In conclusion, our study revealed the unique biomolecular signature of mitophagy in HPV-associated HNSCC, which may contribute to the development of precise treatment regimens for HPV-associated HNSCC patients.Head and neck squamous cell carcinoma (HNSCC) is a common tumour type in otorhinolaryngology, and its occurrence is related to long-term exposure to tobacco and alcohol. Recently, HPV infection has become an increasingly important contributor to HNSCC, and HPV-associated HNSCC has a different clinical course and better prognosis than non-HPV-associated HNSCC. However, the exact molecular mechanism of HNSCC is unclear. Here, we obtained data from The Cancer Genome Atlas (TCGA) and gene expression omnibus (GEO) to analyse the mitophagy process and related influencing factors of HPV-associated HNSCC via the integration of bioinformatics analysis and experimental validation. We found that in HPV-associated HNSCC, process of mitophagy affects tumour development, immune cell infiltration and prognosis. In the mitophagy process of HPV-related HNSCC: NOS2, IL17REL, TMSB15A, TUBB4A and other hub genes showed significantly higher expression levels than in non-HPV-related HNSCC. Furthermore, this was also confirmed by quantitative real-time PCR (qRT‒PCR), which was used to detect the expression of differentially expressed genes in HNSCC tissues. Furthermore, we found that the unique immunological characteristics by expressing CD8 HPV is a circular double-stranded DNA, and more than 200 genotypes have been discovered, all of which are divided into high- and low-risk HPV2. It is currently believed that the occurrence of HNSCC is mainly related to high-risk HPV infection, especially HPV-16. Persistent viral infection affects the cell cycle and causes carcinoma through the expression of oncoproteins3. The survival of patients with HPV-positive head and neck cancer is significantly higher than that of patients with HPV-negative cancer5, and different studies have shown that change is associated with multiple factors9.Head and neck squamous cell carcinoma (HNSCC) is generally associated with tobacco consumption, alcohol abuse or both. However, it has recently been increasingly attributed to infection with human papillomavirus (HPV)10. The mechanism of mitophagy in carcinoma cells is significantly more complex than that in normal cells. In colorectal cancer studies, inhibition of mitophagy enhanced the sensitivity of tumour cells to chemotherapeutic drugs11. However, inhibition of mitophagy may also contribute to bone metastasis of breast cancer12. Maybe, mitophagy plays different roles in the occurrence and development of tumours, and the relationship between mitophagy and HPV-associated HNSCC has rarely been studied; the main research is on the effect of the motiphagy of combination of antitumour drugs on HNSCC14. We speculate that infection with high-risk HPV alters the process of mitophagy, and the overall prognosis of HPV-associated HNSCC is better than that of non-HPV-associated HNSCC.Mitochondria are important organelles in eukaryotic organisms. Normal mitochondria play an important role in ATP synthesis, calcium buffering, immune monitoring and other processes. When mitochondria are damaged, they can be removed by mitophagy to maintain mitochondrial quality control and mitochondrial quantityTo investigate the relationship between mitophagy and HPV-related HNSCC, we determined the differentially expressed genes related to mitophagy and the effects of the mitophagy score on the prognosis and immune cell infiltration by obtaining HPV-related and non-HPV-related HNSCC human specimen datasets from the Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases. To obtain the relationship between mitophagy and HPV-related HNSCC, differential gene screening was performed on HPV-related and non-HPV-related HNSCC samples obtained from TCGA database, and confirmed the differential genes by basic experiment. Then autophagy-related differential pathways were obtained according to differential gene analysis. Then, we combined TCGA and GEO database HPV-related HNSCC samples to construct the mitophagy score model and analyse the effect on immune cell infiltration and prognosis. We found differential gene expression and molecular pathways between HPV-associated and non-HPV-associated HNSCC samples during mitophagy. Furthermore, the infiltration of CD4-positive memory T cells and M2 macrophages was reduced in the group with a higher mitophagy score, and the mitophagy score was more accurate in predicting long-term prognosis.15, and TCGA-HNSCC HPV-related phenotype information was obtained by Genomic Data Commons (GDC) software. HPV-negative and HPV-positive tissues were distinguished according to p16 test results. After the invalid samples were removed, the remaining samples were classified as 72 HPV-negative tissues and 31 HPV-positive tissues for gene expression analysis. Gene set enrichment analysis (GSEC) was used to identify the mitophagy regulatory gene set GO: 1901524, and 12 functional mitophagy regulatory genes were selected for expression analysis in HNSCC data and to build a mitophagy score. The HNSCC-related dataset GSE6585816 was obtained from the GEO database by the GEOquery package17, and 196 HPV-negative controls with p16-negative samples and 60 HPV-positive samples with p16-positive samples were identified; all were human samples.HNSCC gene expression profile data were obtained from the TCGA database using the R package TCGA biolinks package (version 2.22.4)18 was used for differential analysis based on the new grouping, and 76 upregulated differentially expressed genes(DEGs) and 321 downregulated DEGs were finally obtained. We set the genes with logFC > 1 and adjP value < 0.05 as upregulated differentially expressed genes and the genes with logFC < − 1 and adjP value < 0.05 as downregulated differentially expressed genes. Mitophagy regulation and DEGs were displayed in heatmaps by the pheatmap package.The tumour samples from the HPV-negative group and HPV-positive group in the TCGA database were analysed by the R package rgl package (version 0.107.10) for dimension reduction. Then, the R package ggplot2 package (version 3.3.5) was used to determine the expression differences in mitophagy-regulating genes. Tissue samples from the TCGA database were redivided into 70 A and 33 B types according to mitophagy regulatory genes. The R package DESeq2(version 1.34.0)We selected 6 cases of human HPV-negative HNSCC tissue and 6 cases of human normal tonsil tissue  for random combination. Each case of HNSCC tissue corresponding to a case of normal tonsil tissue was divided into 6 groups for control experiment analysis of some differentially expressed genes in mitophagy. The same experimental procedure was performed for each group: the rice grain-sized tissues were removed from liquid nitrogen and ground into powder by a grinder. RNA extraction was performed using TRIzol , and the RNA was measured with a 2000 spectrophotometer. The concentration and purity of the extracted RNA were determined according to the instructions of the HiScript III RT SuperMix for qPCR kit. The extracted RNA was reverse transcribed into cDNA, and 2 × ChamQ Universal SYBR qPCR Master Mix reagent was used for PCR on the StepOnePlus PCR system . The following steps were performed under the following conditions: first, predenaturation at 95 °C for 30 s, then 95 °C for 10 s, 60 °C for 30 s, and 95 °C for 15 s, 60 °C for 60 s, 95 °C for 15 s, for a total of 40 cycles. The primer sequences for PCR amplification were as follows: IL17REL, forward: 5′-GCTGTGGGACACGGTCTACTA-3′, reverse:5′-GCTGGATTGCTGCAGCTTACG-3′; NOS2, forward: 5′-CCTGGCAAGCCCAAGGTCTA-3′, reverse: 5′-CGCACATCCCCGCAAACATAG-3′; TMSB15A, forward:5′-CCTCCCAACAGCAGATTTCGAC-3′, reverse: 5′-TCCGAAGACGCCTAAAATCTCTACA-3′; TUBB4A, forward:5′-TCGATGCCAAGAACATGATGGC-3′, reverse: 5′-TGTTCTTGCTCTGCACGCTCA-3′; GAPDH, forward: 5′-GCACCGTCAAGGCTGAGAAC-3′, reverse:5′-TGGTGAAGACGCCCAGTGGA-3′. The relative mRNA expression level was calculated by the 2–11 Ct method and standardized to GAPDH.19 were used for Gene Ontology(GO) annotation analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG)22 pathway enrichment analysis of differentially expressed genes, and a critical value of FDR less than 0.05 was considered to be statistically significant. To differentiate the biological processes of the two groups, according to the gene expression profile database of TCGA HNSCC samples, gene set enrichment analysis (GSEO)23 was used for gene set enrichment analysis. "c2.cp.kegg.v6.2. -symbols" gene set was obtained from the MSigDB database24 for GSEA. A false discovery rate (FDR) < 0.25 was considered to be significantly enriched. Using the R package GSVA 25, single-sample gene set enrichment analysis (ssGSEA) was used to calculate the scores of related pathways based on the gene expression matrix of each sample, and differences in enrichment functions (or pathways) were screened by the limma package(version 3.50.1)26.Cluster Profiler packages (version 4.2.2)27 was used to construct the scoring model with the differential genes obtained by typing, and the parameters were set as follows: seed (2), family = "binomial". Based on the screened TCGA-HNSCC and GSE65858 datasets, univariate Cox regression analysis and LASSO regression analysis were used to further screen the prognostic genes and then establish the prognostic model. First, we used univariate Cox regression analysis to calculate the association between the expression of each differential gene and OS  and retained the genes with a P value < 0.05. Then, the combined dataset was randomly divided into a training set and validation set, and the Lasso algorithm was used to eliminate multicollinearity in the training set and select the meaningful variables in univariate Cox regression analysis. Finally, the risk score formula was calculated by considering optimized gene expression and the correlation estimation Cox regression coefficient: 28 to calculate area under the ROC curve values to measure prognosis or prediction accuracy. Validation sets were tested using scoring formulas to assess survival prediction and prediction accuracy.The glmnet packet (version 4.1.3)P < 0.05, and obtained the immune cell infiltration matrix. A histogram was drawn using the R language ggplot2 package to show the distribution of 22 immune cell infiltrates in each sample.We uploaded the expression matrix data of the training set and validation set to CIBERSORTx, combined with the LM22 characteristic gene matrix, filtered the output samples with P values were bilateral, and P < 0.05 was considered statistically significant.To compare two sets of continuous variables, the independent Student’s t test was used to estimate the statistical significance of normally distributed variables, and the Mann‒Whitney U test (or Wilcoxon rank sum test) was used to analyse the differences between nonnormally distributed variables. The receiver operating characteristic curve was plotted using the R time ROC package, and the area under the curve (AUC) was calculated to assess the accuracy of the risk score in estimating prognosis. All statistical The study was performed in accordance with the ethical standards of the Declaration of Helsinki (1964) and its subsequent amendments. All experimental protocols were approved by the Medical Ethics Committee of the First Affiliated Hospital of Shandong First Medical University & Shandong Provincial Qianfoshan Hospital. All participants provided written informed consent before participating, all minors have the consent of their guardians and the guardian signed the informed consent.Seventy-two HPV-negative tissue samples and 31 HPV-positive tissue samples obtained from the TCGA-HNSCC dataset were subjected to principal component analysis (PCA) dimensionality reduction. The results showed that HPV-grouped samples could be clearly distinguished  to transform the expression matrix of genes between different samples into the expression matrix of genes between different samples to evaluate whether different metabolic pathways were enriched between different samples. Then, the pheatmap package was used to visualize the results Fig. A–B. The P value < 0.1 were retained. Then, the combined dataset was randomly divided into a training set and a validation set. The Lasso algorithm was used to eliminate multicollinearity in the training set and select meaningful variables in univariate Cox regression analysis.Finally, the risk score formula was calculated by considering optimized gene expression and correlation estimation Cox. According to the given score, the patients in the training set and validation set were divided into a high-score group and a group, respectively.To increase the reliability of the mitophagy regulatory scoring model, 31 HPV-positive TCGA-HNSCC data were combined with 60 HPV-positive GSE data, and the SVA package was used for batch removal. The PCA dimension reduction cluster diagram before and after batch removal is shown Fig. A–B. The Kaplan‒Meier analysis and logarithmic rank test were performed using the survival package, and the results showed that the overall survival rate of the high-rated and low-rated samples in the training concentration could be well distinguished Fig. C, and thTo analyse the difference between the mitophagy regulation score model and the immune infiltration degree in the training set and the validation set, we calculated the infiltration degree of 22 immune cells in the training set and validation set. After removing the cells with total immune abundance values of 0, we analyse and include 15 kinds of immune cells, they were B cells naive, B cells memory, Plasma cells, T cells CD8, T cells CD4 memory resting, T cells follicular helper, T cells regulatory (Tregs), NK cells resting, NK cells activated, Monocytes, Macrophages M0, Macrophages M2, Dendritic cells activated, Mast cells resting, Mast cells activated  in cells or mitochondria, increased apoptosis, or enhanced mitophagy. However, this study targeted all HNSCCs and did not distinguish HPV-related HNSCC from non-HPV-related HNSCC. Therefore, whether this study on mitophagy enhancement of HNSCC induced by tumour drugs can be applied to HPV-related HNSCC remains to be verified. Our study identified DEGs related to mitophagy in HPV-associated HNSCC. How these genes affect mitophagy and how they further affect the development of HPV-related HNSCC requires further research. Moreover, through the construction of a mitophagy score model, we characterized the infiltration of immune cells in HPV-related HNSCC, which initially showed a low level of CD4-positive memory T cells and M2 macrophages in the low-score group. We believe that this is meaningful and may aid in in the future development of immunotherapy against HPV-related HNSCC. At present, there are very few studies on mitophagy in HPV-related HNSCC, so the directions for future research are unclear. We hope that our study can provide aid in future HPV-related HNSCC research.Related studies have shown that combined melatonin and rapamycin treatment of HNSCC can induce changes in mitochondrial functionIn general, we investigated the link between mitophagy and HPV-related HNSCC by analysing DEGs. We believe that mitophagy can affect the prognosis of HPV-related HNSCC patients and propose that the high level of HPV expression may provide guidance for the development of precision medicine for HPV-related cancer patients.Supplementary Information.


Oral lichen planus (OLP) is a chronic inflammatory disorder with cell‐induced immunopathological responses and is considered a potential malignancy disorder in the oral cavity. Due to the high prevalence of OLP as well as the potential for malignancy, human papillomaviruses (HPVs) may play an important role in it. Although previous studies have explored the possible relationship between HPV and OLP, the findings have been conflicting and nonconclusive. This study aims to review the studies that investigated HPV‐16 and HPV‐18 in OLP.The research protocol followed the Preferred Reporting Items for Systematic Reviews (PRISMA2020) checklist. The online databases Pubmed, Scopus, Embase, Google Scholar, and Cochrane were searched using the following individual keywords: “OLP” OR “Oral Lichen Planus” OR “HPV” OR “Human Papillomavirus.” The search strategy resulted in the selection of 80 articles. The articles were evaluated, and after duplication removal, 53 abstracts were reviewed, resulting in the selection of 25 studies according to inclusion and exclusion criteria. The risk of bias assessment was done by using the Modified Newcastle–Ottawa quality assessment scale. The overall prevalence of HPV in OLP lesions varied from 2.7% to 70%, depending on the type of diagnostic method used.Despite the studies conducted on the relationship between OLP and HPV infection, there is still no conclusive evidence that HPV can play a role in the etiopathogenesis of OLP, either in clinical manifestations or in the malignant transformation of lesions. The common term (OPMD) of potentially malignant oral disorders is suggested for oral premalignancy, including premalignant lesions  and premalignant conditions . All oral mucosa lesions with the risk of malignant changes are included under the term OPMD.OLP mostly occurs in middle‐aged adults and may occur in any area in the oral cavity, but the buccal mucosa, tongue, and gingiva are the most common areas of involvement.The factors that initiate OLP are unknown, but its mechanism includes several steps, including an initiating factor or a phenomenon (exogenous/endogenous antigenic stimulation), local release of regulatory cytokines, upregulation in vascular adhesion molecules, accumulation, and retention of T lymphocytes and cytotoxicity in basal keratinocytes regulated by T lymphocytes.In 1997, the World Health Organization classified OLP as a potentially malignant disorder with a nonspecific risk of malignant transformation and suggested that patients should be closely monitored. In a meta‐analysis conducted by Syrjänen, a strong relationship between HPV and oral squamous cell carcinoma and other premalignant conditions has been suggested. HPV16 levels were higher in OLP patients compared to healthy controls. These findings suggest that HPV may play a role in the etiology and malignant changes of lichen planus.HPV is a member of the Papillomavirus family, with a diameter of 50–500 nm and no capsular coating. Different types of HPV are recognized based on the degree of nucleic acid sequence homology. Different types of HPV, including HPV‐16, HPV‐18, and HPV‐31, have been associated with certain types of malignant and premalignant lesions.The common role of HPV has been reported in OLP and OSCC, but there are wide differences in its prevalence depending on different geographic populations. In addition, data from the International Biological Study of Cervical Cancer has shown that phylogenetic clusters of HPV16 are in direct association with specific racial groups and geographic locations. Most HPV infections are asymptomatic, although the lesions can occur at any time after infection. The period of HPV infection depends on its subtype and related factors such as smoking, nutritional status, immune status, and hormonal effects.Due to the high prevalence of OLP and the painful and irritating nature of erosion atrophic types of this disease, as well as the potential of malignancy and the formation of oral squamous cell carcinoma in some types of OLP, HPV‐16 and HPV‐18 may play an important role in it. This review study aims to review the studies that investigated HPV‐16/‐18 in OLP.22.1Searching in Electronic databases, Ovid, Embase, Pubmed, Google Scholar, Scopus, and Cochrane were searched from 1960 to March 2022 with the keywords “OLP” OR “Oral Lichen Planus” AND “HPV” OR “Human Papillomavirus”. In addition, all the references of these articles were checked manually to identify other relevant articles that were not identified in the initial search. Ethics approval and patient informed consent were not required as this study is a review based on published literature.2.2The studies selected in this study included case–control studies and clinical trials with the topic of examining the HPV‐16/‐18 virus in tissue samples of oral mucosa and saliva of people with OLP. Only studies published in English were included in this study. Studies were excluded from our study due to the unavailability of the full text, irrelevant results, lack of WHO criteria to approve the OLP, studies without a defined control group, and systematic review articles.2.3At this stage, both researchers (FA and KHM) individually examined the obtained articles in three steps. In the first step, using Endnote 20 software and then manual review, duplicate sources were removed. In the second step, articles were screened by examining their titles and abstracts to exclude studies not related to the present study. In the third full‐text step, the articles were studied in full, and their eligibility was checked based on the determined entry and exclusion criteria.A data extraction sheet was developed by using Microsoft Excel 2013 software. Two authors have independently extracted study characteristics from the included article including: author name, publication year, aim of study, study design, sample size, detection methods of HPV, and subtype of OLP if applicable.3n = 55). At this stage, by screening studies based on title and abstract, two studies were excluded due to not being in English (n = 53).A total of 80 articles were obtained from five electronic databases: Pubmed, Scopus, Embase, Google Scholar, and Cochrane, and by an additional manual search of the sources of the selected articles, an article was added in total. After removing duplicate articles, 55 articles entered the next stage of Screening (n = 25). Of a total of 25 studies, 23 were case–control studies and 2 were cohort studies, all of which were performed with the aim to investigate the prevalence of HPV in OLP.From the remaining 53 studies that were examined to verify eligibility for full‐text reading, 16 studies were excluded . From the remaining 37 studies, 12 studies were excluded due to the lack of a control group, the lack of WHO criteria to confirm OLP, and the indirect effect of HPV on OLP  articles with PCR method, 4 (16%) articles with immunohistochemistry (IHC) method, 3 (12%) articles with in situ hybridization (ISH) method, 3 (12%) articles with both PCR and Southern blot methods, and 1 (4%) article with Luminex‐based method had identified the HPV virus. In terms of the type of samples, the results showed that 56% of the studies were conducted on fresh biopsy of the oral mucosa and 40% of the studies on formalin‐fixed paraffin‐embedded (FFPE) blocks, and 4% were conducted with the brushing method of oral mucosa cells.3.1Risk of bias assessment was done by the two authors using Modified Newcastle–Ottawa quality assessment scale4OLP is a chronic inflammatory disorder with periods of relapse and remission and with the potential of malignant transformation (MT).For the first time in the 1987s, the relationship between HPV and OLP was proposed.HPV is the second most common sexually transmitted infection, of which more than 200 types are known to date. This virus can cause cancer of the vagina, penis, anus, and some cancers of the head and neck, especially the oropharynx. Infection with HPV can cause various skin manifestations, including (1) common warts, (2) stringy warts, and (3) warts on palms and soles. HPV16 accounts for approximately 95% of HPV‐positive oropharyngeal carcinomas.Among the studies we examined in this review, the overall prevalence of HPV in OLP lesions has been reported from 2.7%To better understand the process of HPV carcinogenesis, we must know that HPV shows a preference for squamous epithelium, and the abraded and scraped epithelium transmits the virus. To develop a fixed infection with HPV, the virions must reach the basal lamina in the squamous epithelium through the microlesions in the epithelium. The main capsid protein L1 and the partial capsid protein L2 deliver the virus genome to the nucleus of epithelial cells.E6 and E7 proteins are oncogenic, which by affecting p53 and pRb, respectively, gradually help to progress toward malignancy by inducing genomic instabilities.Many studies have shown that changes in p53 expression are essential for carcinogenesis and deregulation of p53 is considered a valuable biomarker in the malignant changes of premalignant oral lesions.Retinoblastoma protein (pRb) plays the role of the cell proliferation controller by stopping the cell cycle in the G1 phase and maintaining chromosomal stability, which is done by stopping E2F transcription factors.However, as some studies show, the results regarding the role of HPV in the pathogenesis of OLP and its malignant changes have been contradictory.A wide variety of assay methods have been used to detect HPV in OLPIHC is widely used to detect abnormal cells and identify their origin based on the binding of specific antibodies to antigens in the target tissue. The use of IHC in the diagnosis of cervical HPV infections through the detection of p16 expression is considered to be a highly sensitive method.Despite extensive studies regarding the role of HPV in oral cavity lesions, there are still no specific clinical guidelines for testing for HPV infection in oral lesions. In terms of the type of diagnostic method, according to Table Regardless of the numerous articles that have investigated the relationship between HPV and OPMD, there are few articles related to the mechanism of interaction between HPV and OPMD. To date, two hypotheses have been proposed regarding the cause of the susceptibility of potentially malignant oral lesions to HPV infection. The first theory is that the presence of an ulcer in the mucosa can make the oral cavity more susceptible to HPV, and this is why EA‐OLP and inhomogeneous leukoplakia are more associated with HPV.The findings of this review study show that the prevalence of oral HPV infection is higher in men than in women. The rate of orogenital HPV transmission from women to men has been reported to be higher than other methods. A reasonable theory is that the risk of oral HPV infection in men is higher among heterosexuals than among homosexuals, and a weaker immune response against HPV occurs in men.The prevalence of HPV in OLP varies based on different parameters such as geographical differences in the population, HPV genotype, clinical type of OLP, and HPV identification method. There are considerable differences in the geographical distribution of HPV in OLP in different regions of the world. Among the studies examined in this review, only two studies were conducted with the aim of investigating the prevalence of HPV in OLP patients, considering the geographical distribution. In the first study, the authors stated that the prevalence of HPV in the Thai population with OLP is low.51.The most common HPV genotypes reported in OLP are genotypes 16 and 18.2.In terms of virus detection methods, PCR and IHC methods have been widely used in the articles, but few studies are using the ISH method.3.In terms of the type of examined samples, it is recommended that future studies be performed using fresh biopsy from the oral mucosa.4.In none of the studies examined in this review, the prevalence of HPV virus in OLP, before and after treatment with topical steroids, has not been investigated.5.The prevalence of HPV in OLP has not been widely discussed in terms of the clinical type of OLP lesions, and further studies are recommended to investigate the relationship between this virus and the clinical types of OLP.The conclusion of this review study is as follows:Despite the studies conducted on the relationship between OLP and HPV infection, there is still no conclusive evidence that HPV can play a role in the etiopathogenesis of OLP, either in clinical manifestations or in the malignant transformation of lesions. The association between HPV infection and OLP should be interpreted with caution because it is not a constant phenomenon and could be just an incidental finding. More prospective cohort studies are needed to demonstrate the potential role of HPV infection in the incidence of OLP.Farzaneh Agha‐Hosseini: Conceptualization; investigation; project administration; supervision. Kimia Hafezi Motlagh: Conceptualization; data curation; formal analysis; investigation; methodology; writing—original draft; writing—review and editing.The authors declare no conflict of interest.Supporting information.Click here for additional data file.


Head and neck cancers of the oropharyngeal subsite can be driven by the human papillomavirus (HPV). In countries such as the United States, the incidence of HPV-associated oropharyngeal cancer has exceeded that of HPV-associated cervical cancer. HPV vaccination is currently the main preventative approach for HPV-associated oropharyngeal cancer. Globally, both the epidemiology of HPV-associated oropharyngeal cancer and HPV vaccine policy vary. This paper aims to describe regional variations in HPV-associated oropharyngeal cancer, variations in gender-neutral vaccine policy, and future areas of policy-relevant research.Global trends in human papillomavirus (HPV)-associated head and neck cancers (HNC), specifically in the oropharynx subsite, have been dynamically changing, leading to new staging and treatment paradigms. Epidemiologic studies have noted regional variations in HPV-associated oropharyngeal squamous cell carcinoma (OPSCC). While HPV vaccination remains the main preventative approach, vaccination policy in relation to gender neutrality is heterogeneous and particularly sparse in low- and middle-income countries, where the burden of global cancer cases and HPV-associated HNC are not well-characterized in certain regions. This review summarizes the existing literature on regional variations of HPV-associated OPSCC and gender-neutral vaccine policies. Based on available data, the incidence of HPV-associated OPSCC is highest in North America, Europe, and Oceania. As of 2022, 122 of 195 (63%) World Health Organization (WHO) member states had incorporated HPV vaccinations nationally; of these, 41 of 122 (34%) member states have introduced gender-neutral vaccine coverage. Future research is needed to describe continued evolving trends in HPV-associated OPSCC, understand underlying risk factors leading to regional variation in disease, and implement gender-neutral policy more broadly. The number of cases of human papillomavirus (HPV)-associated oropharyngeal cancers has surpassed that of cervical cancer in high-income countries such as the United States . Across With the growing epidemic of HPV-associated OPSCC globally, there is an opportunity to expand the current coverage of gender-neutral HPV vaccinations. HPV vaccination is the best preventative approach but requires strategic implementation prior to sexual debut and exposure to sexually transmitted HPV to be most effective . StudiesIn this review, we examine the literature to characterize the epidemiology of HPV-associated OPSCC by region and compare the current policies for gender-neutral HPV vaccination. Characterizing gaps in both epidemiologic data and policies is needed to influence future areas of research and tailor public health interventions.The incidence of HPV-associated OPSCC varies both regionally and between sexes . Across The North American region has the highest incidences of HPV-associated OPSCC in the world. The ASIR is estimated to be 3.41 per 100,000 in males and 0.71 in females, with an estimated 63% AF . Gi. Gi51]. Only recently has there been increased implementation of gender-neutral vaccination strategies worldwide. Australia became the first country to introduce HPV vaccination for boys in 2007 . In 2009Additionally, there are disparities in HPV vaccine coverage between high-income countries (HICs) and low-income countries (LICs) ,54,59. AThis observed trend of lower HPV vaccine coverage in low- and middle-income countries (LMICs) compared to high-income countries (HICs) can be attributed to several factors including cost and implementation strategies. In 2011, Gavi, the Vaccine Alliance and UNICEF, international public health partnerships and organizations, respectively, reached an agreement to provide HPV vaccines for USD 4.50 per dose for LICs . HoweverCoverage has also been impacted by limitations in global supplies of vaccines. The introduction of Gavi support to LICs is contracted through only two manufacturers (GlaxosmithKline and Merck), which has outpaced their production capacity and impacted the supply of HPV vaccines to these regions . ConsequInnovative approaches in implementing the HPV vaccine globally have led to improved vaccine coverage. Vaccination administration programs in low-resource settings in schools are one approach to reaching adolescents, who tend to use health services to a lesser extent ,66. In AIn addition to delivery systems, the use of one- or two-dose vaccination schedules, has shown promise in reducing dropout rates ,71. SpecStudies have indicated that relying solely on girls-only HPV vaccination programs, which primarily aim to prevent cervical cancer and achieve herd immunity, may not effectively reduce the increasing incidence of HPV-associated OPSCC . Female-The epidemiology of HPV-associated OPSCC is changing, and future research is critically needed to characterize the changing patterns and importantly to support the implementation of gender-neutral HPV vaccination. This review highlights the gaps in our understanding of the changing trends of HPV-associated OPSCC. Specifically, existing data emphasize the need for representative and robust sources of data; this will require increased availability of HPV histopathologic testing, investment in data collection systems such as cancer registries, and funding of studies from regions of the world in which data are currently sparse. Variations in available data also highlight an opportunity for increased HPV association testing at a clinical, patient-care level. Additionally, studies have even shown changes in trends over a 10-year span; as a result, continually updated data are also needed. Further research into understanding regional variations may additionally provide insight into the underlying biology and have implications for public health policies. Overall, the quality of data is central to policy recommendations and decisions.In addition to a more comprehensive understanding of the changing epidemiology of HPV-associated OPSCC, there is a critical gap in public health policy research. While there has been implementation of HPV vaccination programs in over 122 countries/territories, only 41 countries/territories have gender-neutral vaccination policies . LeveragThere are regional trends in HPV-associated OPSCC that are changing and will require additional epidemiologic research moving forward. HPV vaccinations are the primary mode of prevention for HPV-associated OPSCC, and there exist gaps in the coverage and gender neutrality of HPV vaccination policies. Given the burden of HPV-associated malignancies beyond the head and neck, more efforts are needed to promote the global expansion and implementation of gender-neutral HPV vaccination programs.


Address the importance of organized screening for cervical cancer.Indicate new technologies in screening.Anogenital warts are caused by HPV, mainly 6 and 11.The diagnosis of condyloma acuminata is clinical with several effective therapeutic options.Low-grade squamous intraepithelial lesion (LSIL) and atypias of undetermined significance in squamous cells (ASC-US).As the risk of histological high-grade squamous intraepithelial lesion (HSIL) in a patient with cytological LSIL is significant, the quality of the cytopathological examination is essential.Cases of cytological HSIL should be systematically referred to colposcopy.“See-and-treat” is a diagnostic and therapeutic procedure and must be performed according to rules established for non-pregnant women over 25 years of age.“See-and-treat” should not be performed in adolescents given the future possibility of obstetric comorbidities.Screening should be organized based on population data records with wide coverage.The HPV DNA test must be offered for screening.The therapeutic method for warts should be changed if there is no significant improvement within four weeks of treatment.Podophyllin, 5-fluorouracil and imiquimod should not be used during pregnancy.Conservative therapy for HSIL is preferred in patients ≤ 25 years of age.In patients ≥ 25 years of age, risk estimates such as immunosuppression, HPV DNA positive and previous altered cytology and/or biopsy results should be considered.The cytopathology of HSIL should be referred to colposcopy, it is not acceptable to repeat the cytopathology exam.The endocervical canal should be evaluated with cytological brushing or curettage when the squamocolumnar junction (SCJ) is not visible.After treatment of HSIL, follow-up with cytology and colposcopy is recommended every six months for two years then, annual cytology until completing five years. If possible, perform HPV DNA test on high-risk patients after six months and annually for three years; if tests are negative, return to triennial screening for 25 years.Human papillomavirus (HPV) is the most common sexually transmitted infection in the world. The risk of being infected with the virus at least once in your life is of 50%.Cervical cancer is a public health problem worldwide and the fourth most common type of tumor among women, with 500,000 new cases and 250,000 deaths per year.According to Brazilian Guidelines for the Screening of Cervical Cancer ,The Brazilian Guidelines recommend the periodic performance of conventional cytology, although this is characterized by low reproducibility among observers. Liquid-based cytology (LBC), created with the aim of reducing unsatisfactory smears, is an alternative method of screening. Cells are deposited in a fixative suspension, which allows their uniform distribution on the slides after processing. Another advantage is the possibility to carry out new tests on the material residue in the liquid medium, such as HPV DNA test, although LBC has not shown a gain in sensitivity, compared to conventional smears.Evidence of the causal relationship between infection by oncogenic HPV types and the onset of precursor lesions and cervical cancer favored the creation of new HPV DNA detection technologies.In 2018, Febrasgo published the dossier proposing strategies to add to the 2016 Brazilian Guidelines :Population based screening;Insertion of the HPV DNA test as a primary test;Refer women positive for HPV-16/18 directly to colposcopy;HPV DNA self-sampling for women who reject the professional exam or live in remote areas.What is condyloma acuminata?They are exophytic lesions caused by HPV, mainly types 6 and 11. They can be painful and/or itchy. In women, they are located in the vulva, perineum, perianal region, vagina and cervix. Less often they develop in extragenital areas, such as nasal and oral mucosa.How is the diagnosis of condyloma? Clinical diagnosis; biopsy is only indicated in the following cases:- Doubt in the diagnosis or suspicion of neoplasia;- Lack of response to conventional treatment;- When located in the transformation zone of the cervix or anal canal;- Immunocompromised patients.What are the therapeutic modalities?They can be self-administered, outpatient or associated.Imiquimod cream 5%:triggers local cellular immune response. Apply to the vulva at night, three times a week on alternate days, washing the area after six to ten hours. It can be used for up to 16 weeks, with an average response in eight weeks.Podophyllotoxin cream 0.15%:has antimitotic action. Self-application twice a day for three consecutive days, followed by a four-day pause, repeated weekly for four weeks.5-fluorouracil 5%:for vulvar lesions, biweekly application on lesions and removal after 4 hours is recommended. There may be erythema, burning and itching. Treatment in the vagina with application of 2.5g weekly or biweekly should be restricted to selected cases with strict monitoring. Indiscriminate use in the past has caused ulcers and vaginal adenosis.80%-90% Trichloroacetic Acid (TCA), solution:promotes chemical coagulation of the condyloma protein content. Apply a small amount to lesions at weekly intervals for eight to ten weeks. A burning sensation occurs at the time of application.Podophyllin 10%-25% solution:vegetable resin with cytotoxic action that inhibits cell metaphase. Apply weekly on each wart, washing after four hours; it is a good option in keratinized lesions. Use must be careful because it is neurotoxic and nephrotoxic.Cryotherapy:promotes thermo-induced cytolysis with liquid nitrogen. It is useful in keratinized lesions. Side effects include pain, erythema and blisters on the spot.2laser:COpromotes tissue evaporation with little deleterious effect. The advantage is the aesthetic and functional result. When epithelial planes are not respected, hypochromia, retraction and alopecia of the treated area may occur. Disadvantages include the cost of equipment and the need for professional training.Exeresis:lesions can be removed with a scalpel, scissors or even with electrocautery. There is a risk of scarring and retraction.Podophyllin, 5-fluorouracil and imiquimod should not be used during pregnancy; trichloroacetic acid, laser or cryotherapy are good options. In case of massive lesions, electrocoagulation or tangential exeresis (shaving) are alternatives. There is no indication of cesarean delivery due to the presence of lesions, except in obstruction of the birth canal or bleeding.The natural history of cervical cancer involves cellular changes that can lead to invasive carcinoma.LSIL corresponds to the cytological manifestation of HPV infection and has a high potential for regression.On the other hand, the reliability of oncotic cytology is important, since the diagnosed LSIL can be associated with the presence of histological HSIL, demonstrating that most of these lesions already existed previously and did not correspond to the evolution of less severe lesions.The advent of HPV DNA testing allowed risk stratification in patients with LSIL/ASC-US.Recommendations for patients with a cytopathological diagnosis of LSIL/ASC-US range from immediate referral to colposcopy, repeat cytology or HPV DNA testing with referral to colposcopy if the result is positive.Several protocols recommend similar approaches for the cytopathological diagnosis of LSIL and ASC-US:<25 years of age: repeat cytology in three years;25-29 years of age: repeat the cytology in 12 months;≥30 years of age: repeat the cytology in six months;Treat infectious processes or atrophy;If the repeat cytology is negative in two consecutive exams, return to routine screening;If one of subsequent cytologies is positive, perform colposcopy;Indicate colposcopy for immunocompromised women with cytopathological examination showing LSIL.In a recent protocol (2019), the recommended that patients under 25 years of age presenting LSIL cytology, HPV-positive ASC-US or ASC-US without HPV DNA test should repeat isolated cytology one and two years after the initial abnormal result.In patients with ASC-US and negative HPV DNA test, repeat the cytology in three years. After two negative cytological tests, return to routine age-based screening.For patients ≥ 25 years of age, if the initial colposcopy is compatible with low grade , repeat cytology and colposcopy in one year. In addition, risk estimates should be used:HPV DNA standard;Patient's age;Immunosuppression;Changed results of previous cytology and/or biopsy.For patients aged 25 years or older with histology of LSIL diagnosed in consecutive visits for up to two years, observation is preferred, but treatment is acceptable. If treatment is chosen and SCJ and limits of lesions are completely displayed in colposcopic examination, excision or ablation treatments are acceptable. Considering that the biological behavior of smears corresponding to ASC-US/HPV + and LSIL are similar, the approach can be the same.The importance of HSIL diagnosis is based on the fact that 70-75% of women with this result will have diagnostic histopathological confirmation of 1-2% of invasive carcinoma.The diagnosis of HSIL is made through cytology; referral to colposcopy is mandatory and cytological examination should not be repeated. Biopsy is indicated in the presence of major or discrepant colposcopic findings.When there is cytocolposcopic agreement (HSIL cytology and major findings) in non-pregnant patients over 25 years of age with visible transformation zone (types 1 or 2), without suspicion of invasion or glandular disease, the “See-and-Treat” procedure is indicated. This method avoids unnecessary returns and reduces costs.When the lesion is not fully visible, try to expose the SCJ with a good examination of the vagina to exclude the presence of lesions and indicate excision of the type 3 transformation zone (EZT) with a length of 1.5-2.5 cm.The main objective of excisional treatment is to rule out stromal invasion and assess the state of the surgical margins.Post-treatment follow-up should be done with the HPV-based test (HPV DNA or co-test) six months after the procedure and then annually, until there are three consecutive negative tests. If one of the tests is positive, colposcopy is indicated. If the margins are positive with HSIL (CIN 2+), patient is older than 25 years and formed offspring, repeated excision or observation are acceptable. If recurrent histological HSIL (CIN 2+) is identified and another transformation zone excision does not present technical conditions or is not desired, hysterectomy is recommended.HPV infection is universal in the female genital tract, and can compromise both skin and mucous membranes, causing a series of important nosological manifestations, including genital warts, intraepithelial neoplasms and cancers. Broad coverage of the population through organized screening and vaccination could substantially decrease HPV-induced diseases. The World Health Organization issued a call in 2018 for the elimination of cervical cancer as a serious public health problem : primary prevention strategies, such as the HPV vaccine, and secondary prevention strategies, such as screening, should be strengthened in the coming years. By 2030, the goals are: 90% of girls vaccinated by the age of 15, 70% of women screened with a high-effectiveness test at 35 and 45 years of age; 90% of precursor lesions and invasive cancer treated. Febrasgo, through its National Specialized Commissions on the Lower Genital Tract, Oncology and Vaccines endorses and supports the call.National Specialty Commission for Lower Genital Tract of the Brazilian Federation of Gynecology and Obstetrics Associations (FEBRASGO)President:Neila Maria de Gois SpeckVice-President:Márcia Fuzaro Terra CardialSecretary:Mila de Moura Behar Pontremoli SalcedoMembers:Adriana Bittencourt CampanerAna Katherine da Silveira GoncalvesCláudia Márcia de Azevedo JacynthoFernanda Kesselring TsoGustavo Rubino de Azevedo FochiIsabel Cristina Chulvis do Val GuimarãesJosé Humberto Belmino ChavesNeide Aparecida Tosato BoldriniRaquel Autran Coelho PeixotoRita Maira ZanineSilvana Maria QuintanaYara Lucia Mendes Furtado de Melo


Understanding the role of naturally acquired  human papillomavirus (HPV) antibodies against reinfection is important given the high incidence of this sexually transmitted infection. However, the protective effect of naturally acquired antibodies in terms of the level of protection, duration, and differential effect by sex remains incompletely understood. We conducted a systematic review and a meta-analysis to (1) strengthen the evidence on the association between HPV antibodies acquired through past infection and subsequent type-specific HPV detection, (2) investigate the potential influence of type-specific HPV antibody levels, and (3) assess differential effects by HIV status.We searched Embase and Medline databases to identify studies which prospectively assessed the risk of type-specific HPV detection by baseline homologous HPV serostatus among unvaccinated individuals. Random-effect models were used to pool the measures of association of naturally acquired HPV antibodies against subsequent incident detection and persistent HPV positivity. Sources of heterogeneity for each type were assessed through subgroup analyses stratified by sex, anatomical site of infection, male sexual orientation, age group, and length of follow-up period. Evidence of a dose-response relationship of the association between levels of baseline HPV antibodies and type-specific HPV detection was assessed. Finally, we pooled estimates from publications reporting associations between HPV serostatus and type-specific HPV detection by baseline HIV status.E = 11) and persistent HPV positivity  mainly for HPV-16 among females, but not among males, nor for HPV-18. Estimates from 8 studies suggested a negative dose–response relationship between HPV antibody level and subsequent detection among females. Finally, we did not observe any differential effect by baseline HIV status due to the limited number of studies available.We identified 26 publications  reporting associations between baseline HPV serostatus and incident HPV detection, mainly for HPV-16 and HPV-18, the most detected HPV type. We found evidence of protective effects of baseline HPV seropositivity and subsequent detection of HPV DNA (0.70, 95% CI 0.61–0.80, NWe did not find evidence that naturally acquired HPV antibodies protect against subsequent HPV positivity in males and provide only modest protection among females for HPV-16. One potential limitation to the interpretation of these findings is potential misclassification biases due to different causes.The online version contains supplementary material available at 10.1186/s13027-023-00546-3. Human papillomavirus (HPV) is one of the most common sexually transmitted viruses, affecting 50% of sexually active individuals at least once in their lifetime . InfectiMost HPV infections clear within 1–2 year by cell-mediated immune response and generation of serum neutralizing antibodies (IgG) against the capsid L1 protein of HPV . HoweverThe evidence base regarding risk of subsequent infections with HR-HPV is mixed. Several studies found no effect of naturally-acquired antibodies in reducing the risk of subsequent HPV detection in men –17 and iCurrent evidence points to multiple interactions between HIV and HPV infections, due to similar risk factors, and biological and immunological factors . StudiesThe objectives of this study were threefold. First, we updated the existing evidence on the association between naturally acquired immunity and subsequent type-specific HPV detection through a systematic review and meta-analysis. We also performed several subgroup analyses to further investigate sources of heterogeneity in the association between natural immunity and risk of subsequent HPV detection . Second, we investigated the influence of HPV antibody levels to type-specific HPV detection risk. Third, we examined if HPV detection risk differ by HIV status. We performed analyses on all HPV types where possible but reported results for HPV-16 and HPV-18 in the main results.All results of this study are reported according to the PRISMA guidelines  independently screened titles and abstracts of potentially eligible articles after removing duplicate records and resolving any discrepancies in the selection. Full texts of all potentially relevant articles were then screened. We excluded publications that did not report estimates of association between baseline HPV serostatus and type-specific HPV incident detection at follow-up, or that did not provide sufficient data to derive them. Finally, the reference lists of all included publications were screened to identify additional relevant studies .self-calculated) from reported counts and/or incidence rates  and any discrepancies were resolved by consensus. We extracted the reported crude and adjusted estimates, or the data to derive it, of any measures of association between type-specific serostatus of any reported HPV-type and incident detection of HPV DNA at follow-ups. These include the incidence rate ratio (IRR), cumulative incidence ratio or risk ratio (RR), hazards ratio (HR), or the odds ratio (OR) and their 95% confidence intervals (95%CI) or the data to derive unadjusted RR or IRR  and 95%CI using inverse variance weights and the DerSimonian-Laird random-effect method on the logarithmic scale  . To maxistimates . To avoiFor the first objective, we used two different outcomes: 1) incident type-specific HPV detection, defined as the first detection of homologous HPV DNA at follow-up among HPV DNA-negative individuals at baseline (as defined by included studies), and (2) persistent HPV positivity, defined as the detection of homologous HPV DNA at two or more consecutive follow-up visits within a 6- or 12-month interval among HPV DNA-negative individuals at baseline. For both outcomes, we focused on HPV-16 and HPV-18 in our main meta-analyses, since these HPV types were the most commonly reported . We pres incidentTo assess evidence of a dose–response relationship between baseline type-specific HPV antibodies and same type HPV detection, we plotted the measures of association by levels of HPV antibodies  from publication reporting this data for HPV-16 and HPV-18. We also performed meta regression using the estimates from the publications above as individual data point. Given heterogeneity in the categorization of antibody titres, we compared and pooled the risk of detection in the highest category  to the lowest one .Finally, for the third objective, we pooled estimates reporting associations between HPV serostatus and type-specific HPV detection by baseline HIV status. Differences between HIV positive and HIV negative individuals were assessed using the Q statistic. Again, these analyses were restricted to HPV-16 and HPV-18 only, based on the availability of extracted data from studies.We tested the influence of each individual study estimate on the overall pooled estimates using leave-one-out sensitivity analyses. We also performed several sensitivity analyses to assess whether study estimates varied due to potential biases. Specifically, we compared (1) crude versus adjusted estimates; (2) reported versus estimates calculated from raw data reported in publications; (3) types of measures of associations ; (4) methods used for serotyping ; (5) primers set used for HPV-DNA detection  ; and (6)Newcastle–Ottawa-Scale (NOS) for cohort studies  and HPV-18 (Np = 14) were the two most common HR-HPV types documented in these studies  focused on females and HPV detection at cervical or cervicovaginal sites, while 6 publications focused on males, and 1 publication included both sexes as participants. Most publications reported on multi-country studies but only one study included cohorts from sub-Saharan Africa . The most common infection sites assessed among males were penile and/or scrotal sites (Np = 4), followed by oral (Np = 3) and anal (Np = 2) sites. A total of 8 publications also provided estimates by baseline levels of HPV antibodies. One publication was restricted to HIV-positive females, and another four reported estimates stratified by HIV status among females or both sexes. SPF10 was the most common DNA primer set used for HPV DNA detection (Np = 14), and most of the studies used non-neutralizing serologic assay for the detection of serum HPV antibodies (Np = 24).Of the 16 included independent studies, 9 were prospective observational cohort studies and 7 were randomized clinical trials on HPV vaccines . In reverse, for males, the risk of incident detection among baseline-HPV seropositive individuals was increased compared to those baseline-seronegative   . Among males, the associations remained positive when restricting to specific sites: penile/scrotal , anal , and oral sites    and for HPV-18 . Out of nine reported estimates of association among female participants for incident detection at cervical/cervicovaginal site, 4 estimates for HPV-16 and 4 estimates for HPV-18 showed negative associations between increasing baseline antibody levels and the likelihood of HPV detection. We found signs of a positive association between the highest antibody threshold categories and HPV detection risk among males for HPV-16 , however, the results were inconclusive. There was only one study reporting males’ estimates by tertiles for HPV-18  among female individuals without HIV and 0.97  among female individuals living with HIV. For HPV-18, the results were also not statistically significant  .For both HPV-16 and HPV-18 incident detections, we did not observe large differences between crude and adjusted estimates, methods used for serotyping, and by study quality, after stratifying by sex , there was a reduction in risk of persistent HPV positivity (two or more detections of HPV-DNA during consecutive follow-ups). Current knowledge points to multiple interactions between HPV infection and HIV status , 43, 44.Assessing the association between naturally acquired HPV antibodies and subsequent re-infection, as well as synthesizing the evidence of this association, are subject to challenges and limitations. First, it is challenging to differentiate between true re-infection and detection of an activated latent HPV or deposition when HPV DNA is detected –47. TherThis review synthesises current knowledge on naturally acquired immunity against HPV. It substantially increases the precision of effect sizes estimates, especially for HPV-16. Compared to the previous review, we added 12 additional publications and nearly 35,000 new participants to the pooled analysis. Our results suggest differences in the effect of HPV antibodies between males and females and a potential negative dose–response relationship between antibody titre levels and subsequent detection for HPV-16 and HPV-18 in females. Although our results were inconclusive regarding differential impacts of naturally acquired immunity by HIV status, PLHIV could be at greater risk of reinfection, and consequently, at higher risk of developing HPV-attributable cancers.We did not find evidence that naturally acquired HPV antibodies protect against subsequent HPV positivity in males and provide only modest protection among females for HPV-16. Being the first study to evaluate the dose–response relationship between antibody titre levels and subsequent detection of HPV-DNA, we found some evidence of a potential negative dose–response. Finally, we did not find conclusive evidence of differential impacts of naturally acquired antibodies by HIV status, and further investigation is warranted.Additional file 1. Association of naturally acquired type-specific HPV antibodies and subsequent HPV re-detection: Systematic review and meta-analysis.


Worldwide, about 20–25 million people are affected by sickle cell disease (SCD), with 60% of patients living in sub-Saharan Africa. Despite recent therapeutic advancements resulting in improved life expectancy among SCD patients, the prevalence of high-risk human papillomavirus (hr-HPV) and cervical lesions have not been studied in women with SCD. We determined the prevalence of hr-HPV and cervical lesions among women with SCD and recommended strategies for reducing cervical cancer incidence in this cohort.Through the mPharma 10,000 Women Initiative, women with SCD attending routine visits at the Ghana Institute of Clinical Genetics SCD clinic were screened by trained nurses. Screening was performed via concurrent MA-6000 hr-HPV DNA testing and enhanced visual assessment (EVA) mobile colposcopy from mobileODT.Among 168 participants screened , the overall prevalence rates of hr-HPV infection and cervical lesions were 28.6%  and 3.6% , respectively. The hr-HPV prevalence rates stratified by haemoglobin genotype were 29.4%  and 28.6%  for genotypes SS and SC, respectively. None of the five women with the SF genotype tested hr-HPV positive, and the only patient with Sbthal genotype tested hr-HPV positive. Two women were EVA ‘positive’ but hr-HPV negative, whereas four were EVA positive and hr-HPV positive. Exploratory analysis revealed no significant associations between hr-HPV positivity and age, education level, marital status or parity.In the absence of a comprehensive national cervical screening programme aimed at including women with SCD as a special population, cervical cancer may increase in frequency among SCD patients. Thus, there is a need to build capacity and expand the scope of screening services for women with SCD. Streptococcus pneumoniae and type b Haemophilus influenzae and hydroxyurea administration [Sickle cell disease (SCD) is a common haemoglobinopathy that results from a genetic mutation in the β-globin gene. SCD occurs in 1.8% of Ghana-born newborns, and approximately 300,000 babies are born each year in sub-Saharan Africa –5. In thstration , 8. As pstration –12. Studstration , 14.In SCD, sustained red blood cell sickling may result in a vicious cycle of impaired blood flow, hypoxia, premature breakdown of red blood cells with iron release and serious sequelae of irreversible damage to end organs with severe long-term consequences . ChronicIn Ghana, an estimated 2,797 women are diagnosed with cervical cancer annually, and more than 1,699 women die from the disease yearly . DespiteTo answer these questions, we determined the prevalence of hr-HPV infection and cervical lesions in a cohort of women who were enrolled in an SCD clinic in Accra, Ghana, and underwent cervical precancer screening. Specifically, we compared their estimated prevalence to the general female population of Ghana, stratified the prevalence according to sickle cell genotype, and discussed potential strategies for targeting this cohort.We conducted the present retrospective descriptive cross-sectional study to investigate the prevalence of hr-HPV infection and cervical lesions among women living with SCD who underwent cervical screening by way of concurrent hr-HPV testing and visual inspection using the EVA mobile colposcope from MobileODT, Tel Aviv, Israel, in an outpatient setting in Accra, Ghana. The screening exercises were undertaken as part of the mPharma company’s 10,000 women campaign to screen 10,000 women in Ghana and Nigeria using HPV DNA testing.This study involved the screening of 168 women with SCD aged ≥20 years who volunteered to be registered and screened during March–May 2022. These women were regular attendees of the SCD clinic held at the Ghana Institute of Clinical Genetics (GICG), Korle-Bu Teaching Hospital, Accra, Ghana. The GICG provides care to patients with SCD on an outpatient basis. As the premier adult SCD clinic in the country, the GICG SCD clinic sees patients from all over Ghana, with a large majority coming from Southern Ghana. As of 2022, the clinic had registered more than 25,000 adolescents and adults with SCD . The cliEthical clearance was given by the Scientific and Technical Committee/Institutional Review Board of the Korle-Bu Teaching Hospital . All study participants provided verbal consent before questionnaire administration, cervical screening and sample collection.We did not calculate an optimum sample size before initiating the study primarily because the screening exercise and procedures were not initially performed in the context of a research study but as service provision. Further, we did not find any epidemiological studies of cervical cancer risk in women with SCD; thus, no baseline data could objectively guide such a calculation. Instead, we included all women attending the SCD clinic who were eligible, willing and able to consent to screening.Each woman was provided with detailed information on the need for cervical screening, as well as the procedures to be performed and their associated benefits and risks. After giving verbal informed consent to participate, a nurse collected data on sociodemographic and clinical information using a structured questionnaire routinely used at the Cervical Cancer Prevention and Training Centre (CCPTC), Battor, Ghana. Then, cervical screening was performed via cervical sampling for hr-HPV DNA testing and EVA mobile colposcopy during the same visit. Cervical specimens were then submitted to the central laboratory of the CCPTC for testing with the MA-6000 hr-HPV DNA platform  within 7 days after sample collection. Data collected using the questionnaire and screening outcomes were entered into REDCap version 11.0.3  and stored in secure databases managed by the CCPTC. The databases were then queried, and data were extracted and anonymised before the statistical analyses. The outcome of interest in this study was the presence of a clinically-relevant lesion(s) on EVA mobile colposcopy or a positive hr-HPV DNA test.Well-trained and experienced nurses from the CCPTC performed cervical sampling and mobile colposcopy professionally. After placing the woman in the dorsal lithotomy position, the nurse inserted a sterile vaginal speculum to expose the cervix and used a cytobrush to take the cervical specimen in gentle motion. The sample was then placed in a specimen collection tube, capped and sent for laboratory processing and typing. The nurse performed a colposcopy within the same screening session using the EVA device. The results of EVA colposcopy were interpreted as ‘positive’ in the presence of a clinically relevant lesion or ‘negative’ otherwise. Women with cervical lesions were managed either conservatively if they had minor changes or offered the option of thermal ablation onsite. Women with lesions not amenable to ablation were offered a loop electrosurgical excision procedure (LEEP) performed by a specialist gynaecologist. A prior publication has described the algorithms used for cervical screening and triaging with EVA mobile colposcopy .Cervical specimens obtained for MA-6000 testing were processed per the manufacturer’s instructions  and as dWe used frequencies and percentages to summarise the study participants' categorical sociodemographic  and clinical  variables. Symmetrically-distributed continuous variables are presented as means and standard deviations (SDs), whereas skewed variables are summarised using medians and interquartile ranges (IQRs). Box plots describe the age distribution of women with SCD by hr-HPV positivity status. The chi-squared test of independence was used to explore the relationship between hr-HPV positivity and selected sociodemographic factors. The overall prevalence rate of hr-HPV infection and prevalence rates stratified by sickle cell genotype with their 95% confidence intervals (CIs) are presented as percentages. Due to the low number of women with cervical lesions, the exact binomial 95% CI is presented for the overall prevalence of cervical lesions without stratification. All statistical analyses were performed using Stata 14.2 . Hypothesis tests were performed at a two-sided significance level of 5%.n = 85, 50.6%); 77 women (45.8%) had SC genotype, 5 (3.0%) had SF genotype and 1 (0.6%) had Sbthal genotype.A flow chart of patient recruitment and screening is shown in The average age of the women was 43.0 years  with a median parity of 1 . Forty percent of the women were married, 35.1% were single, 10.1% were widows, 8.9% had a steady partner and 6.0% were divorced. A majority of participants (60.7%) had at least a secondary level education, and most (78.6%) earned an income . A minorThe hr-HPV positivity rate among the 168 SCD patients screened via concurrent hr-HPV DNA testing and EVA mobile colposcopy was 28.6% . When stratified by sickle cell genotype, the hr-HPV prevalence rates were 28.6%  among women with SC genotype and 29.4%  among those with SS genotype . None ofOn EVA colposcopy, six women showed clinically relevant lesions; thus, the overall prevalence of cervical lesions was 3.6% . Two women were EVA ‘positive’ but hr-HPV negative, whereas four were EVA ‘positive’ and hr-HPV positive. As of the time of this report, one of the six women with cervical lesions in the latter group had been treated using LEEP, four were managed conservatively and one had treatment pending despite being contacted multiple times to come for treatment.t-test p = 0.3064)). Compared to married women with SCD, unmarried women were more likely to test hr-HPV positive (odds ratio (OR) = 1.58; 95% CI, 0.75–3.43; p = 0.1973). Further, relative to women with an education level below the secondary school, those with at least a secondary school education showed an OR of 1.43  for hr-HPV positivity. On the other hand, compared to women with a parity of ≤1, those with a parity of ≥2 showed an OR of 0.78  for hr-HPV positivity. Thus, in this cohort of SCD patients, hr-HPV positivity was not significantly associated with age, education level, marital status or parity.Next, we explored the association between hr-HPV positivity and selected sociodemographic characteristics among the study participants. hr-HPV-positive SCD patients tended to be slightly younger than their hr-HPV-negative counterparts ; howeverThis study aimed to estimate the prevalence of hr-HPV infection and cervical lesions among women with SCD who underwent cervical screening via concurrent hr-HPV DNA testing and EVA mobile colposcopy at an outpatient SCD clinic in Ghana. To the best of our knowledge, no prior research has been conducted in such a cohort or attempted to assess the risk of cervical precancer and cancer among women with SCD. Our study draws on the need to document the epidemiology and risk of cervical cancer in patients with SCD, given that many survive into adulthood due to therapeutic advancements that have improved their life expectancy. Here, we found an overall hr-HPV prevalence of 28.6% with only minor variations among sickle cell genotypes . In addition, 2.4% tested both hr-HPV and EVA ‘positive’, while EVA colposcopy revealed clinically relevant cervical lesions in 3.6%. Exploratory analysis revealed no significant associations between hr-HPV positivity and age, level of education, marital status or parity.Although we could not identify any prior screened SCD cohorts to compare our findings, the overall and haemoglobin genotype-stratified hr-HPV prevalence rates among our study participants were higher than the expected prevalence of 21.3% reported by the World Health Organization (WHO) for West African women , 26. StaIn addition to the elevated cervical precancer risk (judged solely based on hr-HPV estimates in this study), a number of physiologic abnormality characteristics of SCD could potentially worsen the prognoses of cervical precancerous and cancerous lesions among SCD patients. First, erythrocyte deformability may reduce in the presence of acidosis, as frequently observed in cervical cancer and other solid malignancies , 31. HypApart from the possible changes in the precancer tumour microenvironment mediated by chronic inflammation and oxidative stress , these dOur study had some limitations. First, the participants were drawn from a convenience sample of women who presented for routine visits at the clinic. Thus, they may not represent the entire clinic population or all women with SCD in Ghana. Second, we did not differentiate between transient and persistent hr-HPV infections among the participants. Third, we did not perform full genotyping for hr-HPV-positive women due to logistic and resource limitations constraints, thereby preventing complete hierarchical analysis of genotypes according to their known associations with cervical cancer. Further, due to the small sample size and having performed the screening exercise outside a research setting, we could not screen adequate numbers of women with SF and Sbthal genotypes to estimate hr-HPV prevalence rates among them accurately.This work was part of the mPharma 10,000 Women Initiative which was to give 10,000 women in Ghana and Nigeria free HPV DNA testing. Unfortunately, we were not given enough funds for HIV testing and biopsies of all lesions, so the plan was to follow up with women with minor changes (thin acetowhitening) suggestive of CIN 1 in 6–12 months unless there was a high risk of loss to follow up. This was to prevent overtreatment. Women with major changes (dense acetowhitening) suggestive of CIN II/III were treated.Due to therapeutic advancements in the care of SCD patients, increasing their life expectancy, and in the absence of a comprehensive national cervical screening programme that aims to include women with SCD as a special population, we posit that cervical cancer may increase in incidence among SCD patients. Thus, there is a need to build capacity and expand the scope of screening services offered for women with SCD.The authors declare that they have no conflict of interest.This work was part of the mPharma 10,000 Women Initiative which was to give 10,000 women in Ghana and Nigeria free HPV DNA testing. None of the authors has any financial interests to declare.Conceptualization and study design: KE, ET, CMW, YDA, and JEA. Screening and data collection: ET, CMW, GT, FT, SC, EACM, SK, SD, and KE. Data management and formal analysis: JEA, SD, CMW, ET, NOE, and KE. Writing – original draft: NOE, YDA, JEA, ET, KE, PKA, and EVA. All the authors read and approved the manuscript in its current form.This work has been partially presented as an abstract poster at the 35th International Papillomavirus Conference in Washington, DC, from 17 to 21 April 2023.


Although the role of viral agents, such as human papillomavirus  in colorectal cancer (CRC) has been previously investigated, results remain inconclusive.To further evaluate the involvement of oncogenic HPV types in CRC, 40 frozen neoplastic and 40 adjacent colonic tissues collected from Italian patients were analyzed by Luminex-based assays that detect a broad spectrum of HPV types, i.e. Alpha (n = 21), Beta (n = 46) and Gamma HPVs (n = 52). In addition, 125 frozen CRC samples and 70 surrounding mucosal tissues were collected from Czech patients and analyzed by broad spectrum PCR protocols: (i) FAP59/64, (ii) FAPM1 and (iii) CUT combined with Next Generation Sequencing (NGS).Using Luminex-basedassays, DNA from HPV16 was detected in 5% (2/40) CRC tissues from Italian patients. One HPV16 DNA-positive CRC case was subsequently confirmed positive for E6*I mRNA. Cutaneous beta HPV types were detected in 10% (4/40) adjacent tissues only, namely HPV111 (n = 3) and HPV120 (n = 1), while gamma HPV168 (n = 1) and HPV199 (n = 1) types were detected in adjacent and in tumor tissues, respectively. The NGS analysis of the CRC Czech samples identified HPV sequences from mucosal alpha-3 (HPV89), alpha-7  and alpha-10 species (HPV11), as well as cutaneous beta-1  beta-2 (HPV23), beta-3 (HPV49) and gamma-1 species (HPV205).Our findings indicate that HPV types belonging to the mucosal alpha, and the ‘cutaneous’ beta and gamma genera can be detected in the colonic mucosal samples with a low prevalence rate and a low number of HPV reads by Luminex and NGS, respectively. However, additional studies are required to corroborate these findings. Colon rectal cancer (CRC) represents the third most common cancer and is a major cause of mortality worldwide . TobaccoSeveral case–control studies have shown a higher HPV prevalence, e.g., mucosal types HPV16 and HPV18, in CRC tumor tissue compared to controls . A receni.e., Luminex-based assay) and next generation sequencing using a relatively large number of CRC specimens collected from Italian (n = 40) and Czech (n = 125) patients, and to; (ii) compare the HPV diversity in CRC to that of adjacent non-malignant mucosa. In addition, the Italian cohort has been fully characterized for HPV DNA and E6*I mRNA to evaluate the role of mucosal HPV in CRC.This study aimed to: (i) characterize the mucosal and cutaneous HPV diversity in CRC using a highly sensitive molecular screening technique  and Italy (n = 80 specimens), were analyzed for the presence of HPV DNA by NGS or a multiplex Luminex genotyping assay, respectively.CRC frozen tissues (n = 40) and matched surrounding healthy tissues (n = 40) were collected at Istituto Tumori “Giovanni Paolo II” IRCCC Hospital, Bari, Italy between 2017 and 2019. The 40 patients were 26 male and 14 female, with an average age of 67.3. Tumor stage and other baseline characteristics are indicated in Table The simultaneous purification of DNA and RNA from frozen tissue was performed at the International Agency for Research on Cancer  using AllPrep DNA/RNA/Protein Mini Kit  following the manufacturer’s instructions. Extracted DNA, RNA and proteins were stored at −80 °C until use.The prevalence of a broad spectrum of cutaneous and mucosal HPV types was determined by using type-specific multiplex genotyping (TS-MPG) assays, which is based on multiplex polymerase chain reaction (PCR) and bead-based Luminex technology , as described elsewhere . BrieflyRT-PCR was carried out using the QuantiTect Virus Kit , in a total volume of 25 μl containing 5 μl of 5 × QuantiTect Virus Mastermix, 0.25 μl of 100 × QuantiTect Virus RT Mix, 0.4 μM of each oligonucleotide, and 1 μl RNA as described previously . HPV speISH) using the HPV Probe   for the qualitative detection of HPV DNA, on the automated Leica BOND‐III system, according to the manufacturer’s instructions.The 3-µm thick sections of formalin-fixed, paraffin-embedded (FFPE) tissue were tested by in situ hybridization .The CRC patients were recruited from the Department of Surgery, Teaching Hospital and Medical School, in Pilsen, Czech Republic, between January 2008 and November 2011.Collection, processing of tissues and data acquisition of the Czech samples, was performed as previously described . The cli (iii) CUT protocols as previously reported [Total DNA was extracted from frozen CRC (n = 125), and available surrounding tissues (n = 70) using the DNeasy Blood and Tissue Kit following the manufacturer’s instructions . DNA samples were sent to IARC for HPV DNA characterization by PCRs combined with NGS. Total extracted DNA was amplified using three degenerated primer sets (i) FAP59/64 (FAP), (ii) FAPM1 andreported –22.PCR amplicons of the expected size (about 480 bp) were generated from 41 and 78 CRC samples using FAP and FAPM1 primers, respectively. In adjacent CRC specimens, PCR amplicons were generated from 16 and 45 specimens using FAP and FAPM1 primers, respectively. Finally, amplicons from 98 CRC and 37 adjacent samples were obtained using CUT primers and. PCR amplicons of the expected size were purified as previously described  and diviISH) (Data not shown).HPV prevalence was determined in a series of frozen surrounding (n = 40) and CRC matched tissues (n = 40) from Italy, using Luminex-beads based assays. DNA from HPV16 was found in two CRC tissue samples  from male patients, and in one adjacent tissue Table . One of Cutaneous beta HPV111 n = 3) and HPV120 (n = 1) DNA was detected in 10% (4/40) of surrounding tissue samples. Cutaneous gamma HPV DNA from HPV168 and HPV199 types was found in adjacent and tumor tissues, respectively  of reads align to the host human genome, while 18,893 reads (0.1%) were classified into the PCR protocols generated a total of 2214 reads related to alpha HPVs, of which 1,815 reads, all assigned to HPV68, were found in tumor samples, while 391 reads assigned to HPV70 were found in adjacent tissue  and gamma-1 HPV205 (n = 57) and HPV-mDysk3 (n = 2) observed in tumor and non-neoplastic tissues, respectively. Altogether, a total of 18 different HPV sequences were detected in colorectal tissues, although some of these had very low read numbers  versus specific primers and probes which constitute the backbone of the Luminex-based assays. Interestingly, the presence of alpha-7 HPV in the colonic mucosa has been reported by other studies using different strategies. The presence of oncogenic HPV18 was previously reported in CRC  as well We also detected DNA sequences from several beta HPV types from species beta-1, beta-2 and beta-3  and from gamma-1 (HPV205) (Tables Using different methodologies for detecting HPV DNA, the current study provided evidence for a low HPV prevalence in Italian CRC specimens, and a low number of NGS HPV reads in the Czech cohort, in agreement with previous studies , 29–32. However, the strength of this study is the use of both a highly sensitive molecular screening technique and a deep sequencing for a broad spectrum of cutaneous and mucosal HPV types in a substantial number of CRC and adjacent tissue samples.In agreement with a previous study conducted on healthy individuals , we deteThis study investigated the presence of a broad spectrum of HPV types, some of which are oncogenic, in the colonic mucosa using validated Luminex and NGS DNA assays. The low prevalence of HPV types by Luminex and the low number of HPV reads by NGS do not support an appreciable role of HPVs in CRC development. However, since an etiological role of infectious agents in CRC cannot be completely excluded, further and larger epidemiological investigations in diverse settings are needed to evaluate the impact of HPV and other oncogenic viruses in CRC samples.