3. The stages were as follows: Stage 1—20%; Stage 2—31%; Stage 3—35%, and Stage 4—1%. The most common type of histology which is presented in Of the 114 tumors, only 75 had detectable internal control; therefore, the rest were rejected. Out of the 75 valid tests, 2 were found to be positive (2.7%). One of the cases was positive for the high-risk HPV16 genotype, while the other was positive both for high-risk HPV39 and low-risk HPV6.A strong expression of p16 was detected in the case positive for HPV16 but not detected in the case that was dual positive for HPV39 and HPV6. The expression of p16 across our cohorts is summarized in In this study, we explored the presence of 19 high-risk HPV types  and 9 low-risk HPV types . The Anyplex Multiplex qPCR technology for detection and genotyping of HPVs were utilized. We detected the high-risk HPV DNA 2/75 (2.7%) of the study participants from Ethiopian breast cancer patients. Researchers from different countries reported an overall prevalence of 2–74% of HPV in breast cancer as presented in the paper by Lawson et al. . Our obsVarious types of HPV have been identified from different countries with HPV16 reported as the most common HPV followed by the HPV18 and HPV33 . In our Additionally, the high-risk HPV39 which is detected in our study was not found in any of the 24 studies reviewed by Lawson et al. However, it was the major high-risk HPV detected in the recent UK study by Salman et al. , 21.The p16 (cell cycle protein) expression is detected both in HPV-positive and negative cases in our cohorts. There is evidence that indicates strong expression as opposed to low or medium expression of p16 is associated with HPV biological activity and high expression of p16 can be used as a surrogate for an indication of transcriptional activity of HPV . In our The prevalence of HPV is low in Ethiopian breast cancer patients, but the role played by HPV in breast carcinogenesis among Ethiopian breast cancer patients cannot be commented based on these observations.


This paper highlights the low levels of vaccine coverage and high levels of reported vaccination hesitancy in Yerevan, Armenia, that present profound challenges to the control of disease through routine vaccination programmes. We draw on investigations of hesitancy towards the introduction of new vaccines, using the Human Papillomavirus (HPV) vaccine Gardasil as a case study, to interrogate underlying challenges to vaccine acceptance. We analyse primary data from the introduction of Gardasil, first used in Armenia in 2017, to investigate how levels of medical knowledge amongst physicians in 20 health facilities in Yerevan, Armenia, regarding vaccine science influence attitudes towards the introduction of a newly developed vaccine. A questionnaire-based cross-sectional study was completed by 348 physicians between December 2017 and September 2018. The responding physicians displayed a respectable level of knowledge and awareness regarding vaccination with respect to some characteristics  but low knowledge and poor understanding of other key issues such as the age at which women were most likely to develop cervical cancer (only 15% answered correctly), whether or not the vaccine should be administered to people who had already been infected (27% answered correctly) and whether sexually active young people should be treated for infection before vaccination (26% answered correctly). The study suggests that the drivers of vaccine hesitancy are complex and may not be consistent from vaccine to vaccine. The Armenian healthcare sector may need to provide additional training, awareness-raising and educational activities alongside the introduction of new vaccines to improve understanding of and trust in vaccination programmes. Armenia has consistently displayed lower levels of vaccine confidence than surrounding countries and Europe as a whole ,2,3, witPapillomaviridae that affect epithelial tissue, the layer or layers of cells that form the covering of most internal and external surfaces of the body and its organs. More than 150 types of HPV have been identified [Human papillomaviruses (HPVs) are a group of viruses belonging to the family of entified . In womeentified ,11,12.Invasive cervical cancer (ICC) is one of the leading causes of cancer in women and, according to 2018 global estimates, results in approximately 570,000 new cases and 311,000 deaths annually . In the In Armenia, the cumulative incidence rate of ICC has increased from 9.5 (1985) to 16.6 2017) per 100,000 women. Annually, about 120–130 women die from cervical cancer in Armenia . About 50% of women with ICC are not diagnosed until the third and fourth stages of cancer, when the treatment outcome is significantly less certain [ per 100,Currently, there are three HPV vaccines available, bivalent (HPV 16 and 18), quadrivalent , and 9-valent , which can protect against up to nine of the most prevalent HPV genotypes associated with cancer and genital warts ,20.Despite recommendation for routine HPV vaccinations from the World Health Organization (WHO), some countries nevertheless remain hesitant about vaccinating their young people against HPV . ArmeniaSince the quadrivalent vaccine Gardasil was introduced in Armenia in December 2017 , uptake 2*pq/d2 formula [Between December 2017 and September 2018, a questionnaire-based, cross-sectional, quantitative study was conducted in 20 Armenian healthcare facilities to identify respondents’ awareness of and attitudes toward HPV-related cervical cancer and the Gardasil vaccine. The questionnaire did not contain questions specifically related to practice, as this is known to be sub-optimal; the aim of the study was to determine the reasons why. The questionnaire was adapted from a survey previously used to determine the knowledge and attitude of registered nurses towards HPV and HPV vaccines in the USA . This wa formula .The inclusion criteria for the study respondents were fully qualified medical staff from four medical specialities considered most likely to have direct interaction with HPV-infected patients or with patients eligible for HPV vaccination, namely, paediatricians, family-practice doctors, gynaecologists, and oncologists. Exclusion criteria were student doctors, nurses, residents, and doctors from other specialities. This yielded a possible survey size of 405 physicians across the 20 facilities. Trained researchers from Yerevan State University School of Public Health visited each of the healthcare facilities included and personally invited physicians to participate in the study. Of the 405 eligible staff, 385 (95%) were approached in person to participate (the remaining 20 were not available on any of the days the researchers visited) of whom 348 (90.3%) gave consent and completed the survey. The survey was conducted by face-to-face interview in each physician’s usual place of work. This is above the number required to be considered a representative sample .Data were collected on the socio-demographic characteristics of the respondents . The questionnaire also addressed the respondents’ existing knowledge and attitudes across four areas: (1) knowledge/information about HPV; (2) knowledge/information about the HPV vaccine; (3) attitude towards the HPV vaccine; (4) attitude towards other vaccines. Respondents could answer the survey questions “True”, “False” or “I don’t know”, numerically coded 1, 2, and 88, respectively.p < 0.05 was considered to be statistically significant. The analyses were performed using the SPSS software (Version 16.0). Chi-square analyses were used for the categorical variables which compared the age groups of respondents, their lengths of experience as doctors, and the fields they specialised in. For all the analyses conducted in the study, an association of https://www.rdocumentation.org/packages/pheatmap/versions/1.0.12/topics/pheatmap accessed on 22 February 2021) based on the percentage of respondents who answered “yes” to a series of questions.A hierarchy heatmap on the physicians’ knowledge of and attitudes toward the HPV vaccine was constructed using the R statistical programme language package “pheatmap”  and 95% confidence intervals (CI) for physicians answering at random: (1) HPV is relatively uncommon ; (2) Almost all cervical cancers are caused by HPV (true); (3) HPV is most common in women in their 30 s (true); (4) Cervical cancer is one of the most prevalent cancers among women (true); (5) Most people with genital HPV are symptomatic ; (6) Genital warts are caused by the same HPV types that cause cervical cancer ; (7) Sexually active adolescents should be tested before HPV vaccination  (8) The HPV vaccine is available for both males and females (true); (9) Men and women who have been diagnosed with HPV should not be given HPV vaccine . Factors that were considered in the model were physician speciality, age, and experience.A total of 348 participants (90.3% of those eligible) from the 20 randomly selected health facilities  completed the survey and were included in the final analysis . The respondents were predominantly female (95%), reflecting the demographics of the professions . The gender inequality among the respondents  is not a limitation of this study. According to recent statistics, in Armenia, female doctors are more likely to specialise in paediatrics, family-practice, and gynaecology; hence, the high levels of female respondents in this study. A report published by the Ministry of Health, Armenia, in 2016 shows that 40–60% of physicians were female and, of these, 91.8% were paediatricians, family-practice physicians, and general practitioners; 95.2% of all paediatricians were female . As prevIn response to some questions, study participants displayed strong awareness of the issues surrounding HPV infection and its related health outcomes; for instance, most knew that cervical cancer is one of the most prevalent types of cancer among women (87.2%) and nearly three-quarters (73.4%) knew that most cervical cancers are caused by the HPV virus . AwareneHowever, 44.4% of the participants were unaware that HPV is a relatively common sexually transmitted infection and 29% of respondents were unaware that the vaccine is available to both females and males. More than half (53.5%) answered that the same types of HPV that cause genital warts also cause cervical cancer, which is incorrect. Only 27% of the respondents were aware that both male and female patients diagnosed with HPV should still be encouraged to receive the vaccine and only a similar proportion (26%) knew that sexually active adolescents do not need to be tested before receiving their HPV vaccinations. Fewer than two thirds (61%) of physicians knew that the HPV vaccine consists of recombinant HPV protein. p > 0.05). A higher number of correct answers came from paediatricians (65%) than from other groups.The proportion of correct answers varied among the physicians according to speciality, although the differences were not statistically significant overall (p = 0.000824), although, as only 40.9% of gynaecologists answered correctly, knowledge was low over all specialties. However, gynaecologists were more likely to answer incorrectly that genital warts are caused by the same strains of HPV, with 65.9% giving an incorrect answer compared with 24.6% of paediatricians (OR 2.17 p = 0.0246)  . This sup = 0.03). There was no significant difference between the specialties in their understanding of whether people with HPV are likely to be symptomatic  (p = 0.187), that cervical cancer is one of the most common cancers amongst women, which most answered correctly (p = 0.08), and whether or not HPV is a relatively common infection (p = 0.643), which just over half of family physicians, oncologists and paediatricians believed to be true and just under half of the gynaecologists (46.5%). For the other questions, differences between specialities were not statistically significant, though paediatricians (74.8%) and oncologists (74.2%) returned a higher rate of correct responses than family practitioners (70.9%) and gynaecologists (68.2%) for the question, “All common cervical cancers are caused by HPV” (p = 0.004). Respondents in the age group 45–59 years old returned a higher rate of correct answers overall. Physicians in the age group under 44 years old were more likely (69.1%) to give a correct answer for the question regarding how common HPV is  (p = 0.018) and the over 60 group.The proportion of correct answers was significantly different across the age groups (n HPV is  comparedctively) . The 45–p = 0.003). This difference may be linked to mid-career physicians being more likely to still attend courses and to engage in more educational activities than those nearing the end of their careers, which provides them with more opportunities for keeping up to date with new developments. The proportion of correct answers was significantly different when compared across the number of years of service. Respondents with 15–19 years of service answered questions correctly more often (50%) than those who had been in the profession for both a longer (30%) and a shorter time (44.6%)  supported the national vaccination programme for general childhood vaccination but fewer than two-thirds (63.8%) agreed that the introduction of the HPV vaccine into the national vaccination programme was appropriate. More than half (53.3%) expressed concern over the efficacy of the HPV vaccine, 61.0% expressed concerns over its safety, and 58.50% agreed with the statement that the vaccine is “too new and hasn’t been around long enough”. Just under half (42.9%) agreed with the statement that 13 is too young for a child to receive the vaccine .p < 0.0001) attitudes to the introduction of the HPV vaccine into the national vaccination programme. A higher proportion of paediatricians (75.2%) agreed with it, which was statistically higher than the other groups . The high likelihood of paediatricians to agree with the need for the HPV vaccination programme is consistent with a study from Serbia, in which nearly two-thirds of the paediatricians were willing to recommend the vaccine (60.2%) [Different specialties displayed statistically significant ( (60.2%) .p < 0.0001), with a higher percentage of family physicians and gynaecologists showing concern  than paediatricians and oncologists (52% and 67%). Physicians over 60 years old showed a statistically significantly different level of concern about efficiency and the side effects than those under 60   also differed significantly among different specialties (= 0.002) .p = 0.01) but there was with regard to the age at which it should be given (p < 0.0001). Family physicians were more concerned than other specialities that the vaccine was too new  and that a 13-year-old was too young to receive the vaccine , suggesting that this speciality is more prone to vaccine hesitancy than others, the reasons for which require further investigation. There was no statistically significant difference in the speciality of physicians who were concerned about the newness of the vaccine (Respondents who were concerned about efficacy were more likely to hold the opinion that the HPV vaccine is too new and “has not been around long enough” (58.8%) . p = 0.003). The proportion of physicians over the age of 60 who were concerned that 13-year-old females were too young for the vaccination was higher (55.4%) than in younger physicians (39.2%), suggesting that attitudes can differ among demographic groups. The differences in answers to this question among the different age groups were statistically significant , specialist literature (63%), colleagues (58%), and self-guided internet searches (57%).No specific questions were asked regarding physicians’ practice in administering the vaccine or not to eligible patients, as information on practice is taken from figures on vaccination coverage in Armenia recorded by existing studies, which show that it is very low compared to other countries ,2,6,7,8.Our study shows that there are knowledge gaps in the understanding of Armenian physicians with regards to some basic aspects of HPV infection and the HPV vaccine. These knowledge gaps affect physicians’ understanding of disease transmission, symptoms, and presentation, as well as who benefits from the vaccine and vaccine safety and efficacy. This is one of the first KAP  studies to be conducted with regard to physicians’ attitudes towards vaccination in Armenia and its results show that this is a complex area that requires much more nuanced understanding before interventions can be developed. However, it does, suggest that poor knowledge is influencing sub-optimal attitudes and practice.The level of understanding amongst Armenian physicians (their knowledge) with regard to the mechanisms of disease and vaccination efficacy  and beneThe particularly low number of correct answers given by the longer-serving group  suggestsAlmost all respondents were aware of the HPV vaccine but their lack of knowledge impacts on their attitudes to its efficacy and safety and thus its value to patients. Paediatricians were significantly more knowledgeable than other speciality groups, possibly because they were the group most likely to be dealing with the age group that is recommended for vaccination. However, the results show that knowing a vaccine is available does not always correspond to being willing to recommend it, particularly when one is not well informed about it or the disease it can prevent. A total of 62% of physicians were concerned about side effects of the HPV vaccine , suggesting that honest discussions around any likely side effects, including their frequency, treatment, and patient outcomes may help to build healthcare practitioner confidence, change their attitudes towards the vaccines, and enable them to pass this confidence on to patients, thus informing their practice around vaccine administration. In addition, 54% of the physicians were worried about the effectiveness of the HPV vaccine, which, again, could be addressed by in-job training and awareness that would help them to change their attitudes towards the value of vaccination and impart greater confidence to patients, who are likely to look to healthcare professionals to advise them.It is concerning that knowledge gaps exist, despite the fact that almost all respondents reported participating in special events, such as training and seminars, and mentioned specialized literature as one of their main sources of information. This suggests that the current training and methods of information dissemination are ineffective. Further studies are needed to understand the reasons behind these statistics as physicians can act as powerful gatekeepers to vaccines; their level of knowledge influences their attitudes, which, in turn, influence their practice, thus having ramifications for vaccine hesitancy and acceptance (or not) within the communities of patients they attend.We note that cultural assumptions, in particular, influence attitudes and threaten to undermine the scientific basis of vaccine schedules. In Armenia, the routine early childhood immunisation coverage is estimated at 93–97% of all children and physicians strongly support vaccination programmes amongst this age group . Our stuHowever, the age of vaccine administration is not, in fact, determined by the age at which sexual activity is likely to begin. According to the WHO, the recommended priority age for HPV vaccination is 9–13 years old because this is the age at which the immune system response is highest and produces the necessary level of antibodies and immune-system memory to protect against HPV infection for life, not because this is the age at which children may become sexually active. It is important for preteens to get all three doses long before any sexual activity begins . This shThe Armenian physicians expressed considerable concern over the newness of the Gardasil vaccine . This attitude may require targeted education to help healthcare providers understand the development and approvals process for these new vaccines in order to give assurance that the procedures have been conducted safely , thus giThe different levels of confidence in the national childhood vaccination programme compared with the introduction of the HPV vaccination highlight that individual vaccines need to be considered independently of one another, as attitudes may differ between one vaccine and another, or one context and another. Vaccine hesitancy may not be universal to all vaccines nor have the same underlying concerns with regard to each vaccine. Understanding these attitudes more fully would benefit from further research to help determine how different forms of hesitancy can be effectively challenged, how they influence and impact on practice, and what steps might be taken in advance to stop such hesitancy from becoming widespread within certain communities. Refusal of one vaccine does not necessarily point to complete vaccine denial. Whilst we appreciate that HPV and Gardasil are one specific case, we do believe that the above findings highlight issues that are likely to be generalizable to other vaccines and to be transferable to attitudes towards vaccines more widely than the Gardasil vaccine and HPV alone. Firstly, they identify a spectrum of vaccine hesitancy , in whicFurther studies could also usefully determine the extent to which physicians’ poor understanding, misconceptions, and hesitancy may influence their decision to administer vaccines and their ability to sufficiently inform patients of vaccine benefits. Our study is represented by health facilities (and physicians) from Yerevan only, for a single vaccine, and during a specific period of time. Therefore, the findings may not be generalizable to the whole of Armenia, or to physicians in other countries, or to different vaccination programmes. Knowledge, attitudes, and practice of respondents working in medical facilities located out of Yerevan and with different vaccines may vary from our study results. The assumptions we made about the generalisability of these findings to vaccine confidence in general are assumptions only and need to be tested in further research studies. This study shows that the concerns and worries that influence negative attitudes towards vaccines and which may prevent physicians from recommending vaccination are complex. Reasons may differ for different diseases and may be influenced by different underlying factors, all of which suggest the need for stronger vaccine-confidence awareness training that helps physicians—and, by extension, the patients they advise—to navigate these concerns by providing up-to-date and comprehensive information that will enhance confidence and dispel unwarranted concerns. The surveyed healthcare workers were generally not hesitant to the concept of vaccines, more to uncertainties about aspects of vaccine development, efficacy, or side effects that can be answered with easier access to accurate and up-to-date information. The role of the media and its portrayal of vaccination development may also have some influence in this process and should be considered within future research programmes. Differences in the approach of different physicians in Armenia to HPV vaccination was evident in this study, with different approaches noted based on their age, length of service, speciality, and cultural assumptions. Overall, however, the study merely highlights gaps in our understanding of vaccine hesitancy and how this may vary between different vaccines and for the prevention of different diseases; it does not address them or offer interventions. In this regard, priority consideration should be given to further studying and, in the short term, to developing a programme of continuous education for Armenian healthcare professionals on all aspects of vaccination development, safety, efficacy, and societal value, both for HPV infections and for other newly developed vaccines, ensuring such knowledge is not taken for granted when introducing new vaccines.


The global incidence of oropharyngeal cancer (OPC) is increasing. Dental professionals play a key role in the detection of oral lesions that could lead to cancer. However, scientific-based HPV-OPC visual inspection guidelines are underdeveloped and HPV knowledge and awareness has been reported to be low among dental students and professionals. The present study adapted and performed pretesting of a multi-scale survey evaluating knowledge, perceptions, and clinical practices regarding HPV and HPV-OPC for Latin American Spanish-speaking populations.A previously developed questionnaire for English-speaking dental students was translated to Spanish. The questionnaire was administered to first year dental students at two Latin American universities with dental programs. Internal consistencies were measured using Cronbach Alpha. Analyses were conducted in SAS Version 9.4.Data from a total of 114 students, a majority of the which were female (61%), and Hispanic/Latino(a)/Spanish (91%). The HPV, HPV-OPC, and HPV vaccine knowledge subscales demonstrated good internal consistency, the Cronbach’s alpha was 0.83, 0.75, and 0.86 respectively. The Barriers subscale had a Cronbach’s alpha of 0.93, showing excellent internal consistency. The Clinical Procedures subscale, focused on factors surrounding dental students’ hypothetical clinical practice procedures, had a Cronbach’s alpha of 0.86. The Scope of Practice scale had a Cronbach’s alpha of 0.93.Ultimately, this survey demonstrated reliability and applicability for the assessment of dental students’ knowledge, perceptions, and clinical practices regarding HPV and HPV-OPC in Latin America.The online version contains supplementary material available at 10.1186/s12903-022-02108-2. The global incidence of oropharyngeal cancer (OPC) is increasing, especially among those younger than 45 years . An estiDental professionals play a key role in the detection of any oral and OPC lesions that could lead to cancer ; howeverWith sufficient knowledge and adequate training on how to educate patients about HPV vaccination, dental providers have the potential to lead HPV-OPC prevention . In factGlobally, HPV knowledge and awareness has been reported to be low among the dental student, oral health student, and oral health professional populations; however, the construct validity of these instruments is questionable as many were study-specific –21. LikeValidated instruments have been developed in the United States for HPV and HPV vaccine communication, advocacy, attitudes, and clinical practices among dental providers , 24–26. It is crucial that the next generation of Latin American dental professions have the tools needed to improve the health and well-being of their patients through increasing HPV vaccination coverage. This can be achieved through HPV-OPC patient education , HPV vacThe present study aimed to perform a cross-cultural adaptation of a multi-scale questionnaire evaluating Knowledge, Perceptions, and Clinical Practices (KPCP) regarding HPV and HPV-OPC, originally developed for English-speaking US dental and dental hygiene student populations, and evaluate the applicability and reliability of a Latin American Spanish translation . The priThe present study was conducted by researchers from the University of Utah School of Dentistry in Salt Lake City, Utah in the US and the Autonomous University of Baja California’s Faculty of Dentistry in Tijuana, Mexico. Huntsman Cancer Institute and University of Utah School of Medicine also supported this research.A previously developed questionnaire for English-speaking dental students was utilized for cross-cultural adaptation for Spanish-speaking dental students in Latin America. Using Beaton et al. 2000 as a guideline for cross cultural adaption, and described in Additional file The HPV-OPC Knowledge, Perceptions, and Clinical Practices (KPCP) Spanish Version (SV) multi-scale survey consisted of 147 items, of which 106 items were included in the final analysis. Items made up seven subscales: (1) HPV knowledge, (2) HPV-OPC knowledge, (3) HPV vaccine knowledge, (4) Barriers, (5) Clinical Procedures, (6) Scope of Practice, and (7) Curriculum Evaluation. The questionnaire also included demographic questions. The questions had a variety of response types, including true/false/don’t-know, multiple choice, check all that apply, yes/no, and Likert scale. While 30–40 respondents are often considered a sufficient sample size for cross-cultural adaptation studies –30, we eApproval from University of Utah’s and Institutional Review Board (IRB) was obtained prior to the present study. Throughout Latin America, email invitations were sent to eight universities to participate in this pretest of the cross-culturally adapted HPV questionnaire, from which two agreed to participate; one program was located in Colombia (Facultad de Odontología de la Universidad de Antioquia) and the other, in Mexico . The participating two schools are located in geographical regions where dental student’s HPV-OPC knowledge has not been evaluated before. Eligible participants were first year dental (D1) students as they perform oral health educational duties as part of their program requirements. Convenience sampling and study procedures were conducted by two of the study investigators  according to the IRB-approved study protocol in both sites to ensure consistency. First, study investigators gave a short oral presentation about the research project to D1 students. Students were then instructed on how to properly answer the questions and then completed the questionnaire at their own pace. The questionnaire was administered in pencil-and-paper format, and then study investigators manually entered the data into Vanderbilt University’s Research Electronic Data Capture (REDCap) program for data management. Participation was voluntary and completion of the questionnaire served as consent.Students took between 30 to 40 min to complete the questionnaire, after which, they participated in an in-person discussion for about 30 min. During the in-person discussion, issues related to the survey questions were reviewed to gain additional feedback. Participants also provided their overall impressions of the instrument, suggested changes, shared what they liked or disliked about the instrument, and provided feedback on the order of questions. D1 students were incentivized to participate in the study by holding three raffles per University with the opportunity to win clinical dental instruments. Raffles were done immediately after participants finished the questionnaire and discussion.Cronbach’s alpha was used to measure the internal consistency of the HPV-OPC-KPCP-SV instrument. The higher the Cronbach’s alpha, the more related the items of the instrument are to each other; a low Cronbach’s alpha may indicate that the items of the instrument are unrelated, a low number of questions are assessed, or that heterogeneous constructs are included. A Cronbach’s alpha threshold of 0.7 and higher was employed as suggested by George and Mallery (2003) . AnalyseN = 114 Latin American dental students participated in the present study: n = 86 attending Facultad de Odontología de la Universidad Autónoma de Baja California, Tijuana and n = 36 attending Facultad de Odontología de la Universidad de Antioquia in Colombia. The majority of participants were female (61%), Hispanic/Latino(a)/Spanish (91%), and had at least an undergraduate associate and/or bachelor’s degree prior to entering their dental program (100%). Table A total of Table “verdad” in true or false questions, to change it to “verdadero”, as the first word means truth and the latter means true. In Spanish speaking countries, “verdadero” is used for these types of questions. Also highlighted by several participants, important grammatical errors, such as missing accents, which can change the context of a question and can cause the participant to re-read every question, including missing backwards interrogation question marks (¿) applied to the beginning of questions.Qualitative comments from students (data not shown) were also concurrently reviewed. All of the students who participated in this study were in their first year of Dental School and it was a common statement that they had not yet taken the oral pathology class, were they would learn information related oral diseases. It was suggested that instead of using the word The average number of correctly answered HPV knowledge questions  and HPV vaccination knowledge questions  among all participants was low overall . As shown in Additional file The present study performed the initial steps of a cross-cultural adaptation of a previously validated English version HPV survey, and aimed to evaluate internal consistency of the HPV-OPC Knowledge, Perceptions, and Clinical Practices (KPCP) subscales of the Spanish Version (SV) in a sample of Latin American dental students. The two schools that participated in the SV pretest were both good candidates to test the HPV-OPC-KPCP-SV instrument and measure internal consistency, since their HPV-OPC knowledge had not been evaluated before. The main findings indicate high internal consistency of most subscales in the instrument. Culturally, the survey also demonstrated strong practical relevance for students in Latin American countries, as participants expressed interest in acquiring knowledge related to the topic through open-ended questions. Feedback received from students suggest some minor orthographical and grammatical corrections.In general, there was evidence of equivalence between the HPV-OPC-KPCP-SV and the English version.Compared to the alpha coefficients of the English version in our previous work , HPV-OPCPrevious researches in Latin America have evaluated different aspects related to HPV-OPC in dental students . Each reEven though the previous studies listed above were valuable in assessing dental students’ knowledge regarding HPV-OPC, they did not demonstrate any methodological steps needed to claim validity of their new scales. HPV-OPC-KPCP-SV is the first to describe their validation methodology. Besides assessing dental students’ knowledge, perception, and clinical practice, the current survey also assessed the HPV-OPC curriculum in dental schools. The study results reveal a great opportunity for curriculum evaluation and development among Latin American dental schools and can be used to further implement research in Latin American countries regarding HPV-OPC. By comprehensively evaluating dental students’ knowledge, perception, curriculum, and clinical practice, the barriers that prevent or limit the discussion of HPV-OPC and HPV vaccination with patients can be identified and mitigated. Knowing these barriers provides the foundation to propose a guide and/or intervention that would aim to increase HPV-OPC education among patients and increase HPV vaccination rates in Latin America.The HPV-OPC-KPCP-SV survey appears to have been successfully translated from English to Spanish. Moreover, the reliability and cross-cultural applicability is appropriate to be implemented in dental students from Latin America countries. However, in interpreting the results, it is important to take the study’s limitations into account. Despite the success of the pretesting of the HPV-OPC-KPCP-SV, the study was conducted in only two countries: Mexico and Colombia, thus findings may not generalize to other Latin American countries. Nevertheless, people from different cultural and demographic background were included in the present study and reliability was high suggesting that generalizability may be appropriate. Additional research is needed to support this claim.The present study administered one survey in a cross-sectional design meaning Cronbach’s alpha was the only feasible reliability measure that could be assessed. Due to the research team’s limited access to the dental students, funding, and logistical constraints, the students were only asked to complete the survey once and the present study could not measure test–retest reliability. Concurrent validity or discriminant reliability measures were not possible as the students were only asked to complete one survey. Floor and ceiling effects are also possible limitations in surveys of this type. Administering this survey to a wider range of other dental students and professionals, both in experience and knowledge, and at multiple time points would reduce these limitations.Religion and religiosity have been found to be important factors of HPV vaccination receipt, discussions, attitudes, and knowledge. HPV vaccination is often a health decision that is made on behalf of the patient by parents and/or caregivers, not considered required, and is ideally administered starting at age 9–11 years old, prior to sexual debut. HPV is often viewed as a sexually transmitted disease and that HPV vaccination is not needed for individuals who are in, or will only be in, monogamous relationships. Cronbach’s alpha can be considered as a characteristic of the sample and it is plausible that dimensionality of scales may vary by religion and/or religiosity and reported Cronbach’s alphas are under-estimated. Due to sample size, we were unable to effectively determine dimensionality of scales , although a cursory examination of the HPV knowledge subscale did indicate the scale may not be unidimensional   and thatAfter the survey was administered, there was an in-person group discussion. This may have limited the ability of participants to confidently express and share their feedback. Also, it is possible that the participants’ responses could have been influenced by social desirability and conformity bias. Since participants were relatively new to the dental school academic environment, they may have been knowingly or unknowingly responding to items while taking into consideration their peers or the researchers’ perceptions of their responses. This was mitigated by an anonymous comment field that allowed the participants, who may feel uncomfortable to discus in the in-person group discussion, opportunities to provide their feedback. However, these biases could still exist.We observed that participants answered an average (mean) of 44.76% (15.40 SD) of all HPV knowledge subscale questions correctly and 41.39% (18.18 SD) of HPV vaccination knowledge subscale questions correctly. Since the participants were first-year students, we expected knowledge scores to be low and that missingness would not be random as some students might feel uncomfortable answering some questions in this pretest due to the limited patient hours and the knowledge regarding cancer-related topics. Likewise, participants had not completed oral pathology curriculum prior to the pretest, meaning caution should be taken when interpreting the knowledge sections’ accuracy or internal consistency. This limitation of knowledge may also be the reason why the Curriculum Evaluation had a low alpha coefficient.Future research should focus on the evaluation of knowledge that senior dental students (D4 and D5) as well as dentists and dental hygienists have about HPV Knowledge, HPV-OPC Knowledge, HPV Vaccine Knowledge, Barriers, Clinical Procedures, Scope of Practice, and Curriculum Evaluation in order to identify and overcome real or perceived barriers. This could take the form of offering continuing education courses, workshops, or changes in dental school curriculums. Subsequent studies should focus on further validation of the HPV-OPC-KPCP-SV and include assessment of additional reliably measures.Aside from assessing the linguistic and cultural equivalency of the Spanish version of the survey, the present study found high internal consistencies for the majority of subscales examining HPV Knowledge, HPV-OPC Knowledge, HPV Vaccine Knowledge, Barriers, Clinical Procedures, and Scope of Practice.The process of a Spanish translation of the HPV-OPC-KPCP-SV survey demonstrated initial reliability and applicability for the assessment of the knowledge, perceptions, and clinical practices regarding HPV-OPC for dental students in Latin America. However, this is only a first step, the next step would be to do further reliability and validity assessments using a larger sample. The present study has implications for dental HPV curriculum development and may be used to assess needs for improving the quality of dental practice among our future dental health professionals in the area of HPV education, HPV vaccination, and detection of HPV-OPC cancers at early stages.Additional file 1: Survey items and flow chart document.Additional file 2: Subscales analyzed for reliability.Additional file 3: Complete survey.Additional file 4: Cross-cultural adaptation flow chart.Additional file 5: Anonymous participants responses.


Human papilloma virus (HPV) is a viral agent whose transmission occurs mainly by sexual means. It causes different pathological conditions in both males and females, ranging from benign pathologies up to cancers. The introduction of vaccination has certainly had a major impact in terms of reducing the incidence of both HPV infections and diseases but in the European Union and the European Economic Area (EU/EEA) there are still about more than 13,000 deaths due to cervical cancer each year. To date in Europe and in Italy there are three vaccines available . The vaccination campaign started irregularly in Europe and Italy in 2007, with pre-adolescent girls as the primary target. Later, other cohorts were introduced such as 12-year-old boys, additional cohorts of >25-year-old women, women who already underwent cervical surgery and other subjects entitled to free vaccination. The COVID-19 pandemic has strongly impacted on public health services, particularly on vaccinations that, especially during the first pandemic phase, have been often delayed and/or canceled. The most affected vaccinations by the pandemic have been the non-mandatory ones, particularly those addressing the adolescent and adult population, such as immunization against papillomavirus. To date the achievement of the coverage target set by the Italian National Immunization Plan (NIP) has not yet been achieved. The aim of this work is to summarize the current situation in Italy and to discuss the strategies that have been implemented to increase overall vaccination coverage rates. Human papilloma virus (HPV) is an extremely widespread viral agent throughout the world; it includes more than 200 distinct genotypes and its human-to-human transmission occurs mainly by sexual means . HPV cauWorldwide, approximately 70% of cervical cancers and HPV-associated precancerous lesions are caused by the serotypes 16 and 18. Considering that this type of cancer is currently the fourth leading cause of death in the female world population, HPV is responsible for an important burden of disease .,39.4,39]As regards females, the average HPV vaccination coverage is well below the optimal threshold provided by the 2017–2019 NIP (95% in the 12th year of life) . Only onThe full-cycle coverage for 11-year-old girls (2008 cohort in 2020) has shown a decrease compared to the full-cycle coverage for 11-year-olds of the previous years   4,39]..39].For males, the average vaccination coverage rate is very far from the objectives set by the 2017–2019 NIP (95% in 2019) . Only twThe data on vaccination coverage (full cycle) 2020, for both females and males, show a significant decrease compared to those referring to 2019 . The decTargeted interventions would be necessary in specific geographical contexts bearing in mind that HPV vaccination, even not mandatory, is an Essential Level of Assistance and as such should be actively and free offered .The COVID-19 pandemic, with the temporary forced block of many health activities including vaccinations and the fear of contagion in attending healthcare environments , has certainly had a significant impact on vaccination coverage rates. In Italy, vaccination against HPV is recommended but not mandatory: according to the data collected by the Ministry of Health through a questionnaire administered to Local Health Units, the age group 13 months–18 years has been the one with the greatest reduction in vaccinations during the first lockdown period in 2020, consequently, HPV turns out to be the one that has suffered the greatest decrease in coverage. Given the significant impact that the pandemic has had on public health, the Ministry of Health has outlined some specific strategies for the catch-up of vaccination activities such as the hypothesis of making telephone contacts with the families, the preparation of a list of cohorts of subjects not yet vaccinated, the preparation of information and awareness activities on the importance of vaccinations, the strengthening of communication campaigns and the expansion of vaccination services to reach the unvaccinated more easily. With regard to HPV vaccination, a particularly important task is carried out by the school and by the teachers, who during their daily contact with adolescents and their families can have on a central role in communicating and disseminating information about the importance of vaccinations .Other useful tools for the catch-up and the maintenance of coverage rates are the involvement of general practitioners, pediatricians, gynecologists and midwives. A relevant role could be played by the active call of both sexes, the maintenance of the right to free vaccination at least up to 18 years of age and the right acquired based on the cohort of birth, the inclusion of subjects at risk in vaccine offer and the application of the previously listed multi-cohort strategies ,31,41.The role played by social media should also be considered central, especially in light of the infodemic period we are going through. Even before the pandemic, the important role of social media was highlighted as adolescents were the primary target of the HPV vaccination campaign .A recent systematic review has identified the perceived low efficacy of the vaccine, possible side effects, lack of trust in health authorities and inadequate and partial information as the main causes of vaccine hesitancy regarding HPV vaccination in Europe .Catch-up programs are essential to try to reach the coverage target set by the 2017–2019 NIP. According to a study published at the end of 2019, in Europe some of the countries that recorded the best vaccination coverage (>75%) in the period 2010–2017 had implemented mass catch-up strategies in a limited period of time, while in countries with low or very low vaccination coverage this strategy has not been implemented . The tarThe role of general practitioners and pediatricians is very important as they are the health professionals who first come into contact with adolescents and their families. Updated and continuous training on HPV vaccination are essential in order to keep these health professionals focused on this issue, so that they are constantly encouraged to talk and discuss with adolescents and their parents ,45.Vaccination coverage for HPV in Italy, according to updated data as of 31 December 2020, does not reach the 95% target set by the 2017–2019 NIP, both in male and female cohorts. Although over the years and until 2019 there has been an increase in coverage for female cohorts thanks to specific catch-up interventions and vaccination has also been recommended and made free for male cohorts, this has not been sufficient to guarantee the achievement of the target threshold. The use of additional cohorts and the implementation of multi-cohort strategies has certainly helped to increase the rate of citizens protected against HPV, but to date, not enough has been done to ensure effective coverage for the entire at-risk population. It is vital to continue pushing to ensure a gender-neutral approach to HPV vaccination. Providing adequate protection for all adolescents can lead to significant health gains for the general population, greater protection for the male population, especially for men who have sex with men who do not benefit from vaccination of the female population , and finBesides, a more homogeneous approach, both at European and national level, could help to reach the final target of 90% for 15-year-old girls set by the WHO for 2030. In European countries, in fact, a great variation of the HPV vaccination coverage is registered , which iLast data have shown the terrible impact that COVID-19 has had on HPV vaccinal coverage rate. In this historical moment in which vaccines are a topic of daily discussion, it is of fundamental importance to guarantee and disseminate information that is as clear, simple and reliable as possible and that is aimed at both adolescents and parents. Furthermore, given that when we talk about HPV vaccination it is inevitable to also talk about sexual health, there is the opportunity to deliver scientifically correct information on sexually transmitted diseases and sexual education in general.It is therefore essential, especially after the impact of COVID-19 pandemic, to try to implement even more expanded catch-up interventions and to continue with vaccination programs involving more and more professionals close to adolescents and to their families such as general practitioners, pediatricians, gynecologists, midwives, and teachers.


Patients with multiple HPV infection displayed persistent and longer duration of the HPV infection compared to patients with single HPV infection. Multiple HPV infections have distinct clinicopathologic characteristics. Since it is associated with persistent HPV infection, HSIL, and different HR-HPV strains in contrast to single HPV infection, the presence of multiple HPV infection should be reported; close follow up is warranted.Infections with multiple human papilloma virus (HPV) types have been reported, but their role in cervical carcinogenesis has not been fully elucidated. In this study, 236 cases with multiple HPV infection were examined and compared to 180 cases with single HPV infection. HPV genotyping was performed with cervico-vaginal swab specimens using multiplex  polymerase chain reaction (PCR). In multiple HPV infection, the most prevalent HPV genotype was HPV 53, followed by HPV 16, 58, 52, and 68. HPV 33, 35, 39, 51, 52, 53, 58, and 68 were high-risk-HPV (HR-HPV) genotypes that were more frequently detected in multiple HPV infection compared to that in single HPV infection. The association between multiple HPV infection and high-grade SIL (HSIL) was significantly stronger compared to that of single HPV infection and HSIL ( Human papillomavirus (HPV) infection is the major cause of cervical cancers and their precursor lesions ,2. Thus Infection with multiple HPV types is reported in 20–45% of infected women ,16,17, dHPV vaccines are a breakthrough in the prevention of major cervical cancers and greatly reduce cervical cancer rates. At present, three types of commercial vaccines are available: bivalent , quadrivalent , and nine-valent  vaccines ,26. It iAmong 1967 cases wherein HPV genotyping tests were performed, we retrospectively found 236 consecutive cases  with confirmed multiple HPV infection between 2017 and 2019 . For theLiquid-based Papanicolaou tests  were performed for cytological evaluation. Two experienced gynecologic pathologists (JYJ and NJP) at Kyungpook National University Chilgok Hospital interpreted the cytologic findings, as per the 2001 Bethesda System for cervicovaginal cytology reporting.HPV genotyping was performed with cervicovaginal swab specimens using the Anyplex II HPV28 assay kit . DNA extraction was carried out following the manufacturer’s instructions. Briefly, 5 μL of DNA was used in each of two 20 μL reactions with primer set A or B. In the assay, HPV-specific dual priming oligonucleotides were used for multiplex  polymerase chain reaction (PCR). A total of 28 HPV types were tested to simultaneously detect 18 HR types  and 8 low-risk types . Persistent HPV infection was defined as the detection of any HPV genotype in two consecutive genotyping tests (first test and a follow-up test) . The folFrom a total of 416 cases, comprising 236 multiple HPV infections and 180 single HPV infections detected by HPV genotyping tests, 264 cases with abnormal cervical screening tests (162 multiple and 102 single HPV infection cases) underwent colposcopy-guided cervical biopsy . Cervicap < 0.05. For multiple comparisons, an adjusted p-value was applied using the Bonferroni correction. All statistical analyses were conducted using IBM SPSS Statistics v.23 .Relationships between clinicopathologic parameters were evaluated using the chi-square test for categorical parameters and Fisher’s exact test for parameters with an expected frequency of <5. We used the Kaplan–Meier method to evaluate the duration of HPV infection. Statistical differences in the duration of the infection were determined using the log-rank test. Differences were considered significant at p < 0.001), 35 (p < 0.001), 39 (p < 0.001), 51 (p < 0.001), 52 (p = 0.001), 53 (p < 0.001), 58 (p < 0.001), and 68 (p < 0.001) were more frequently detected in multiple HPV infection than in single HPV infection. Detailed information on HPV infection status is presented in The average age of the patients was 56.3 years in multiple HPV infection and 53.8 years in single HPV infection. HPV 53 (27.5%) was the most prevalent genotype in multiple HPV infection, followed by HPV 16 (21.8%), 58 (21.2%), 52 (20.8%), and 68 (20.8%). HPV 16 (15.6%) was the most prevalent genotype in single HPV infection, followed by HPV 56 (11.7%), 53 (8.3%), 52 (7.8%), and 68 (7.2%). Among HR-HPV, HPV 33 (p = 0.002). LSIL was also more frequently found in multiple HPV infection  compared to that in single HPV infection , although it was not statistically significant. There was no major difference in the proportion of SQCC or adenocarcinoma (ADC) between the single  and multiple HPV infection groups . SIL was found to be more frequently negative in single HPV infection group  compared to that in the multiple HPV infection group . Most of the HSILs, SQCCs, or ADCs were associated with HR-HPV infection . Furthermore, we investigated if there was any correlation between multiple HPV infection and the duration of HPV infection. The median follow-up period was 26 months. The infection lasted significantly longer in patients with multiple HPV strains compared to patients infected by a single HPV strain  a diverse distribution of HPV genotypes occurs in multiple HPV infections in contrast to single HPV infections, (2) multiple HPV infections are more associated with HSIL and the duration of their infections is longer than that of single HPV infections, and (3) various HR-HPV strains contribute to HSIL or SQCC in addition to HPV 16.The role of HPV infection in cervical carcinogenesis has been well-established . Althougp < 0.001), 35 (p < 0.001), 39 (p < 0.001), 51 (p < 0.001), 52 (p = 0.001), 53 (p < 0.001), 58 (p < 0.001), 59 (p = 0.009), 66 (p = 0.003), and 68 (p < 0.001) were associated more frequently with multiple than single HPV infection. As most of these genotypes were identified in a similar range of 10–20% without specific patterns of co-infection or phylogenetically related groups, it is difficult to determine whether this is the result of the synergistic or competitive effects of different HPV genotypes or the mere sum of incidental cumulative HPV co-infection.The prevalence and distribution of multiple and single HPV infections vary widely worldwide and are affected by diverse factors, including age, socioeconomic status, immune status, and vaccination status ,32,33. Ip = 0.002). LSIL was also more frequently detected in multiple HPV infection , although statistically not significant. A study by Schimitt et al. also reported that multiple HPV types were prevalent in LSIL and HSIL [In our study, HSIL 76/236, 33.2%) was significantly more frequent in multiple HPV infection than in single HPV infection (33/180, 18.3%, , 33.2% wand HSIL ; howeverand HSIL ,12,37, wAlthough this study shows that HPV16 is the major cause of HSIL and SQCC, patients with multiple HPV infection also exhibited comparable amounts of other HR-HPV genotypes, including HPV 33, 35, 39, 53, 58, 66, and 68. Previous studies have demonstrated that the diverse distribution of prevalent genotypes and co-infection patterns of HR-HPVs in HSIL and SQCC depends on demographic and socioeconomic factors ,39,40. BAnother noteworthy finding of this study is that patients with multiple HPV genotypes had persistent HPV infection during follow-up periods compared to patients with single HPV infection. Some previous studies have pointed to the possible association between multiple HPV genotypes and persistence of infection ,20,23,41This study has some limitations. First, the frequency of multiple HPV infections and the distribution pattern of HPV genotypes being influenced by the detection method. AnyplexTM II HPV28 assay is a real-time PCR-based HPV detection method, which is more sensitive compared to the HPV DNA chip assay or the HPV hybrid capture DNA test . TherefoIn conclusion, we demonstrated distinct clinicopathologic characteristics of multiple HPV infection in comparison to single HPV infection regarding the progression of the infection to SIL and the duration of the infection. Multiple HPV infections were also enriched with diverse HPV genotypes in contrast to single HPV infections. As understanding the distribution pattern and infection mechanism of multiple HPV genotypes is critical for establishing the prevention plans and vaccination programs of HPV, subsequent studies are warranted to further validate the results of this study.