Cervical cancer screening has enabled a decrease in the incidence and mortality of cervical cancer. Various screening modalities have been studied to date. In many countries, screening is still based on cervical cytology, where cervical cells obtained either on glass or in a liquid medium are examined under a microscope. However, the fact that the vast majority of cervical cancers are a result of persistent infection with high-risk human papillomaviruses (hr-HPV) has led to the implementation of primary HPV screening in many countries. Taking into consideration the fact that the majority of HPV infections are transient and do not cause cervical precancer, effective triage methods are needed to prevent an increase in colposcopy referrals. Among these, the most extensively investigated are HPV genotyping, HPV methylation, and p16/Ki67 dual staining. In this manuscript, we briefly summarize the current knowledge regarding different screening strategies for the prevention of cervical cancer, with a focus on p16/Ki67 dual staining. In addition, we provide an explanation regarding the rationale for the use of various screening modalities based on the molecular biology of cervical cancer and cervical precancerous lesions. For many decades, cervical cancer screening was based on cervical cytology . This caTo decrease the number of unnecessary colposcopy referrals, an appropriate triage test for HPV-positive women is needed. In the majority of countries, cervical cytology is used as a triage test . HoweverWe performed a systematic review of the literature on the role of p16/Ki67 dual staining in cervical cancer screening. The literature search was conducted using the MEDLINE electronic database. After screening for relevant content among the 120 identified papers, we selected 102 papers until September 2023. Peer-reviewed articles published in the English language and containing an abstract were considered, and reference lists were screened for additional relevant citations. Some additional manuscripts were added due to their close association with the content of the manuscript. Full-text versions of all manuscripts were obtained. The systematic review was conducted in accordance with PRISMA guidelines.In this review, we briefly summarize the current knowledge regarding the most commonly used screening techniques for cervical cancer. In the first part, we focus on the studies that evaluated the effectiveness of cytology and HPV testing in the prevention of cervical cancer. In the second part of the manuscript, we describe in depth the molecular biology of cervical cancer development as a result of hr-HPV infection. In the final part, we summarize current knowledge regarding p16/Ki67 dual staining and propose further research regarding the use of p16/Ki67 dual staining in cervical cancer screening.Cervical cancer screening based on CC by the method of Papanicolaou (PAP) significantly decreased the incidence and mortality of cervical cancer . The smeCervical smears are classified according to the Bethesda system . AbnormaThe main characteristic of this lesion is the presence of koilocytes. LSIL lesions often spontaneously regress. HSIL , on the The abnormalities of glandular cells include ‘atypical glandular cells’ (AGC), endocervical adenocarcinoma in situ (AIS) , and adeThe sampling of glandular cells may be difficult due to the often multifocal nature of AIS and the possible occurrence of the lesion in the base of the endocervical crypts, which can preclude the detection of glandular lesions .The disadvantage of cervical cytology is its low sensitivity, which is in the range of 20–70%, and its high interobserver variability in the interpretation of cervical smears ,13,14. TThe latest systematic review published in the Cochrane library included 61,099 women with ASC-US+ . The senThe alternative to Pap smears is LBC, which has, in many countries, replaced CC as it offers good cost benefits due to the lower rate of inadequate smears  ,13,18. TThese two methods have been compared in many comparative studies that provided conflicting results ,25,26,27The majority of cervical cancers develop after an infection with hr-HPV, and therefore testing for the presence of hr-HPV has been incorporated into the screening protocols of many countries ,34. PrimThere are many commercially available assays for HPV testing. However, only tests that show a consistently low false-negative result rate for the detection of CIN2+ or CIN3+ and at the same time detect minimal rates of transient, clinically irrelevant infections should be used in clinical practice . Hr-HPV To summarize, HPV testing offers high sensitivity and lower specificity for the detection of cervical precancerous lesions. If screening was based solely on HPV testing, this would result in high rates of colposcopy referrals. Therefore, many screening strategies have been studied to date to overcome this limitation, such as HPV genotyping, HPV methylation, and p16/Ki67 dual staining.About 40 HPV genotypes, among more than 200 identified, are able to infect the cervix. Among these, HPV 16, HPV 18, and HPV 45 account for the majority of cases . HPV 45 As a result of persistent infection with hr-HPV, many epigenetic changes occur in both viral and host DNA. The expression of these genes is changed without a change in the DNA sequence ,46. DNA The vast majority of cervical cancers are HPV-positive. However, less than 5% of all HPV infections progress to CIN3 lesions ,70. HPV p16 is a regulator of the normal cell cycle and functions as a CDK inhibitor. Its expression is increased in squamous epithelium through the E7/pRb pathway . As prevA large prospective study including 27,349 women who attended routine cervical cancer screening was conducted in five European countries . All womA retrospective study analyzed the diagnostic performance of p16/Ki67 dual staining on a sample of 776 women with ASC-US or LSIL cytology results . The resA German study investigated the clinical utility of p16/Ki67 dual staining in a Pap-negative/HPV-positive population . This waA Dutch retrospective analysis of 847 women with Pap-negative/HPV-positive results reported a 3-year sensitivity of 73.3% and a specificity of 70.0% for the detection of CIN3+ with dual staining. The reported 5-year cumulative incidence rate for the detection of CIN3+ was 6.9% in women with normal cytology and positive-HPV, and this percentage fell to 3.3% if these women were tested with p16/Ki67 dual staining, provided the test was negative . These fRecently, a British cohort study compared the diagnostic accuracies of LBC, p16/Ki67 dual staining, and HPV 16/18 genotyping on a sample of 61 patients with CIN2+ compared to 279 controls with CIN1 or less . The autDual staining was also found to have high sensitivity and specificity values for the detection of CIN2+ and CIN3+ compared to HPV genotyping in a recent American study, indicating that this method could be used as triage for HPV-positive women . p16/Ki6Taken together from studies conducted so far, p16/Ki-67 immunostaining combines high specificity and sensitivity for the detection of CIN2+ when used as a triage test for HPV-positive populations or for abnormal cytology ,91,92,93There are two promising aspects of p16/Ki67 screening that need further research, namely the role of the number of p16/Ki67-positive cells in the detection of high-grade cervical precancerous lesions and the possible use of dual staining in the triage of atypical glandular cells .p16/Ki67 dual staining is interpreted as positive when one or more cells with positive-p16/Ki67 immunoreactivity are found . Our resThe utility of p16/Ki67 dual staining for the detection of cervical adenocarcinoma was studied in a recent Japanese study . The stuAn important aspect of p16/Ki67 dual staining and its role in cervical cancer screening is its cost-effectiveness ,97. A FrAnother important aspect of cervical cancer screening is the impact of HPV vaccination on screening strategies ,101. HPVThis manuscript covers all relevant research regarding the use of p16/Ki67 dual staining in cervical cancer screening. This method is promising in terms of its capability to decrease the number of unnecessary colposcopies. In addition, it could be helpful in predicting the regression or progression of CIN2 and could also be useful in triaging abnormal glandular cells.


Objective To evaluate the accuracy and patient acceptability toward self-sampling using a new device - SelfCervix® - for detecting HPV-DNA.Methods A total of 73 women aged 25–65 who underwent regular cervical cancer screening from March to October 2016 were included. Women performed self-sampling followed by a physician-sampling, and the samples were analyzed for HPV-DNA. After that, patients were surveyed about their acceptability of self-sampling.Results HPV-DNA detection rate of self-sampling presented high accuracy and was similar to physician-collection. Sixty-four (87.7%) patients answered the acceptability survey. Most patients (89%) considered the self-sampling comfortable, and 82.5% preferred self-sampling to physician-sampling. The reasons cited were time-saving and convenience. Fifty-one (79.7%) reported that they would recommend self-sampling.Conclusion Self-sampling using the new Brazilian device SelfCervix® is not inferior in HPV-DNA detection rate compared with physician-collection, and patients are supportive of the method. Therefore, it might be an option to reach under-screened populations in Brazil. Cervical cancer is the fourth most common cancer in women worldwide and is responsible for ∼311,000 deaths per year.Persistent infection with high-risk Human Papillomavirus (mainly HPV types 16 and 18) is the etiologic cause of cervical cancer development.Self-sampling of cervicovaginal specimens is feasible, can be done at a convenient location and time, is cost-effective, avoids the need for a professional-based sampling, and enhances women's empowerment for their health. The combination of self-sampling with HPV DNA testing has similar accuracy compared with professional-based collection,We developed a Brazilian self-collector of cervicovaginal samples, SelfCervix®, to collect enough cells to perform HPV DNA testing, liquid-based cytology, and analysis of several sexually transmitted diseases. Our study aims to evaluate self-sampling acceptability and accuracy using the SelfCervix® for detecting HPV DNA in a Brazilian cohort.Women aged 25–65 years who underwent previous Pap testing for cervical cancer screening at USP Clinic Hospital , USP University Hospital , and Citoclin  from March to October 2016 were invited to participate in the study. Pregnant women, women who have not yet started the sexual activity, or those who underwent chemotherapy or radiotherapy for cervical cancer were not eligible for the study. Patients presented the following results in the previous Pap testing: no histology alteration in one (1.6%) sample, cervicitis in 13 samples (20.2%), atypia in one (1.6%) sample, Cervical intraepithelial neoplasia I (CINI) in 13 (20.2%) samples, CIN2/CIN3 in 31 (48.4%) samples, adenocarcinoma in two (3.2%) samples, condyloma/warts in one (1.6%) sample, and squamous metaplasia in two (3.2%) samples. From 9 patients, the histology information was missing due to insufficient samples to perform the test.Women who provided informed consent performed self-sampling followed by a physician-sampling with a vaginal swab. Before the physician-sampling, the SelfCervix® and verbal instructions explaining how to carry out the cervical self-sampling were provided to each patient by the physician. The SelfCervix® comprises a plunger with a soft material in the distal portion. After inserting the device into the vagina, women depressed the plunger, and through rotational movements, the device collected the cells from the cervicovaginal area. Once collected, the material was placed in a tube containing PreservCyt®  medium. The material obtained was used for carrying out HPV-DNA tests and liquid-based cytology when the HPV-DNA was positive.After the self-sampling using SelfCervix®, the physician collected cervical smears with a vaginal swab, and the patient underwent a standard professional exam. The sample was placed in a tube containing PreservCyt®  medium for carrying out HPV-DNA test and liquid-based cytology. Our study's gold-standard analysis of cervical samples was the HPV-DNA detection rate by physician-based sampling. The samples were classified according to the International Federation for Cervical Pathology and colposcopy 2011.The HPV-DNA test was performed using the hybrid capture technique II Qiagen . Oncogenic probes were used to identify HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68. For HPV-DNA-positive women, liquid-based cytology was performed on the same cervical sample.When the cytology was positive for any cervical abnormality ), the woman was referred for colposcopy and classified according to Bethesda classification: CIN 1 corresponds to low-grade squamous intraepithelial neoplasia and CIN 2 and 3 to high-grade squamous intraepithelial neoplasia.The HPV-DNA testing, liquid-based cytology, and colposcopy were analyzed at the Gynecological Oncology Research Institute (IPOG) laboratory based in São Paulo.The participants were invited to complete the acceptability of vaginal self-sampling survey following the self and physician-sampling. The survey consisted of 15 questions regarding sexual activity, prior experience with Pap testing, tolerability of both methods, preferences for self or physician-sampling, reasons for preferring one approach, and if they will indicate the self-sampling for other women.Continuous variables were presented as mean and standard deviation and frequency and percentage for categorical variables. The comparison between self-sampling and physician collection samples was analyzed using the Kappa index. Value 0 was considered poor agreement, between 0 - 0.2 reasonable agreement, 0.2–0.4 agreement, 0.4–0.6 moderate agreement, 0.6–0.8 substantial agreement, and above 0.8 excellent agreement. Fischer's exact test and chi-square were used to assess the relationship between each technique and colposcopy and biopsy results. The SPSS version 19.0  was used. A p-value < 0.05 was considered significant with a 95% confidence interval.This study was performed according to The Code of Ethics of the World Medical Association (Declaration of Helsinki) ethical guidelines and was approved by the ethics committee of the USP University Hospital under the number CAAE 56311616.6.0000.0076  and USP Clinic Hospital CAEE 38719314.2.0000.0068 . All patients signed the informed consent form to participate in the study.A total of 73 women from 3 different institutions were included in our study. Twenty-two (30.1%) women underwent cervical sample collection at Citoclin, 17 (23.3%) at the University Hospital of São Paulo (HU), and 34 (46.6%) at Clinic Hospital of São Paulo (HC). The median age of the patients was 33 years.p = 0.1)  .Discrepancies in HPV-DNA detection between self and physician-collected samples occurred in 9 (12.3%) cases. Of these samples, 7 were positive for HPV-DNA as determined in the physician-sampling and the other 2 samples corresponded to cases classified as negative for HPV-DNA in physician-sampling and positive in self-sampling. All HPV-DNA-positive samples in physician and self-sampling were referred to liquid-based cytology. In the discordant results, the physician-sampling was the gold-standard for liquid-based cytology. If the sample was positive for HPV in physician-sampling and negative in self-sampling, only the physician-sampling was referred to liquid-based cytology; and if the sample was HPV negative in physician-sampling but positive in self-sampling, both samples were referred to liquid-based cytology. “These results are summarized inIn our study self-sampling presented 87% accuracy, 86% sensibility, and 84% efficiency for HPV-DNA detection. For HPV-DNA-positive women, liquid-based cytology was performed on the same cervical sample to analyze cellular alterations and also presented 87% accuracy. The HPV-DNA detection rate of the samples collected by the physician was used as the gold standard for comparisons .Of 73 patients, 64 (87.7%) answered the survey. The mean age was 35 (18–65) years old. Approximately half of the patients, 31 (48.4%) started sexual activity between 18 and 20 years, and 38 (59.3%) did not use a condom in sexual relationships. All the patients underwent prior Pap testing at least once in life, 71.4% perform the screening annually, and 22.2% every three years. Fifty-seven patients (89%) considered self-sampling tolerable/painless, and 29 (45.3%) reported self-sampling was more comfortable to use than physician-sampling. The majority (82.5%) preferred self-sampling to physician-sampling, and the reasons cited were convenience (54.7%) and time-saving (30%). Most patients (79.7%) reported that they would recommend self-sampling to other women.To our knowledge, this is the first Brazilian self-collector product, SelfCervix® (ANVISA registry 80525329009), that allows the analysis of HPV and liquid-based cytology with a unique collection.HPV testing as a primary screening method for cervical lesions and cancer is approved in different countries, including the USA, England, and the Netherlands.In the present study, we demonstrated that self-sampling of cervicovaginal samples using the SelfCervix® presented high accuracy, sensibility, and good specificity to detect HPV-DNA and cellular alterations in liquid-based cytology for positive HPV-DNA samples. Corroborating our findings, previous studies presented similar HPV-DNA detection accuracy on self and physician-based sampling, good acceptability, and preference for self-sampling in populations under-screened and women attending routine cervical screening.We observed an HPV detection concordance of 87.7% between the SelfCervix® and physician-based sampling. In accordance, previous authors showed over 90% of HPV detection concordance between self and clinician-collected samples.We compared the results of a meta-analysis already published in the literature with the results of a Pap testing collected by a health professional to our findings of HPV-DNA detection rate by the SelfCervix® device. The Selfcervix® presented a sensitivity of 86% to detect HPV-DNA compared with the meta-analysis results that demonstrated 59% sensitivity of the Pap testing for cervical cancer screening strategy. The two methodologies showed a similar specificity (90% versus 94%).We also performed a survey with the participants to evaluate women's perception of the use of self-sampling. They reported a preference for self-sampling compared with physician-sampling, considering convenience the most important perceived benefit, and almost 80% would recommend it. Nine women did not answer the survey claiming a lack of time. These findings follow previous literature showing positive feedback from women and good acceptability of self-sampling.Our study presented limitations, including the sample size, the possible difference in the professional-sampling and cytology evaluation from the 3 different institutions, and the use of professional-sampling as the gold-standard methodology instead of anatomo-pathological analysis.The self-collector of cervicovaginal samples, SelfCervix®, demonstrated high performance in HPV-DNA detection and patient acceptability in a Brazilian cohort. The SelfCervix® is not inferior in the detection of HPV compared with physician-collection. We suggest that the use of SelfCervix® in combination with HPV testing might be an option to reach under-screened populations and increase the coverage of cervical cancer screening in Brazil.


Every year, large number of women are suffering from cervical cancer. Particularly women living with HIV are at high-risk of being suffered with it. Early testing of high-risk human papillomavirus infection can significantly reduce the incidence of cervical cancer. However, lack of early and regular testing has been identified as one of the major problems among risky populations.p-value < 0.05 was considered significant.Institutional-based cross-sectional study design was conducted among women living with HIV in Shashemene town public health facilities with a total sample size of 406 from February 1–March 30, 2022. Systematic random sampling technique was employed to select the study subjects. A structured questionnaire and checklist was used to collect data. The collected data were cleaned, coded, and entered into Epi-info version 7.2.5 and exported to statistical package for social science version 24 for analysis. Finally, bi-variable and multivariable logistic regression analyses were performed to identify determinants of high-risk human papillomavirus. Odds ratio with 95% confidence interval was used to test association between exposure and outcome under study and The prevalence of high-risk HPV infections among study participant was 173(35.2%) with 95% CI (30.5%-40.1%). Prevalence of high-risk HPV type 16, 18 and other high-risk HPV types were 62(15.3%), 23(5.7%) and 58(14.3%), respectively. Having history of sexually transmitted infections , Endline CD4 count < 200 cells/mm3 , Endline HIV viral-load ≥ 50 copies/ml  and more than one-lifetime sexual partner  were significantly associated with high-risk HPV infections.More than one third of women living with HIV had high-risk HPV. Having history of STI, low CD4 count, high viral load and multiple sexual partners were associated with high risk HPV. HIV positive women with these risk factors should be given special consideration in clinical and public health intervention. Similarly, women who had > 1-lifetime sexual partners were about 2-times higher to be infected with Hr HPV than those who had only one-lifetime sexual partner, . And also WLWH whose end line CD4 count was < 200 cells/mm3 about 3-times greater to be infected with Hr HPV than women whose endline CD4 count was greater than or equal to 200 cells/mm3, . WLWH whose end line viral load was >  = 50 copies were about 3-times to be infected with Hr HPV than women whose viral load was < 50 copies,  , 95% CI (30.5%—40.1%). The identified prevalence of Hr HPV in this study was close with study conducted in Amazons which is 31.1% (95% CI: 25.8%-36.4%) of HIV positive women infected with Hr HPV . The pre.2%, 95% Having history of STI, Endline viral load ≥ 50 copies/ml, Endline CD4 count < 200 cells/mm3 and having more than one life time sexual partner were identified as the predictors of Hr HPV infections.In this study, WLWH who had a history of STI were significantly associated with Hr HPV infections which is 3-times more than those WLWH who did not have STI infections and it is consistent with study conducted in Brazil . The posThis study revealed that WLWH whose end line CD4 count < 200 cells/mm3 were strongly associated with Hr HPV infections than those whose End line CD4 count were ≥ 200 cells/mm3. This is line with previous study finding in the amazons, Brazil and Cameroon , 33. TheThis study also revealed that WLWH who had more than one-lifetime sexual partner were about two times more likely to be infected with Hr HPV than those WLWH who had an only one-lifetime sexual partner. This finding is in line with a similar study conducted in the rural Eastern Cape of South Africa, Denmark, and Brazil , 23, 37.Further more WLWH whose endline HIV viral ≥ 50 copies/ml were about 3 times more likely to be infected with Hr HPV than those WLWH whose HIV vial load is < 50 copies/ml. This finding is line with previous study finding conducted in Bahamas . This maSeveral significant strengths were present in this study. To my knowledge, it's the first study to measure Hr HPV prevalence among WLWH throughout the nation, which can be useful for other researchers. Availability of serology and genotyping in the study area. The collected data were both primary and secondary which keeps the quality of the data.The study design  not explain any causal relationship between any of the factors involved in the study. Some variables were collected from records and it was difficult to as required. Social desirable bias . Recall bias .The results of this study indicate that there is a high prevalence of Hr HPV infection among WLWH in Shashemene town. A WLWH who have a lower end-line CD4 count, a high viral load, a history of STI, and/or more than one lifetime sexual partner were more likely to develop Hr HPV infection. These findings suggest that Hr HPV testing for cervical cancer screening in HIV-infected women would be necessary in Shashemene town, particularly for women who have identified factor.Shashemene town health office and health bureau should have to strengthen ART drug availability and early initiation, follow-up adherence, increase access to healthcare facilities. To healthcare providers and health extension workers, have to provide health education on the proper use of ART drugs and advice eating balanced diet, encourage correct and consistent use of condom and advice to limit number of sexual partner and in collaboration with community religious leaders and community leader need to mobilize and educate the health risk of having multiple sexual partners on the women and her family. Women living with HIV/AIDS encouraged to use ART drug properly, limit their life time sexual partner, consistent and proper use condoms, make regular screening of cervical cancer. Further researcher should be conducted using strong methodological approach to identify the underlying cause of Hr HPV.


HPV infection can cause cancer, and standard treatments often result in recurrence. The extent to which liquid biopsy using HPV circulating tumor DNA (HPV ctDNA) can be used as a promising marker for predicting recurrence in HPV‐related cancers remains to be validated. Here we conducted a systematic review and meta‐analysis to assess its effectiveness in predicting treatment response.We conducted a systematic literature search of online databases, including PubMed, Embase, Scopus, and the Cochrane Library, up to December 2022. The goal was to identify survival studies that evaluated the potential of plasma HPV ctDNA at baseline and end‐of‐treatment (EoT) in predicting recurrence of related cancers. Hazard ratios were estimated directly from models or extracted from Kaplan–Meier plots.The pooled effect of HPV ctDNA presence on disease recurrence was estimated to be HR = 7.97 . Subgroup analysis showed that the risk of recurrence was HR = 2.17  for baseline‐positive cases and HR = 13.21  for EoT‐positive cases. Significant associations were also observed between recurrence of oropharyngeal squamous cell carcinoma ) and cervical cancer ) in plasma HPV ctDNA‐positive patients.The study found that HPV ctDNA detection can predict the rate of relapse or recurrence after treatment, with post‐treatment measurement being more effective than baseline assessment. HPV ctDNA could be used as a surrogate or incorporated with other methods for detecting residual disease. The study showed that detecting HPV ctDNA can predict the rate of relapse or recurrence after treatment, with post‐treatment measurement appearing to be superior to baseline assessment in recurrence prediction. This highlights the potential of HPV ctDNA as a surrogate or in combination with other methods for detecting residual disease. This virus is the culprit for about one‐third of infection‐related cancers and plays remarkable roles in different steps of cancer evolution.Nowadays in the setting of major HPV‐related cancers like cervical cancer (CC), head and neck squamous cell carcinoma (HNSCC), and anal squamous cell carcinoma (ASCC) the preferred standard‐of‐care therapeutic approach includes the combination of platinum‐based chemotherapy and radiation.HPV‐related tumor cells usually contain several copies of the HPV genome, as a single genome and multiple tandem head‐to‐tail repeats in humans, as well as in episomal form.22.1Online databases of PubMed, Embase, Scopus, and the Cochrane Library were systematically searched to find all relevant studies on HPV ctDNA and CC, HNSCC, and ASCC up to December 2022. A combination of subsequent MeSH terms, including “Human Papillomavirus Viruses”, “Circulating Tumor DNA”, “Cell‐Free Nucleic Acids”, “Uterine Cervical Neoplasms”, “Anus Neoplasms”, “Head and Neck Neoplasms”, “Disease‐Free Survival”, “Progression‐Free Survival”, “Minimal Residual Disease”, “Survival Analysis” and “Kaplan–Meier Estimate” were used to search the literature. Moreover, search terms that had no available MeSH terms included “HPV ctDNA”, “Relapse‐Free Survival”, “Recurrence‐Free Survival”, “Locoregional Relapse‐Free Survival”, “Distant Metastasis‐Free Survival (DMFS)”, “Liquid Biopsy Analysis”, “Prognostic Value”, “Prognostic Factor”, “Prognostic Indicator”, “Prognostic and Predictive Biomarkers”, “Recurrence Risk”, “Predictive for Outcome”. The flow diagram of the meta‐analysis and exclusion criteria are shown in Figure 2.2The following information was obtained from each study: first author, year of publication, cancer type, HPV ctDNA detection/quantitation methods, number of patients in the time‐to‐event analysis, treatment strategies, and recurrent event endpoints data. Chemotherapy with radiation therapy (CRT), chemotherapy only, and radiation therapy only were chosen among other standard treatment regimens. To assess the efficacy of treatment strategies on recurrent event endpoints, we assumed that the widely used endpoints for tumor assessments—progression‐free survival (PFS), disease‐free survival (DFS), recurrence‐free survival (RFS), and event‐free survival (EFS)—represent the same concept, based on studies documenting their interchangeability. The Food and Drug Administration (FDA) defines DFS and PFS as below for approval of cancer drugs and biologics; DFS is defined as the time from randomization or treatment until disease recurrence or death from any cause. PFS is defined as the time from randomization or treatment until objective tumor progression or death, whichever occurs first.Based on the assumptions of these documents, we conclude that DFS, PFS, RFS, and EFS are the time elapsed between the date of initiation or end of treatment and the date of cancer relapse or tumor recurrence. All endpoints are estimated by the Kaplan–Meier (K–M) plots and survival curves. Plasma/serum HPV ctDNA values of pre‐therapeutic (baseline), end‐of‐treatment (EoT), and recurrence at follow‐up were used for estimations of these endpoints across studies.We use two types of analysis for time‐to‐event data: one for the positivity of HPV ctDNA at EoT and the second for HPV ctDNA value at baseline. To have homogeneous Kaplan–Meier (K–M) data, all reported values were converted to hazard ratio (HR). To minimize underestimation of HPV ctDNA detection, documents that used ddPCR and HPV sequencing (HPV‐seq) methods were chosen for final analysis.Titles and abstracts of the papers were carefully reviewed, as articles with similar keywords but content unrelated to the purpose, articles without an appropriate treatment strategy, unclear HPV DNA detection methods, case report studies, and papers assessing only the primary efficacy endpoint of overall survival (OS), and paper having HPV ctDNA value just in recurrence time were excluded from the study.2.3getdata‐graph‐digitizer.com) to extract original data from the K–M curves and then used the Cox PH model to estimate HRs and corresponding SEs. As indicated by heterogeneity indices, a random‐effects model was used to calculate pooled HR and corresponding CI. Pooled estimates and related plots were obtained by Review Manager software (RevMan) version 5.4.To conduct a meta‐analysis on survival studies, we needed estimates on hazard ratios comparing the groups and their standard errors (SE). However, survival studies report different information and most of the time, required data for meta‐analysis are not directly reported. We retrieved data on HR and SE directly or by back‐calculation from confidence intervals (CI) for those studies that had reported this information. For those that had reported the K–M curve along with risk sets in groups at specific time intervals, we estimated the required information by using approximation methods available in the literature.33.1The initial search, according to the detailed list of the search terms yielded the detection of 3798 records Figure . After t3.2Of 13 studies included in the meta‐analysis, 5 studies were related to cervical, 3 to anal, and 6 to head and neck cancers. Both squamous cell carcinoma (SCCA) and anal squamous cell carcinoma (ASCC) are considered neoplasms of the anal canal. In the case of HNSCC, most of the records were HPV‐positive oropharyngeal squamous cell carcinoma (OPSCC). Only one study indicated HPV16‐related HNSCC patients treated by radiotherapy with or without chemotherapy.In the included studies, different surrogate endpoint assessments including PFS, DFS, EFS, RFS, and RDFS  indicated the same idea and concept for treatment response assessment, thanks to the same meaning, all of them were considered as an endpoint assessment in the final analysis. Of 13 studies, 11 studies used EoT [at end of treatment/post‐treatment], 3 studies used baseline, and one study reported both EoT and baseline presence/absence HPV ctDNA for estimating disease relapse/recurrence prediction. Two studies used HPV ctDNA 16 levels in response assessment and outcome. Since the HPV16 ctDNA biomarker is highly specific and sensitive to the detection HPV positive cancer; we considered these studies in the final analysis too. Twelve studies used high‐throughput assays of ddPCR and HPV sequencing for HPV ctDNA quantitation. As shown in Table 3.3A total of 662 patients were included according to time‐to‐event analysis data from 13 HPV‐related studies. The pooled estimate of the effect of HPV ctDNA presence on disease recurrence in HPV‐related cancers was HR = 7.97   3.4I2 was reduced from 79%, 35%, and 58% to 62%, 35%, and 0% for total, baseline, and EoT studies, respectively. Removal of the other studies showed negligible effect on heterogeneity (<2% reduction in I2). For OPSCC incidence . Moreover, PET/CT is a routine tool for response evaluation after RT and follow‐up imaging for cancers with a high negative predictive value. Some unresolved issues, such as optimal uptaking of the probe, optimal cut‐off values , and the optimal timing of PET/CT during RT in predicting the radiotherapy response of different tumors have diverse results and are still under research.According to the risk ratio we found, patients with fast HPV ctDNA clearance and low recurrence risk after treatment could be considered for adaption of CRT with less‐intensive therapy in the last weeks of CRT, to avoid harmful side effects and unnecessary medications. Repeated time‐points may be needed to better predict relapse. As reported previously, two consecutively positive plasma HPV ctDNA detection in HNSCC patients during post‐treatment surveillance brought high (above 90%) positive and negative predictive values indicating its potential in facilitating the decision for salvage therapy.Question: If a tumor is predicted to have a good response to RT and/or CRT, it seems to be worth performing CRT or RT only as a treatment modality. Otherwise, if a tumor is predicted to be radio‐resistant, how we can predict the radiosensitivity of tumors using HPV ctDNA measurement remained to be evaluated.4.1Since anal, penile, vaginal, and vulvar cancers are relatively rare, the potential role of HPV ctDNA in monitoring these cancers has yet to be fully evaluated. We were also unable to analyze cancer surveillance data according to tumor stage. No data were available regarding detectible HPV ctDNA at several time points, especially in mid‐treatment or weeks after treatment for real‐time ctDNA monitoring. Also, there were no qualitative attributes regarding HPV DNA fragments and HPV types in most of the studies and their implementation in clinical practice. Included studies had no relevant information regarding the immunohistochemical detection of p16INK4a (p16) as a reliable marker for active HPV infection.5In conclusion, ultrasensitive detection of HPV ctDNA can predict relapse or recurrence rate after treatment. Based on the obtained data, the end‐of‐treatment measurement of HPV ctDNA has superiority over baseline measurement in treatment monitoring and response assessment of HPV‐associated cancers. Also, OPSCC patients can ultimately benefit from HPV ctDNA measurement; so it is essential to incorporate HPV ctDNA measurement into the decision‐making of postradiotherapy management in OPSCC treatment settings to avoid unnecessary invasive procedures. Although the end‐of‐treatment timepoint sensitivity exceeded relapse prediction, however, a positive result might not guarantee subsequent recurrence prediction and could necessitate repeat testing or complemented with PET/CT scan. Future studies are needed for later time points measurements to guide real‐time treatment decision‐making of HPV‐associated cancers.Abbas Karimi: Conceptualization ; data curation ; investigation ; methodology ; project administration ; supervision ; writing – original draft ; writing – review and editing . Tohid JafariKoshki: Data curation ; formal analysis ; investigation ; methodology ; validation ; writing – review and editing . Mojtaba Zehtabi: Data curation ; investigation ; writing – review and editing . Farzaneh Kargar: Investigation . Tarik Gheit: Conceptualization ; investigation ; supervision ; writing – review and editing .The authors declare that there are no conflicts of interest.Where authors are identified as personnel of the International Agency for Research on Cancer/World Health Organization, the authors alone are responsible for the views expressed in this article and they do not necessarily represent the decisions, policy, or views of the International Agency for Research on Cancer/World Health Organization.


HPV vaccine uptake rates are suboptimal in Hong Kong. A multi-disciplinary school-based HPV health-promotion programme (MDL-SHPVP) aimed at raising HPV knowledge levels and increasing vaccine uptake has therefore been developed to address vaccine hesitancy. This qualitative study was conducted to collect user feedback and identify the strengths and limitations of the educational resources developed for the programme among key vaccination stakeholders including adolescent girls and their mothers. Twenty-six participants including eight mother-daughter dyads, four teachers, three social workers, two school principals and one school nurse were recruited. To cater to the diverse audience, ten educational videos, three animations, a digital game and one booklet were developed for the programme and distributed to the participants for viewing. Semi-structured interviews were then conducted to collect feedback on the acceptability and effectiveness of the resources. Interviews were audio-recorded, transcribed verbatim, and resulting data were thematically analysed. Three themes and six sub-themes emerged. The educational materials were well-received and effective in raising HPV-knowledge levels, generating confidence in vaccine safety and effectiveness, and boosting vaccination intention. Some doubts regarding vaccine necessity remained, and recommendations for improving resource presentation and accessibility were provided. Our findings suggest that the MDL-SHPVP has the potential to boost HPV vaccine uptake. Future studies may explore educational interventions which target to increase not only HPV vaccination intention but also the sense of urgency so as to encourage timely vaccination for adolescents at the ideal age. Study findings may also provide directions for the development of future health education interventions. Human papillomavirus (HPV) infection is the most common sexually transmitted infection in the world. It is also the necessary cause of cervical cancer, the fourth most common cancer among women worldwide and the seventh most common in Hong Kong (HK) , 2. StudHealth education is a potentially effective solution to encourage vaccine acceptance and combat hesitancy. In a previous study, it was found that misconceptions and low knowledge levels of HPV are common among various stakeholders in Hong Kong . By highTo address this research gap, we aimed to assess the effectiveness of a novel multi-disciplinary school-based HPV health-promotion programme (MDL-SHPVP) which we developed, in raising knowledge levels regarding HPV, improving perceptions of HPV vaccination, and consequently, increasing vaccination uptake among adolescents in HK. The Health Belief Model (HBM), which has been used in previous HPV vaccination–promotion interventions, was employed as the underlying framework to develop the programme . The HBMA qualitative exploratory approach was adopted to conduct an in-depth investigation of the attitudes of adolescents, family and school staff .To assess user feedback on the quality, acceptability and effectiveness of MDL-SHPVP educational resources.A purposive sampling strategy was used to recruit participants in order to accurately represent the views of potential users and facilitators of the MDL-SHPVP. As HPV vaccination is more urgent for females due to the high risk of developing cervical cancer and because prior evidence suggests that mothers are the main decision-maker in accepting and initiating HPV vaccination , the priThe MDL-SHPVP was formulated with the aim to increase participants’ understanding of HPV and HPV vaccination and promote vaccine uptake among adolescents. The programme primarily targets adolescents and parents and also involves school staff such as teachers, school nurses and principals. According to the literature, a lack of information on HPV and HPV vaccination is frequently linked with non-vaccination, acting as a significant obstacle in vaccine uptake . To addrIn total, six interview videos with health care professionals (HCPs), three animations, four sharing videos by vaccinated students and their parents, an information booklet and a digital game were developed after conducting an extensive literature review of academic articles and local news publications which provided evidence on current attitudes, barriers and facilitators of HPV vaccination. Paediatric health and education experts were also consulted and involved in resource development. A summary of the programme resources is depicted in Table Intervention materials which included six HCP interview videos, three animations, three sharing videos and a booklet were distributed to participants for their evaluation. After viewing the resources, individual semi-structured interviews were carried out over the telephone by a trained research assistant from January to March 2021 to collect participant feedback. Interviews were conducted over telephone instead of face-to-face due to social distancing requirements arising from the COVID-19 pandemic. Interviews took place in Cantonese and ranged from 15 to 48 min. All interviews were audio-recorded with consent. An interview guide was developed and questions were designed to explore whether the resources met participants’ personal learning needs, to identify potential improvements in knowledge of HPV and HPV vaccination, changes in attitudes and intentions toward HPV vaccination, views of other programme components such as small group discussions and suggestions for improving the resources. Regarding the evaluation of knowledge, the participants were asked to describe the main message they received from each resource. If they were able to describe the messages accurately (descriptions matched with the information provided in the resources), the resources were considered to be effective in raising participant knowledge levels. Examples of interview questions include: “Could you explain how the MDL-SHPVP resources were able to enhance your knowledge and clear up misconceptions about HPV infection and HPV vaccination?”, “If you have the option to promote HPV-related information and HPV vaccination to other students and parents, which components of the MDL-SHPVP resources and key concepts will you use and why?”, and “What is one thing that you would change regarding the MDL-SHPVP resources?”. Data collection was stopped once the point of thematic saturation was identified during data analysis.Interviews were transcribed verbatim and translated into English by a bilingual research team member. Translations were checked and verified by bilingual research team members to ensure accuracy in meaning and message. The six-phase framework devised by Braun and Clarke (2006) was applied for thematic analysis . TranscrA total of 26 potential users and facilitators of the MDL-SHPVP were interviewed. Three main themes and six sub-themes emerged from the data as summarized in Table I never really paid attention to this topic so I don’t know what the HPV vaccine is for. (Student 2)I’d always see promotions for the vaccine but didn’t know how long the protection would last. I also heard about really horrible kinds of negative side effects so it felt like the risks involved with the vaccine were very high. Prior to the study, most participants had little to no knowledge about HPV and HPV vaccination. Some had heard about them vaguely from television advertisements but only possessed superficial knowledge.These resources are definitely helpful. Although we have HPV vaccine promotions on TV, I don’t know if young people nowadays actually watch those. So, if they are provided with such comprehensive materials, it can answer a lot of questions that people might have, misconceptions can be cleared, and parents can be more informed. (Teacher 3)A lot of people mix up concepts of prevention and cure so I felt these were explained clearly in the resources. (Student 2)After viewing the programme resources, they felt that they were able to gain extensive information about the virus and the vaccination, as well as clear up any misconceptions.If you see a commercial [about HPV] you would just watch and think it’s unrelated to you and just move on, but after seeing these materials you would know more and perceive it as a more personal problem and give it more thought. (Teacher 1)I think [the resources] are quite useful especially for parents since they are the ones making the vaccination decision… it can help them understand the situation more and let them evaluate their options. (School nurse)Specifically, the participants noted that they learnt about the difference between the 2-, 4-, and 9-valent vaccines, their efficacy rates, the ideal age for vaccination, diseases caused by HPV infection, low occurrence of negative side effects, applicability to both women and men and more. One participant also noted that after watching the videos, they had more confidence in their ability to explain the topic to others while some suggested that it was able to illustrate the personal relevance of the vaccination.I liked the booklet because it summarized everything and if people miss any points from the videos, they can catch up on those easily. (Student 2)I think the booklet is a must as people can take it home and digest it slowly. (Teacher 5)The booklet on HPV, however, was the most popular resource, as it was considered to be the most comprehensive and convenient source of information. The participants often preferred the booklet over the videos as they felt it already contained information mentioned in the videos, could be viewed at their own pace and was not as time-consuming as the videos.The videos might be better than the booklet because sometimes the things we distribute do not necessarily end up in parents’ hands. (Teacher 3)I prefer the animations because the length was appropriate and the information was relevant as it mentioned the usefulness of the vaccination. (Student 7)Among the videos, views towards the animations and HCP interviews were also favourable. The animations were thought to be suitable and engaging for students while the HCP interviews were more attractive to parents.The sharing videos weren’t really helpful because I could just ask those around me and probably get the same opinions. (Student 5)The sharing videos with the vaccinated students and parents, however, were poorly received. Most felt that the videos did not add any further information and that if they wanted to hear about vaccination experiences, they could simply inquire about it from their own friends or family.I’m open to vaccination because the HCPs in the videos also consider it safe. (Teacher 1)The HCP interviews mentioned that the vaccines have undergone various studies and are already being used in other countries so I feel more confident about them. (Student 4)The resources had a favourable effect on the participants’ attitudes towards HPV vaccination. They suggested that the content was able to increase their confidence in the safety of the vaccines and improve vaccination intention. The boost in confidence was frequently attributed to the content in the HCP interviews, as the advice of health experts was considered trustworthy and persuasive.Since I’m now 14 years old, it would be good for me to get vaccinated soon since it’s recommended for 9-14 years old and the immunity generated would be higher. It makes me want to get vaccinated. (Student 3)[The resources] made me feel like I must get vaccinated…I think it’s worth it for adolescents to get vaccinated as soon as possible as it will have higher effectiveness. Moreover, the importance and personal relevance of early vaccination was also successfully conveyed as several participants expressed a newfound realization of the urgency of vaccination.The materials didn’t convince me that the vaccination needs to be done sooner. Didn’t feel like I need to arrange it immediately. (Parent 4)One parent, however, noted that although they were convinced of the necessity of the vaccination, they remained unsure regarding the urgency.[Group discussions] might be a bit difficult as you need people to proactively ask questions, answer and talk to each other, so you might need to assess whether or not people are enthusiastic about the topic and willing to communicate. (Student 2)Besides the videos and booklet, the participants were also asked to comment on the feasibility of small group discussions including parents, students and health care workers at schools. Responses to the suggestion were largely lukewarm. Although most believed that interactions between the participants could encourage greater exchange and retention of information, there were frequent concerns regarding parent availability and students’ willingness to participate actively.If [participants] don’t know much about HPV, they won’t really know which questions to ask. You need to have some sort of basic awareness in order to come up with follow-up questions… but also, if they are given some information right before the discussion, I don’t know if they will have enough time to digest it. (Teacher 5)Besides issues of participation, it was also suggested that the participants may find it difficult to ask suitable questions due to their lack of background knowledge on the issue and felt that more questions may emerge after the intervention.I think it’s a good method since parents and students can directly ask questions from the HCP instead of finding incorrect answers from their own research. (Student 4)“[Group discussions] can be effective as they are direct, face-to-face, and you could get to know information first-hand. It would be good as long as they have one part where they give out information and one part for Q&A.” (Parent 6)However, some participants believed that group discussions may be effective as the participants could directly discuss their concerns with HCPs and possibly encourage each other to get vaccinated.Despite overall satisfaction with the resources, some doubts about the vaccination still remained among participants. The most common concerned the 10-year protection duration of existing vaccines. Though it was explained that the current estimation of the duration is based on available research which only extends to about 10 years, some adults still appeared sceptical of the value of early vaccination as they were worried that adolescents would not be protected after the 10-year period.I saw that the protection duration lasts ten years so I’m unsure about what time is actually best for vaccination. They said the protection duration is at least 10 years, but if my daughter is 14 or 15 and already vaccinated, then what will she do when she is 25? Is [the vaccination] meaningless then? Will she need to get a booster dose? (Parent 7)In addition, the participants also raised the issue of whether the vaccination was genuinely required for boys and whether booster shots were necessary after getting low-valence vaccines in order to provide protection against more HPV subtypes.The resources say that boys can also be infected but don’t talk about the diseases that they could get… so this doesn’t clear up whether or not boys actually need the vaccination. (Parent 8)For people who’ve already had the low-valence vaccines, I wanted to ask if a booster shot with the 9-valent vaccine is needed? If people aren’t focusing, they might miss key points. If there are subtitles and we can see what they’re talking about, it’ll be easier to remember. (Parent 6)Since the videos didn’t have subtitles, I wasn’t really able to get the message unless I really paid attention. The most common barrier encountered by the participants was a lack of subtitles which they felt hindered their ability to fully grasp video contents, as they might not be able to hear all the points, or miss out due to natural lapses in concentration.Did the videos talk about any negative side effects? (Teacher 3)In fact, a few responses indicated that some participants indeed failed to grasp the key messages from the resources. For instance, although potential negative side effects were described in the videos and mentioned in the booklet, this aspect seemed to have been overlooked by a teacher.Since we have started having meetings online during the pandemic, I think it might be easier to recruit people to join online sessions. (School nurse)I’m not sure if parents would be willing to attend in person… maybe they’d be more willing to attend on Zoom. If it’s just a presentation and a Q&A section, the response may be better if it’s arranged online. (Parent 1)Challenges in intervention delivery, particularly in the context of the COVID-19 pandemic, were also identified. As school suspensions have been frequent and classes are often shifted online, some participants suggested that the programme could be delivered through online communication tools. Particularly for parents, it was believed that the convenience of online meetings may lead to increased participation.Though it might be a bit easier to contact parents through Zoom these days, it’s still difficult to get them registered [to attend]. (Teacher 3)“The thing is that the average parent will generally have no idea about this topic and using online communication tools is a bit passive…” (Teacher 5)In contrast, two teachers felt that online meetings would not be entirely effective, as there was a risk of non-attendance, and that interpersonal communication would be significantly affected, therefore reducing the interactive nature of the intervention.As part of a larger randomized controlled trial to evaluate the effectiveness of the MDL-SHPVP in raising HPV vaccination rates among female adolescents in Hong Kong, tailored educational resources were developed following the identification of relevant participant learning needs . ResourcIn terms of the educational value of the resources, the participants believed that their learning needs were met successfully and that they received vital and relevant information on the topic. It was noted that the resources were comprehensive and allowed them to clarify their understanding of certain key concepts such as the differences between strategies of prevention and cure. This is a positive sign, as several studies on predictors of HPV vaccination uptake suggest that heightened knowledge of the virus and vaccination are positively correlated with vaccination intention , 16. MorAdditionally, the participants also reported improvements in their attitude towards HPV vaccination. While previous findings show that a fear of negative side effects among parents and adolescents acts as a major deterrent in initiating HPV vaccination , 19, theBesides the content of the resources, the diverse types of presentation were also well-accepted by the participants. The animations were found to be engaging and appropriate for adolescents, while the HCP interview video was more popular with the parents. The involvement of HCPs was especially compelling as the participants considered them to be authoritative sources of health information and trusted their knowledge on HPV vaccination. This is reflective of findings from past studies which have emphasized the central role played by health providers in vaccination promotion efforts , 22. MorSome limitations with the programme resources and proposed intervention components were noted, however. Regarding the videos, the participants felt that the content in the parent and student sharing videos was repetitive, and that the lack of subtitles hindered comprehension. To address these issues, subtitles will be added to the videos to enhance legibility and facilitate audience understanding, and the sharing videos will be used selectively according to participant needs. As there was also uncertainty regarding the effectiveness of the small group discussion component, process evaluation will be carried out after programme implementation to identify strengths and potential areas of improvement. However, with the requirement of social distancing during COVID-19 outbreaks, teachers voiced concerns about the possibility of low engagement online, suggesting that though online communication may facilitate contact with parents, there was no guarantee of attendance and overall interactivity may also be reduced. Additionally, some participants remained sceptical of the information provided, including uncertainty about the duration of protection offered by the vaccine, and whether it was necessary for boys to also receive the vaccination.This study has limitations. As a purposive sample was recruited for this study, the representative nature of the sample may be affected. Since interviews were conducted over the telephone instead of face-to-face, the possible presence of mothers during their daughters’ interviews may affect response quality. Moreover, the investigation could have benefited from the supplemental use of quantitative data collection, particularly in identifying and comparing participants’ views towards each separate educational resource.The participants demonstrated high acceptability of the MDL-SHPVP resources. They appreciated the quality and content of the programme resources and felt that they were effective in raising knowledge levels and promoting HPV vaccination. Recommendations provided to further improve the materials have been implemented accordingly. Study findings may aid the development of other education-based health interventions targeted at adolescents and generate a positive impact on youth health in society.


Over 95% of cervical cancers are caused by the human papilloma virus (HPV). Locally advanced HPV-related cancers release tiny bits of tumour containing HPV-DNA into the blood (cHPV-DNA). The presence of cHPV-DNA in the blood can serve as a potential marker of cancer and should be absent in pre-cancerous conditions . We have developed an ultra-sensitive and specific test to measure blood cHPV-DNA levels. We performed a feasibility study to confirm our expectation that cHPV-DNA is not found in the blood of patients with CIN but is found in the blood of patients with very early cancers. In this study, we have confirmed that cHPV-DNA is absent in the plasma of women with CIN. In early cervical tumours, there was a low detection rate of cHPV-DNA. Therefore, more sensitive tests are required before cHPV-DNA can be used for the detection of very early cervical cancers.n = 52) and FIGO stage 1A-1B CC (n = 12) prior to treatment and at follow-up. DNA extraction from plasma, followed by NGS, was used for the detection of cHPV-DNA. Results: None of the patients with pre-invasive lesions were positive for CHPV-DNA. In invasive tumours, plasma from one patient (10%) reached the threshold of positivity for cHPV-DNA in plasma. Conclusion: Low detection of cHPV-DNA in early CC may be explained by small tumour size, poorer access to lymphatics and circulation, and therefore little shedding of cHPV-DNA in plasma at detectable levels. The detection rate of cHPV-DNA in patients with early invasive cervix cancer using even the most sensitive of currently available technologies lacks adequate sensitivity for clinical utility.Background: High-risk HPV infection is responsible for >99% of cervix cancers (CC). In persistent infections that lead to cancer, the tumour breaches the basement membrane, releasing HPV-DNA into the bloodstream (cHPV-DNA). A next-generation sequencing assay (NGS) for detection of plasma HPV circulating DNA (cHPV-DNA) has demonstrated high sensitivity and specificity in patients with locally advanced cervix cancers. We hypothesised that cHPV-DNA is detectable in early invasive cervical cancers but not in pre-invasive lesions (CIN). Methods: Blood samples were collected from patients with CIN ( In the UK, cervical cancer is the 14th most common cancer in females [Risk factors for cervical cancer are multiple , includiMany cancers are known to shed fragments of DNA  into the bloodstream. Most cervical cancers are causally related to the human papilloma virus (HPV) . In the Various studies investigating cHPV-DNA as a tumour marker in cervical cancer patients have demonstrated between 11% and 45% positivity rates of cHPV-DNA in patients with invasive cervical cancer at diagnosis, suggesting a low sensitivity of the PCR assays ,9,10.cHPV-DNA has also been detected in HPV-positive head and neck cancers, with many of the studies employing PCR methods, which have a sensitivity of 19–79% in locally advanced diseases. The use of digital droplet PCR (ddPCR) using probes for HPV 16 and 33 has been shown to increase the detection of HPV ctDNA in oropharyngeal squamous cell carcinomas (OPSCC) to almost 96% . SimilarHPV16-detect’) to assess the response to chemoradiotherapy in locally advanced head and neck cancer [Traditionally, HPV genotyping involved the amplification of a region of the L1 gene with next-generation sequencing (NGS) using specific primers. Lee et al. developed an amplicon-based next-generation sequencing assay to detect circulating plasma viral DNA (‘k cancer . This nopanHPV-detect) [panHPV-detect in diagnosing the high-risk HPV subtypes prior to treatment of anal squamous cell carcinoma. Furthermore, panHPV-detect was able to identify those patients who either had residual disease post-treatment or relapsed a few months later [In their other study, which included a cohort of HPV-related anal squamous cell carcinoma patients post-chemoradiotherapy, Lee et al. expanded the novel NGS assay to include HPV subtypes 16, 18, 31, 33, 35, 45, 52, and 58 (-detect) . This inhs later .panHPV-detect—for the detection of cHPV-DNA in both pre-invasive cervical lesions and early invasive cervical cancer. As HPV does not enter the bloodstream in its infective phase, we hypothesized that cHPV-DNA would not be detected in pre-invasive cervical disease but would be detectable in early-stage cancers.There has been some work surrounding early detection and follow-up of treated cervical disease, with circulating DNA playing a promising role. However, these studies tend to include advanced cancers rather than early invasive cancers. In this prospective cohort study, we aimed to investigate the use of this highly sensitive assay—+ve, and able to give informed consent. The exclusion criteria were those with a previous diagnosis of cancer, previous CIN treatment, and other HPV-associated lesions (VIN/VaIN/AIN). Following written, informed consent, 20 mL of venous blood was collected in EDTA tubes prior to treatment and then at the next appointment following treatment to assess for cHPV-DNA. The histological findings were correlated with the panHPV-detect result. Women who could not tolerate treatment in the outpatient setting or were undergoing general anesthesia for treatment of a larger lesion were seen in the clinic at the time of consent for their surgery and recruited to this study. A venous blood sample was then taken on the morning of surgery.This study was approved by the institutional board (Ref. no. CCR 4157) and ethics committee (Ref. no. 14/NE/1055). Patients who were referred to the colposcopy clinic at UCLH with cervical smears showing high-grade dyskaryosis or persistent mild dyskaryosis and HPV-positives, and who were attending for cervical treatment in the form of Large Loop Excision of the Transformation Zone (LLETZ) or knife cone biopsy, were recruited. Inclusion criteria were those over the age of 18, referred with HSIL and HPVA second cohort of patients with biopsy-proven early-stage (FIGO 2018 1A/1B) invasive disease was recruited from the UCLH Gynaecological Oncology clinic., in London. These patients were recruited to this study in the outpatient clinic based on radiological staging and prior to definitive treatment, which was typically a simple or radical hysterectomy.HPV16-detect demonstrated 100% sensitivity and 93% specificity in detecting HPV16-positive cancers at diagnosis. To have 80% power to detect a true sensitivity of 99%, we calculated that at least 19 HPV-positive, invasive cancer patients needed to be recruited. As approximately 95% of cervical cancer patients are HPV-positive, we aimed to recruit 22 patients with early invasive carcinoma. To confirm 93% specificity for pre-invasive disease, we required 37 patients with high-grade squamous intraepithelial lesions (HSIL) to be recruited.In the head and neck cancer pilot study, Whole blood sampling and plasma extraction were conducted at the Translational Research Laboratory at UCL, London, UK. The frozen plasma samples were then transported to the Chester Beatty Lab at the Institute of Cancer Research (ICR), London, UK for further analysis.g for 10 min within 48 h of collection. The resulting plasma was then centrifuged again at 1600× g for 10 min to remove any cell sediment, aliquoted, and frozen at −80 °C. DNA was extracted from 5 mL of plasma using the QIAamp Circulating Nucleic Acid Kit (Qiagen) according to the manufacturer’s instructions. DNA was eluted in 50 μL of AVE buffer and stored at −20 °C. Plasma DNA was quantified using a Bio-Rad QX200 ddPCR system , using ribonuclease P (RNase P) as a reference gene, as previously described [Additionally, 20 mL of blood was centrifuged at 1600× We performed an initial validation of panHPV-detect by testing its ability to correctly identify the HPV subtype in cervical cancer tissue samples. We obtained tumour DNA extracted from cervical cancer tissue previously typed for the HPV subtype using polymerase chain reaction (RT-PCR) for E6 and E7 mRNA using validated assays. Five samples for each of the eight high-risk HPV subtypes were obtained from the Scottish HPV archive. Samples from five HPV-negative patients with head and neck cancer were recruited in the head and neck cancer cohort of this study, and 19 patients with breast cancer enrolled in the prospective sample collection study  were used as negative controls. HPV status was confirmed as negative with E7 RT-PCR performed on RNA extracted from the samples. None of the negative controls were known to have pre-cancerous lesions. Written informed consent was obtained from all participants. Following the initial validation, we investigated whether panHPV-detect was able to detect cHPV-DNA in the pre-treatment plasma of patients recruited in this study.+ and HPV− samples using panHPV-detect in tissue, we set a threshold whereby a sample was classified positive if there were 10 reads present from more than 30% of different HPV amplicons for each subtype. To assess the threshold for the number of amplicons needed for a positive panHPV-detect readout in plasma, a ROC analysis was used. In the first step, HPV status and subtype were assigned in tissue using E7 mRNA to separate the two groups. The number of amplicons with greater than 10 reads at baseline was inputted for each patient to find a suitable threshold for this parameter. Sorting the values in both HPV+ and negative groups and averaging adjacent values in the sorted list generated a list of thresholds. Based on the ROC analysis, a threshold that gave the greatest sensitivity and specificity for each HPV subtype was selected as the threshold for the classification of plasma as HPV DNA-positive. Based on the ROC analysis, a threshold of 7 amplicons with more than 10 reads were the thresholds that gave the greatest sensitivity and specificity and were selected as the thresholds for the classification of plasma as HPV DNA-positive.To classify HPVDNA library preparation was performed, and then Ion Torrent qPCR Quantitation with final sequencing was performed at the Tumour Profiling Unit of the ICR. Ion Torrent Libraries were prepared using Ion Ampliseq Library Preparation kit 2.0  according to the manufacturer’s instructions using 5 ng of tissue DNA or 3 ng of plasma DNA per primer pool, except the volumes of the Ion Ampliseq Library Preparation kit and the Custom Primer Pools have been reduced by half. Reads were aligned to an amalgamated reference containing scaffolds for each of the HPV genotypes as well as the reference human targeting genes using default settings in module map4 of the TMAP on the Ion Torrent machine. Bedtools v2.23.0   was used+ and HPV− samples using panHPV-detect in tissue, we set a threshold whereby a sample was classified positive if there were 10 reads present from more than 7 different HPV amplicons for each subtype.In order to classify HPVThe cost of sample analysis was £132 per sample . The bulWe recruited 52 patients between November 2018 and January 2020. The COVID-19 pandemic necessitated the suspension of study recruitment and sample collection, including follow-up of already recruited patients, in January 2020. All non-urgent clinical studies were placed on hold as laboratory services and resources were diverted to the pandemic. In addition, there was a cessation of the cervical screening programme for 2 months, a restriction on colposcopy appointments, and the introduction of social distancing rules, which meant that further recruitment from and follow-up at clinics were halted.None of the recruited patients had a history of prior HPV vaccination. Patient demographics can be found in Of the 52 patients recruited, 12 had early invasive cervical cancer and 40 had pre-invasive disease . The meaUsing the pre-set threshold of 10 reads present from >7 HPV amplicons, only 2 of the 50 baseline samples were HPV positive. HPV reads were detected in 15 other samples but in no more than 6 amplicons per HPV subtype and therefore did not reach the threshold for positivity .Final histology was available after all plasma samples were analysed and DNA sequencing performed (including follow-up samples). Out of the 12 cancer samples, 2 were found to be at a more advanced stage (FIGO 2B) than the inclusion criteria and therefore had to be excluded from this study (1 of these reached the threshold for positivity for HPV). Of the remaining 10 samples, only one was positive for HPV as per threshold criteria. This sample came from a patient who had stage 1A1 cervical cancer and was found to be HPV 18-positive, which correlated with the HPV type on referral cytology. This patient was an ex-smoker, under 30 years old, and had a small tumour. The other five patients whose histology indicated FIGO stage 1A1 tumours, did not reach the threshold for HPV positivity in plasma. None of the four 1B1 tumours had HPV in the plasma at levels that reached the threshold for positivity.None of the plasma samples from patients with final histology showing CIN 1–3 or cervical glandular intraepithelial neoplasia (CGIN) reached the threshold for positivity for HPV . In 10 oOnly eighteen patients out of the cohort of 50 were followed up prior to the COVID-19 pandemic. Two of the patients (9 and 11) had to have follow-up samples analysed despite baseline samples not being available . It was not known at the time of the blood draw that the baseline plasma had failed the PCR and library preparation stages. Of these 18 follow-up samples, 10 had HPV reads detected but none at >6 amplicons, and therefore none of the follow-up samples reached thresholds for positivity . Sample Of the 10 samples that had HPV reads at follow-up, only 2 correlated with the baseline HPV read—in one case, there was no baseline sample to compare. Seven samples detected HPV types that were either new or did not correlate with the baseline HPV types .panHPV-detect could detect cHPV-DNA in the plasma of patients with early-stage cervical cancers, as it has previously been shown to have high sensitivity and specificity for locally advanced HPV-associated cervical, head and neck, and anal cancers [The aim of this project was to determine whether  cancers ,15. UsinThe low sensitivity of this study correlates with other similar studies. Pornthanakasem et al. investigated the diagnostic and prognostic potential of circulating cHPV-DNA in patients with cervical cancer . They coSathish et al. also looked at cHPV-DNA in cervical cancer patients. They collected cervical biopsies and plasma samples from 58 women with invasive cervical cancer. In addition, they also collected samples from 10 women with the pre-invasive disease and 30 control women matched for age. All paired samples for the control group were negative for HPV DNA; however, only eight (11.8%) were positive for cHPV-DNA in the cervical cancer group. In the CIN group, all 10 patients were positive for HPV in cervical biopsies, but none were positive in plasma samples. Of the eight patients who were plasma DNA positive, seven were HPV 16-positive, and six were at stage 3–4 cancer. Cervical tissue and plasma pairs were correlated on sequencing [Both the aforementioned studies used more conventional PCR techniques rather than the novel NGS assay used in this study, which we had expected to achieve greater sensitivity for the detection of disease.A systematic review by Trigg et al. investigated the possible factors that influence the quality and yield of circulating free DNA . The facpanHPV-detect in locally advanced cervical cancers in patients undergoing chemoradiotherapy. This study has demonstrated high sensitivity (88%) and specificity (100%) for our NGS assay in detecting cHPV-DNA at baseline and following treatment completion. This suggests that the stage or volume of tumour plays a role in the amount of circulating tumour DNA that may be released into the plasma.We have carried out a similar study , which eGu et al. published a recent meta-analysis of cHPV-DNA as a biomarker for cervical cancer . They foOn further analysis of each study, the detection of cHPV-DNA in plasma in early-stage cervical cancer showed mixed results, with two studies showing no detection at stage 1B ,23, thre3, or 12.5 mm in diameter, will release enough ctDNA to represent 0.01% of all circulating DNA [Fiala et al. described the challenges of using ctDNA for early-stage cancer diagnosis . There iting DNA ,28. TheyThe strength of this study is that it focused on early-stage cervical cancers only. The low sensitivity for the detection of cHPV-DNA in early-stage cervical cancer may be explained by not only the small size of the tumour but also the inherent difficulties in the design of a sufficiently accurate HPV assay. The high specificity, however, does give some assurance that a positive test is more likely to be a true positive.The limitation of this study is that we were unable to recruit the desired number of patients, particularly in the early cervical cancer cohort, due to the coronavirus pandemic, which also made follow-up difficult. The number did not meet the requirements of the power calculation. However, as only one cervical cancer sample was positive for cHPV-DNA, this likely would not have made a statistical difference. Another inherent limitation of this prospective cohort study was the difficulty of follow-up post-treatment. Again, as all but one of the baseline samples were negative, follow-up of these patients is unlikely to have added a significant finding to this study. Of the patients that were followed up, there were no discrepancies between baseline and follow-up samples, i.e., none became positive after treatment having had a negative baseline result. Most importantly, the sole positive patient was followed up and found to be negative after treatment.HPVdetect as a biomarker for the diagnosis of early-stage cervical cancers, given the low sensitivity demonstrated. However, we have shown that circulating HPV is not detectable in pre-invasive disease, supporting the hypothesis that such lesions do not shed DNA into the circulation. Patients with pre-invasive disease have an effective screening tool based on high-risk HPV detection and cytology in cervical samples. Whilst work is ongoing on the use of HPVdetect as a potential biomarker for monitoring treatment effectiveness and predicting recurrence in locally advanced cervical cancers, further research is needed to establish whether any cDNA assay has sufficient sensitivity for reliable detection of early-stage cervical cancer.Based on this study, we cannot recommend the use of


CβS, MTHFR, NOS3, ACE1, CYBA, HAP, ACP1, GSTT1, GSTM1, and CYP1A1. Notably, the ACE1 gene emerges as a prominent factor with the presence of the I allele offering protection against HPV infection. The association of NOS3 with HPV infection is perceived with the 4a allele showing a protective effect. The presence of the GSTT1 null mutant correlates with increased susceptibility to HPV infection, HSIL and ICC, and ICC. This study also uncovers intriguing epistatic interactions among some of the genes that further accentuate their roles in disease modulation. Indeed, the epistatic interactions between the BB genotype (ACP1) and DD genotype (ECA1) were shown to increase the risk of HPV infection, and the interaction between BB (ACP1) and 0.0 (GSTT1) was associated with HPV infection and cervical lesions. These findings underscore the pivotal role of four oxidative stress-related genes in HPV-associated cervical lesions and cancer development, enriching our clinical understanding of the genetic influences on disease manifestation. The awareness of these genetic variations holds potential clinical implications.Human papillomavirus (HPV) infection is a necessary but not sufficient factor for the development of invasive cervical cancer (ICC) and high-grade intraepithelial lesion (HSIL). Oxidative stress is known to play a crucial role in HPV infection and carcinogenesis. In this study, we comprehensively investigate the modulation of HPV infection, HSIL and ICC, and ICC through an exploration of oxidative stress-related genes:  Cervical carcinoma, a malignancy of the uterus, accounted for approximately 3.1% of all malignant tumors in 2020, ranking as the fourth most prevalent cancer among women and posing a significant global public health challenge . FactorsCβS 844ins68 allele has been observed to normalize homocysteine and folate levels in individuals with the MTFHR C677T variation [Cystathionine beta-synthase (CβS) and methylenetetrahydrofolate reductase (MTFHR) are pivotal enzymes in homocysteine metabolism. Homocysteine, an antioxidant and redox regulator, is associated with aminothiol profiles that are indicative of oxidative stress-related pathologies . CβS medariation .NOS3 gene have been found, one of the most important with clinical importance and associated with variations in the NO levels is the variable number of a 27 bp tandem repeat (VNTR) in intron 4 [NOS3 post-transcriptionally by altering the formation of a small interfering RNA (siRNA). In vitro studies have shown higher siRNA levels in endothelial cells containing five copies (4b), determining lower NOS3 mRNA levels [Nitric oxide synthase (NOS) converts L-arginine into L-citrulline to produce nitric oxide (NO), a central player in reactive nitrogen species (RNS) regulation. NO’s interactions with unpaired electrons generate ROS, such as peroxide nitrite (ONOO–), and its oxidation products may lead to cellular toxicity . Crosstaintron 4 . There aA levels .ACE I/D-variation consists of the insertion or deletion (I/D) of a 287 bp fragment in intron 16. The D allele has been associated with higher ACE activity, accounting for 47% of the total phenotypic variance of serum enzyme levels [Angiotensin-converting enzyme (ACE1) plays a role in oxidative stress by mediating the production of angiotensin II, a potent oxidative stress mediator. Angiotensin II activates membrane NAD(P)H oxidases in vascular smooth muscle cells to produce ROS, such as superoxide and hydrogen peroxide, which are then involved in pleiotropic effects . ACE I/DCYBA gene causes a functional nonconservative substitution from histidine-72 to a tyrosine residue that decreases its activity [The NOX enzyme is a membrane-associated, multi-protein that produce O2- for host defense and other functions . The genactivity .2O2, and heme, per se, can act as a pro-oxidant [Hp1 and Hp2), and the way their gene products interact produces three main phenotypes of haptoglobin—Hp1.1, Hp2.1, and Hp2.2—which may have an impact on the strength of the interaction with hemoglobin; Hp 2.2 is biologically less-effective in binding free hemoglobin [Although haptoglobin is a late acute-phase protein of inflammation, it also binds to free hemoglobin released during intravascular hemolysis. Free hemoglobin can catalyze OH formation from H-oxidant ,34. Two moglobin .GSTM1/T1 results in loss of enzyme activity [Glutathione S-transferases (GSTs) play a vital role in detoxifying electrophilic compounds by generating free radicals. The genetic variation in the genes encoding GST enzymes is, therefore, crucial to regulating oxidative stress. The null genotype of activity .ACP1) is a gene that encodes a low molecular weight phosphotyrosine phosphatase (LMW-PTP), which presents two main enzymatic activities: phosphoprotein tyrosine phosphatase and flavin mononucleotide phosphatase. The ACP1 genotype was found to directly correlate to glutathione reductase activity [ACP1: ACP1*A, ACP1*B, and ACP1*C. The ACP1 alleles differ on single-nucleotide polymorphisms (SNPs), which affect both the total enzymatic activity and the ratio between isoforms F/S [Acid phosphatase locus 1 . DNA extracts were obtained between 1996 and 2003 and are now categorized as archived samples.A total of 308 women infected with HPV , and 44 with low-grade squamous intraepithelial lesions (LSIL)) from the Portuguese Oncological Institutes in Lisbon and Oporto were included in this study. All women had cytologic and histopathologic analyses performed. HPV detection was performed using polymerase chain reaction  variation, was performed. The amplicons were visualized in an agarose gel with 420 bp representing the allele with an additional VNTR (b) repeat and 393 bp representing the allele without an additional VNTR repeat (a).ACE1 Genotyping—rs4646994An amplification with PCR, flanking the I/D (insertion–deletion) variation, was performed. The amplicons were visualized in an agarose gel with 319 bp representing the allele with the deletion (D) and 597 bp representing the allele with the insertion (I).CYBA Genotyping—rs4673RsaI allowed for the facilitated genotyping: CC-348 bp; CT-348, 188, and 160 bp; TT-188 and 160 bp.An amplification with PCR, flanking the C-242T variation, was performed. An amplicon of 348 bp was visualized in an agarose gel. Subsequent restriction with HAP Genotyping—1/2Hap genotyping was determined using the Hp phenotype (Hp–Hb complexes) with PAGE electrophoresis and peroxidase staining that followed a modified version of Linke’s method (1984) .ACP1 Genotyping—A/B/CBsh136I and MspA1I facilitated genotyping: Bsh136I generated two fragments of 225 bp and 175 bp when alleles A or B, while MspA1I generated two fragments of 328 bp and 72 bp for alleles B and C.An amplification with PCR, flanking the A/B/C variation, was performed. An amplicon of 400 bp was visualized in an agarose gel. Subsequent restriction with GSTT1 and GSTM1 Genotyping—1/0GSTT1 gene, 230 bp represented the presence of the GSTM1 gene, and the 157 bp band served as the positive control for the amplification. A null allele was identified by the absence of the corresponding gene amplicon and the presence of the control amplicon.An amplification with multiplex PCR, flanking each gene, was performed. The amplicons were visualized in an agarose gel, where 480 bp represented the presence of the CYP1A1 Genotyping—rs4986884|rs1048943|rs1799814MspI facilitated genotyping: two fragments of 200 bp and 140 bp identified allele C, while the absence of restriction identified allele T. An amplification with PCR, flanking the A4889G (rs1048943) variation and C4887A (rs1799814), was performed. An amplicon of 204 bp was visualized in an agarose gel. Restriction with BsrDI identified two fragments of 149 and 55 bp for allele A (rs1048943), while the absence of restriction identified allele G. Restriction with BsaI identified two fragments of 139 and 65 bp for allele C (rs1799814), while the absence of restriction identified allele A.An amplification with PCR, flanking the T5639C (rs4986884) variation, was performed. An amplicon of 340 bp was visualized in an agarose gel. Subsequent restriction with p-value < 0.005 after correction for multiple testing using the Bonferroni adjustment.All statistical tests were conducted using SPSS 28.0 software. Group differences were assessed using Pearson’s chi-square or Fisher tests. When more than 20% of cells had an expected count inferior to 5, correction for chi-square was conducted using Fisher’s Exact test (2 × 2 tables) or the Monte Carlo simulation method for chi-square (other tables). Statistical significance was defined as a CβS, MTHFR, NOS3, ACE1, CYBA, HAP, GSTT1, GSTM1, ACP1, and CYP1A1).We commenced our analysis by investigating the association between HPV infection and ten distinct genes . Notably, due to the inability of our GSTM1/GSTT1 genotyping methodology to distinguish between 1/1 and 1/0, this gene was excluded from the codominant analysis.Among the genes studied, only NOS3 (p = 0.003) and ACE1 (p = 0.002) exhibited distinct allele distributions. In the case of NOS3, allele 4a demonstrated a protective factor . Regarding ACE1, allele I displayed a protective effect .For a clearer understanding of the risk or protective effects of each allele, we examined the allele distribution between the two populations whenever feasible . NotablyACP1, which comprises more than two possible alleles, two distinct analyses were conducted: one contrasted a single genotype against all others, and the other grouped the genotypes based on functional characteristics. The genotypes were aggregated according to fast/slow ratio ((AA and AB and BB) < (AC and BC)), fast isoform concentration ((AA and AC) < (AB and BC) < (BB)), slow isoform concentration ((AA and AB) < (BB and AC) < (BC)), or total activity ((BB and AB)/(AA and BC and AC)). These isoforms possess differing catalytic and molecular properties, potentially serving distinct cellular functions [In cases where feasible, we evaluated the genotype distribution using the dominant, overdominant, and recessive models . Notablyunctions ,46,47.ACE1 and GSTT1 genes. In the case of ACE1, the presence of allele I (II and ID) were associated with protection . Conversely, the GSTT1 null allele (0/0) was considered a risk factor .The genotype distributions varied for the GSTT1 in this disease context. Once again, the genotype of GSTT1 exhibited significant associations with ICC  and HSIL and ICC   or the most severe phenotypes, namely high-grade squamous intraepithelial lesion (HSIL) plus ICC. Analyzing the genotype distribution within the lesions further underscored the influence of 5–5.366) .We also tested for differences in the genotype distributions between LSIL, HSIL, and ICC, but found no significant results .ACP1(BB)-ACE1(DD)-epistatic interaction when compared to each individual genotype -ACP1(BB) compared to OR = 2.131, CI 1.373–3.309 for ACE1(DD) and no risk for ACP1(BB)). A similar trend was noted for the epistatic interaction ACP1(BB)-GSTT1(0.0) in HPV infection -GSTT1(0.0) compared to no risk for ACP1(BB) and OR = 2.691, CI 1.510–4.794 for GSTT1(0.0)). This last epistasis was also present when the analysis was applied to the population with the most severe lesions (HSIL and IC), in this case an OR = 9.455 (CI 2.615–34.187) was much higher than the risk of having only GSTT1(0.0) , while the BB genotype from ACP1 presented no risk alone.Subsequently, we delved into the most promising epistatic interactions, including those between homozygous genotypes or genotypes featuring the presence of one of the alleles . NotablyIn this study, our investigation revealed associations between HPV infection or cervical lesions and four of the ten candidate genes under consideration.ACE1, where the I allele emerged as a protective factor. The allele’s protective effect could be attributed to its connection with a lower ACE activity and subsequent reduction in ROS levels given that angiotensin II activates NAD(P)H oxidases [ACE1 expression in the tumor microenvironment compared to healthy tissues [The gene most consistently associated with HPV infection was oxidases ,28. Prevoxidases ,49. Rega tissues , a highe tissues , and a l tissues . AlthougNOS3 and HPV infection. Other studies have indicated that lower siRNA levels in endothelial cells carrying five copies (4a) result in elevated NOS3 mRNA levels [NOS3 that favors NO production may be an advantage for HPV clearance. Our findings align with this concept as the 4a variation emerged as a protective factor against HPV infection, suggesting that genetic variations favoring NO production may aid HPV clearance. However, the role of NO in carcinogenesis is complex, as its consequences are dose-dependent and time-sensitive. While low NO levels promote cell proliferation and anti-apoptotic responses, high levels induce cycle arrest, apoptosis, and senescence through oxidative and nitrosative stresses [NOS3 with cancer, phytochemical-induced apoptosis has been linked to the NO signaling pathway activation in cervical cancer [Our study also revealed an association between A levels . As a viA levels . NotablyA levels . In addiA levels ,57,58. NA levels ,59,60,61stresses ,62. Althl cancer .GST genes, the corresponding proteins play a crucial role in the mechanisms against oxidative stress. The homeostasis of a powerful antioxidant, glutathione, is essentially regulated by GST activity. In addition, GST proteins are responsible for the high-capacity metabolic inactivation of electrophilic compounds and toxic substrates. GST-null genotypes result in the loss of the enzyme’s ability to bind genotoxic substrates, leading to a decreased detoxification and an increased tumorigenesis [GSTT1-null genotype with HPV, lesions, and cancer. Also, de Carvalho et al. [GSTT1-null genotype in uterine cervix adenocarcinoma, while the GSTM1-null variant was not implicated in this disease. However, meta-analysis studies revealed that the GSTM1-null variant caused cervix lesions, especially among HPV infection in Indian and Chinese populations [GSTM1 or double mutants with cervical cancer or precursor lesions; GST-null mutants have been reported to be associated with oncologic diseases in general, though [Concerning the igenesis ,65. Our o et al.  verifiedulations ,68. Alsoulations . Our res, though .CβS and MTHFR did not exhibited associations with HPV infection or major lesions. Meta-analyses, other reviews, and original papers have been describing associations of this MTHFR variant with several types of cancers, such as ovarian cancer, breast cancer, colorectal cancer, bladder cancer, lung cancer, childhood acute lymphoblastic leukemia [CβS, our bibliographic survey did not reveal any study of this gene in HPV infection, cervical cancer, or precursor lesions. Nevertheless, this variant has been associated with breast cancer and exhibits altered expression in various cancer forms [Both leukemia ,74,75,76leukemia ,79,80,81leukemia ,83,84,85er forms ,87.ACP1 gene’s relevance. However, the results presented in this work are puzzling since no relevant result concerning functionality was achieved, and the BB genotype was found as a risk factor for HPV infection and severe lesions if in epistasis. Notably, the BB genotype in epistasis with DD (from ACE1) or 0.0 (from GSTT1) indicated risks for these phenotypes. This underscores the interplay of genetic backgrounds in disease contribution. Although lacking prior associations with HPV infection or cervical lesions, the ACP1 genetic variation has been linked to other cancer types [Our investigation highlighted the er types ,89. Curier types .CYBA gene revealed no association with HPV infection, cervical lesions, or cancer. However, CYBA’s involvement has been previously verified in cervical cancer [The l cancer , and thel cancer . This val cancer ,94.HAP was not implicated in tumorigenesis or HPV infection. However, it is known that Haptoglobin’s role in binding free hemoglobin contributes to oxidative damage prevention [Hp 1 allele, particularly in homozygosity, had an increased risk of developing invasive neoplastic lesions. A study carried out at the University of Ghana also obtained similar results, concluding that individuals with the Hp 2.2 phenotype had a lower risk of developing cervical cancer compared to Hp 1.1 individuals [In our study, evention . Regardievention , Mahmud evention  reportedividuals . There iividuals . Nowadayividuals .CYP1A1 gene. There is at least one study able to associate CYP1A1 with susceptibility to HPV infection [Our investigation did not reveal significant associations for the nfection , and allnfection ,107,108.The discrepancies between our study and others could reflect unique genomic features of the Portuguese population. Being one of the oldest countries in Europe, Portugal identity results from the combination of the histories of Iberian tribes, Celtic peoples, the Roman Empire, Germanic kingdoms, Muslim invasions, Christian reconquer, exploration of the “New World”, and emigration and immigration fluxes. Indeed, the Portuguese population has a history of miscegenation with other populations, resulting in a unique genomic background.As limitations to this work, we consider the lack of information on HPV genotyping, sexually transmitted diseases , diet on folic acid, tabaco (immune suppressor), sex hormone intake, and medication with ACE inhibitors. These factors could potentially act as confounders, though gathering such information from archival samples is challenging.In conclusion, our study underscores the importance of four oxidative stress-related genes in HPV infection, cervical lesions, and invasive cervical cancer. While governmental vaccine programs are mitigating this issue in some regions, underdeveloped countries still grapple with HPV-related public health challenges. Further research, especially focusing on functional aspects, will be essential to unravel the molecular mechanisms involved. The clinical significance of these genetic variations should not be overlooked, as they contribute to disease susceptibility in this context.