Journal of Medical Virology

Viability of Self‐Taken Vaginal Swab Samples for RNA‐Based Biomarker Analysis in Cervical Disease

ABSTRACT High risk human papillomaviruses (hrHPVs) cause most cervical cancers. Cervical screening programmes aim to identify precancerous disease using detection of hrHPV nucleic acid using clinician‐taken liquid‐based cytology (LBC) samples, or increasingly, self‐taken samples (STSs). However, STSs are incompatible with traditional cytology triage. Therefore, development of novel triage tests is essential. Quantification of cellular and viral mRNA biomarkers is one option, but little is known about mRNA quality in STSs. We extracted RNA from two sets of STSs (reflecting separate sampling devices) from the Scottish HPV Archive (SHA) and the Swedish Cervical Cytology Biobank (SCCB). We investigated whether the ACTB , GAPDH and p16 RNAs could be amplified by reverse transcription quantitative PCR (RT‐qPCR). RNA was degraded in both sets of samples, but samples from the Scottish HPV Archive were generally suitable for RT‐qPCR analysis, while samples from the SCCB were mostly unsuitable. Genomic DNA contamination was detected in 11.6% of samples. The quantity and quality of RNA derived from STSs was unaffected by storage of the original sample at −70°C for a period of 1 year. These data suggest that feasibility of utilising STSs for mRNA expression work is device‐dependent and that optimisation of collection and storage systems is warranted.

Extended human papillomavirus genotype distribution in cervical intraepithelial neoplasia and cancer: Analysis of 40 352 cases from a large academic gynecologic center in China

Abstract Our aim was to conduct a large epidemiologic analysis of the distribution of human papilloma virus (HPV) genotypes associated with cervical neoplasias and cancers at a major Chinese gynecologic center. The pathologic database was searched for cervical histopathologic diagnoses with prior HPV genotyping from liquid cervical cytology specimens obtained ≤6 months before biopsy. HPV testing was performed by using the Tellgenplex HPV27 or YanengBio HPV23 genotyping assays. A total of 40 352 cases meeting study criteria were identified. High risk human papillomavirus (hrHPV) was detected in 94.1% of squamous cancers compared to in only 83.3% of cervical adenocarcinomas. The prevalence of multiple HPV infections was highest in cervical intraepithelial neoplasia 1 (CIN1) (33.8%) and decreased with increasing severity of squamous lesions. The distribution of HPV genotypes was similar between CIN1 and histopathologic‐negative cases. HPV16 was one of the three most common hrHPV genotypes before all histopathologic abnormalities, ranging from 72.0% for cervical cancers, 38.7% for CIN2/3/AIS, 13.1% for CIN1, and 9.1% for biopsy‐negative cases. HPV16 and HPV18 accounted for over 87.2% of detected hrHPV genotypes for all glandular intraepithelial neoplastic lesions and cancers, whereas squamous lesions did not show this pattern. 80.3% of cervical cancers were associated with genotypes covered by HPV16/18 vaccines and 89.6% with genotypes covered by 9‐valent vaccination.

Increased human papillomavirus viral load is correlated to higher severity of cervical disease and poorer clinical outcome: A systematic review

Abstract Cervical cancer is the fourth most common cancer in women worldwide and is caused by persistent infection with high‐risk types of human papillomavirus (HPV). HPV viral load, the amount of HPV DNA in a sample, has been suggested to correlate with cervical disease severity, and with clinical outcome of cervical cancer. In this systematic review, we searched three databases (EMBASE, PubMed, Web of Science) to examine the current evidence on the association between HPV viral load in cervical samples and disease severity, as well as clinical outcome. After exclusion of articles not on HPV, cervical cancer, or containing clinical outcomes, 85 original studies involving 173 746 women were included. The vast majority (73/85 = 85.9%) reported that a higher viral load was correlated with higher disease severity or worse clinical outcome. Several studies reported either no correlation (3/85 = 3.5%), or the opposite correlation (9/85 = 10.6%); possible reasons being different categorization of HPV viral load levels, or the use of specific sampling methods. Despite variations in study design and populations, the above findings suggest that HPV viral load is correlated to clinical outcome, and may become an important biomarker for treatment selection and response monitoring for cervical cancer.

Cervical/Anal HPV‐Cytology Concordance With Colposcopic‐Anoscopic Pathology: 984 Women Without HIV in China's Largest Lower Genital Tract Center

ABSTRACT The anatomical and immunological parallels between cervical and anal canals raise questions about shared HPV pathogenesis. This study evaluated the concordance of HPV/cytology between sites and their association with histopathology. We retrospectively analyzed 984 women who underwent concurrent cervical/anal cytology and HPV testing (Jan, 2019–Jun, 2024). Subgroups included 706 with colposcopy‐histopatholgoy and 265 with HRA‐histopathology. Consistency was assessed using chi square tests, Cramér's V, and diagnostic performance metrics. HPV‐16 dominated both sites (cervix: 27.54%; anus: 21.44%). Cervical‐anal cytology showed weak‐moderate agreement (Cramér's V = 0.225, p  < 0.001), while HPV genotyping demonstrated stronger concordance (V = 0.458, p  < 0.001), especially for HPV‐16/18 (60%, 180 anal positive in 300 cervical positive). Histopathology correlation was weak (Spearman's ρ = 0.261). Anal HPV predicted anal LSIL+ poorly (PPV = 43.7%, AUC = 0.55). Combined anal HPV/LCT screening improved sensitivity (89.2%) but reduced specificity (22.1%). While cervical and anal HPV infections are moderately associated, the considerable site‐specific disparities in cytological and histopathological findings underscore the need for independent clinical assessment of each site.

Association between vaginal microbiota and the progression of ovarian cancer

AbstractOvarian cancers, especially high‐grade serous ovarian cancer (HGSOC), are one of the most lethal age‐independent gynecologic malignancies. Although pathogenic microorganisms have been demonstrated to participate in the pathogenesis of multiple types of tumors, their potential roles in the development of ovarian cancer remain unclear. To gain an insight into the microbiome‐associated pathogenesis of ovarian cancer and identify potential diagnostic biomarkers, we applied different techniques to analyse the microbiome and serum metabolome of different resources. We found that the vaginal microbiota in ovarian cancer mouse models was under dysbiosis, with altered metabolite configurations that may result from amino acid or lysophospholipid metabolic processes. Local therapeutic intervention with a broad spectrum of antibiotics was effective in reversing microbiota dysbiosis and suppressing carcinogenic progression. As the ovary is situated deeply in the pelvis, it is difficult to directly monitor the ovarian microbial community. Our findings provide alternative options for utilizing the vaginal bacteria as noninvasive biomarkers, such as Burkholderia (area under the curve = 0.8843, 95% confidence interval: 0.743–1.000), which supplement the current invasive diagnostic methods for monitoring ovarian cancer progression and contribute to the development of advanced microbe‐based diagnosis and adjuvant therapies.

Synergy Effects of HPV E6‐E7 Encoding mRNA and Nucleic Acid Immunostimulators Improve Therapeutic Potential in TC‐1 Graft Tumor

ABSTRACT Cervical cancer is the second most common cancer among women globally and the most prevalent cancer in developing countries, which was caused by human papillomavirus (HPV) infection. Messenger RNA (mRNA) vaccines have opened up new avenues for vaccine development and pandemic preparedness with potent scalability, which may possess the potential antitumor effects of an mRNA‐HPV therapeutic vaccine containing nononcogenic E6 and E7 proteins. Here, we reported a lipid nanoparticle (LNP) plus nucleic acid immunostimulators (CPG 1018 and Poly I:C) mRNA vaccine platform. The LNP‐CPG 1018 capsulated HPV E6‐E7 mRNA significantly promoted the maturation of bone marrow‐derived dendritic cells (BMDC) in vitro and were capable of efficiently migrating to lymph nodes (LN) in vivo. In TC‐1 tumor‐bearing mice, the subcutaneous immunization of LNP‐CPG 1018 capsulated HPV E6‐E7 mRNA elicited robust tumor‐specific T‐cell immunity, reshaped the tumor microenvironment, and inhibited tumor growth. In conclusion, the LNP‐CPG 1018 system is a promising delivery platform for facilitating the development of HPV E6‐E7 mRNA cancer vaccines.

Correlation between high‐risk HPV infection and p16/Ki‐67 abnormalities in Pap samples in a South Eastern Europe cohort

AbstractCervical cancer (CC) is the fourth most common cause of cancer‐related deaths amongst women worldwide. CC represents a major global healthcare issue, and Romania ranks the worst in mortality rates amongst EU countries. However, the early detection of CC can be lifesaving. To understand the testing process undergone by women in Romania, we performed a retrospective study, and investigated a cohort of 83 785 cervical cases from Romanian women aged 15−70, obtained in private‐based opportunistic screening. We examined the correlation between Pap smear results, human papilloma virus (HPV) genotyping, and the expression of cell cycle markers p16 and Ki‐67. Analysis of Pap results revealed approximately 10% abnormal cases, of which high‐grade squamous intraepithelial lesions constituted 4.9%. HPV genotyping of 12 185 cases with available Pap results unveiled a range of high‐risk HPV (hrHPV) types associated with cervical abnormalities. Notably, 26% of hrHPV‐positive cases showed no observable abnormalities. In a subset of cases with abnormal Pap and a type of hrHPV, P16/Ki‐67 double‐staining was also positive. This study suggests the importance of an integrated diagnostic algorithm that should consider the HPV genotype, Pap smear, and p16/Ki‐67 staining. This algorithm should enhance the CC screening accuracy and its management strategies, particularly in those regions with a high disease burden, such as Romania.

Detection Rate, Genetic Polymorphism, Viral Load, Persistent Infection Capacity, and Pathogenicity of Human Papillomavirus Type 58

ABSTRACTHuman papillomavirus type 58 (HPV58) poses a substantial burden in Asia; however, the interplay between its genetic variations, viral load, and clinical outcomes remains incompletely characterized. Therefore, we investigated HPV58 detection rates and E6/E7 allele frequency trends, and analyzed the positive selection, viral load, pathogenicity, and persistent infection capacity associated with specific genotypes/mutations using 239 743 exfoliated cervical cell samples. Our results show a gradual increase in HPV58 detection rates over time. Allele replacement occurs slowly, with significant changes manifesting after long‐term accumulation. The E6 A388C(K93N) + E7 prototype enhanced short‐term persistent infection capacity without increasing high‐grade lesion pathogenicity, and its frequency increased. Conversely, E7 C632T (T20I) and G760A (G63S) mutations enhanced high‐grade lesion pathogenicity, whereas G761A (G63D) reduced pathogenicity. HPV58 improves adaptive ability by increasing the persistent infection capacity without increasing the risk of high‐grade lesions. High viral load was positively correlated with both pathogenicity and persistent infection capacity, suggesting its potential as a risk factor for predicting disease progression in HPV58 screening. No correlation was observed between HPV58 viral load and specific gene mutations/genotypes, indicating that alternative mechanisms likely drive allele replacement. This study provides insights to optimize HPV58 screening strategies and deepen understanding of its evolutionary dynamics.

miR‐129‐5p inhibits anchorage‐independent growth through silencing of ACTN1 and the ELK4/c‐FOS axis in HPV‐transformed keratinocytes

AbstractA persistent infection with human papillomavirus (HPV) can induce precancerous lesions of the cervix that may ultimately develop into cancer. Cervical cancer development has been linked to altered microRNA (miRNA) expression, with miRNAs regulating anchorage‐independent growth being particularly important for the progression of precancerous lesions to cancer. In this study, we set out to identify and validate targets of miR‐129‐5p, a previously identified tumor suppressive miRNA involved in anchorage‐independent growth and HPV‐induced carcinogenesis. We predicted 26 potential miR‐129‐5p targets using online databases, followed by KEGG pathway enrichment analysis. RT‐qPCR and luciferase assays confirmed that 3'UTR regions of six genes (ACTN1, BMPR2, CAMK4, ELK4, EP300, and GNAQ) were targeted by miR‐129‐5p. Expressions of ACTN1, CAMK4, and ELK4 were inversely correlated to miR‐129‐5p expression in HPV‐transformed keratinocytes, and their silencing reduced anchorage‐independent growth. Concordantly, miR‐129‐5p overexpression decreased protein levels of ACTN1, BMPR2, CAMK4 and ELK4 in anchorage‐independent conditions. Additionally, c‐FOS, a downstream target of ELK4, was downregulated upon miR‐129‐5p overexpression, suggesting regulation through the ELK4/c‐FOS axis. ACTN1 and ELK4 expression was also upregulated in high‐grade precancerous lesions and cervical cancers, supporting their clinical relevance. In conclusion, we identified six targets of miR‐129‐5p involved in the regulation of anchorage‐independent growth, with ACTN1, BMPR2, ELK4, EP300, and GNAQ representing novel targets for miR‐129‐5p. For both ACTN1 and ELK4 functional and clinical relevance was confirmed, indicating that miR‐129‐5p‐regulated ACTN1 and ELK4 expression contributes to HPV‐induced carcinogenesis.

E6/E7 Similarity to Human Papillomavirus Prototypes and Performance of HPV Testing by Cobas 4800 HPV Test and Anyplex II HPV HR

ABSTRACT Human papillomavirus (HPV) genotype classification relies on DNA sequence similarity to reference (prototype) sequences. Most HPV assays used for cervical cancer screening were clinically validated against European HPV prototypes. However, the impact of HPV sequence polymorphisms on test performance remains unexplored. We evaluated whether sequence variation in E6/E7 relative to HPV prototypes affects test performance by analyzing cervicovaginal samples from 990 women enrolled (2019‐2022) across Belgium, Portugal, Brazil and Ecuador. Samples were tested using cobas, Anyplex, and next‐generation sequencing (Ampliseq/Ion Torrent targeting E6/E7). Sequence variation was defined as the proportion of single nucleotide polymorphisms across E6/E7 relative to reference sequence. Sequence variation was, on average, higher in HPV‐negative than HPV‐positive samples for HPV16 (0.46% vs 0.13%) and HPV18 (0.44% vs 0.37%) using cobas. Similar patterns were observed with Anyplex (HPV16: 0.78% vs 0.13%, HPV33: 0.66% vs 0.40%, HPV58: 0.79% vs 0.53, and HPV66: 1.14% vs 0.25%). For HPV45, sequence variation was, on average, higher in HPV‐positive than HPV‐negative samples when tested with Anyplex (0.87% vs 0.43%). For HPV types 18, 31, 35, 39, 51, 52, 56, 59 and 68, the mean sequence variation was similar between HPV‐negative and HPV‐positive samples using Anyplex. Our findings show that sequence variation relative to prototypes may impact test performance.

Distribution of Human Papillomavirus Genotypes in Real‐World Cervical Self‐Collected Scrapings From the Dutch Cervical Cancer Screening Program

ABSTRACTHigh‐risk HPV (hrHPV) is the necessary cause of cervical cancer with HPV16/18 accounting for around 70% of the cases worldwide, while other non‐HPV16/18 hrHPV genotypes prevail in ~95% of high‐grade lesions. Understanding regional genotype distribution of hrHPV types not covered by the nonavalent vaccine is crucial for evaluating vaccine effectiveness and enhancing population‐based screening (PBS). The objective of the present study is to update hrHPV genotype prevalence in a non‐vaccinated cohort of 1200 hrHPV‐positive women from the Dutch PBS using INNO‐LiPA HPV Genotyping Extra‐II to identify 32 individual HPV genotypes in self‐sampled material. HrHPV prevalence for all 32 genotypes, also grouped by bivalent, quadrivalent, and nonavalent vaccine types (2vHPV, 4vHPV, and 9vHPV), was reported by histologic diagnosis and age. The most common genotypes were HPV16 (394,33%), especially in younger women, followed by HPV31 (216,18%) and HPV52 (199,17%). 2vHPV genotypes were found in 23% (n = 90) of NILM cases, 27% (n = 84) of CIN0/CIN1, 45% (n = 74) of CIN2, 71% (n = 219) of CIN3, and 92% (n = 12) of cervical cancers. In comparison, 9vHPV genotypes appeared in 60% (n = 240) of NILM, 69% (n = 218) of CIN0/CIN1, 88% (n = 145) of CIN2, 94% (n = 289) of CIN3, and all cervical cancers (n = 13). HrHPV types not included in 9vHPV had an overall prevalence of 19% (n = 225), with 88% (197/225) found in NILM or CIN0/CIN1. This study highlights vaccine‐type HPV in all cancer cases and many high‐grade lesions, reinforcing the need for improved vaccination efforts and broader protection.

Killer cell immunoglobulin‐like receptor (KIR) gene contents: Are they associated with cervical cancer?

Abstract Killer cell immunoglobulin‐like receptors (KIRs) are required for natural killer cell function against virus‐infected cells or tumor cells. KIR gene content polymorphisms in Indian women with cervical cancer (CaCx) remain unexplored. Hence, we analyzed the frequencies of KIR genes, KIR haplotypes, and Bx subsets to draw their association with CaCx. The polymerase chain reaction‐sequence‐specific primer method was used for KIR genotyping in three groups of women: healthy controls ( n  = 114), women with human papillomavirus (HPV) infection ( n  = 70), and women with CaCx ( n  = 120). The results showed that the frequency of KIR2DS5 was significantly higher in women with CaCx compared to women with HPV infection ( p  = 0.02) and healthy controls ( p  = 0.01). Whereas the frequency of KIR2DL5B was significantly higher in healthy controls than in women with HPV infection ( p  = 0.02). The total number of activating KIR genes was higher in women with CaCx than in healthy controls ( p  = 0.006), indicating their positive association with CaCx. Moreover, the C4T4 subset was higher in women with CaCx than in women with HPV infection, though not significant. In conclusion, our findings highlight KIR2DS5 , the C4T4 subset, and activating KIR genes are susceptible factors or positively associated with CaCx. Besides KIR2DL5B , this study also reported for the first time significantly high frequency of KIR2DL1 in healthy controls, indicating its possible protective association against CaCx. Further, significantly high frequency of KIR2DL3 observed in HPV‐infected women might be also a promising biomarker for viral infections. Thus, the study confirms the association of KIR genes with cervical cancer in women with HPV infection.

Human Papillomavirus Genotyping and Viral Load as a Predictor of Cervical Lesions: A Prospective Study

ABSTRACTThis prospective study aims to examine the impact of changes in viral load on the occurrence of cervical lesions and to evaluate viral load as a biomarker for predicting cervical lesions and triaging HPV‐positive patients. From September 2022 to August 2023, 1150 women aged 25–60 were enrolled at the Changzhou Maternal and Child Health Hospital. All participants tested positive for HPV and negative for both cytology and pathology. A follow‐up was conducted 6 months later to reassess HPV status and perform colposcopy. BMRT was employed to detect various HPV types and their viral loads. The ROC curve was utilized to determine the viral load cut‐off values for different HPV types to predict cervical lesions. From baseline to follow‐up, women whose HPV infection cleared were significantly younger than those with persistent HPV infection (p < 0.001). At baseline, the viral loads of the virus clearance, maintenance, and progression groups demonstrated an increasing trend (p < 0.001). Among women diagnosed with CIN during follow‐up, the viral load increased significantly from baseline to follow‐up (p = 0.001). The combination of HPV genotyping and viral load in the initial screening of cervical cancer enhances the prediction and identification of cervical lesions.

Patterns of Co‐infection of HPV52 With Other HPV Genotypes and Their Risks of Cervical Precancer and Carcinoma

ABSTRACT Human papillomavirus 52 (HPV52) is the second most frequent HPV type in high‐grade squamous intraepithelial lesion (HSIL) cases in China. However, few researchers have explored the co‐infection of HPV52 with other HPV genotypes and their correlation with cervical lesions. In this study, 13,809 HPV52‐positive patients visiting the Obstetrics and Gynecology Hospital of Fudan University from 2018 to 2023 were included in the first stage to investigate the risk of cervical lesions among different multiple infection patterns. Another 443 HPV52‐positive patients were further included for sequence alignment and phylogenetic analysis. In the current study, the most common HPV52 dual‐infection patterns were as follows: HPV16 + HPV52, HPV52 + HPV58, HPV52 + HPV53, and HPV52 + HPV81. Compared with HPV52 single infection, the risk of HSIL+ was increased in HPV16 + HPV52 (OR = 3.47, 95% CI: 2.56, 4.69) and HPV52 + HPV58 (OR = 1.99, 95% CI: 1.35, 2.92) groups. The most common triple‐infection patterns were HPV16 + HPV52 + HPV53 and HPV52 + HPV53 + HPV81, followed by HPV52 + HPV53 + HPV58. HPV53 was the most common co‐infection type with HPV52 in cases of triple or more multiple infections. However, compared with dual infection, the addition of HPV53 did not affect the risk of HSIL+. Two synonymous mutations, G207A ( p = 0.029) and C1203T ( p = 0.021), showed statistically significant differences in distribution between single and multiple infection groups. Our results demonstrated that HPV52 showed preferences for co‐infection with HPV16, 585,381. HPV52 co‐infection with HPV16 and HPV58 increased the risk of HSIL+, while co‐infection with HPV53 did not increase the risk of HSIL+. Virus variants with certain mutations may be more susceptible to multiple infections.

Diagnostic Accuracy of the Siriraj Portable Digital Cervicography Device Versus Standard Colposcopy for Detecting CIN2 + Lesions

ABSTRACT To improve access to colposcopy for women with abnormal cervical cancer screening results, this study evaluated the diagnostic accuracy of the internally developed Siriraj portable digital cervicography device for detecting cervical intraepithelial neoplasia grade 2 or worse (CIN2 + ) and compared its performance with standard colposcopy. Consenting eligible women underwent examination using both the Siriraj portable digital cervicography device and standard colposcopy. Images from each method were independently evaluated by a physician blinded to the other images and histology results. The primary outcome was the diagnostic performance of both methods for detecting CIN2 + . Diagnostic accuracy was compared using Cochran's Q test, with a significance threshold of p  < 0.05. A total of 450 women participated, with histologically confirmed CIN2 + in 91 cases (20.1%), including 45 women diagnosed with cervical intraepithelial neoplasia grade 3 or worse (CIN3 + ). Incorporating colposcopic impressions, the modified Reid's colposcopic index, and the modified Swede score, the Siriraj portable digital cervicography device demonstrated a diagnostic accuracy of 81.3% for CIN2 + , whereas standard colposcopy ranged from 79.1% to 81.6%. No significant difference in diagnostic accuracy was found among the six evaluative methods ( p  = 0.558). The Siriraj portable digital cervicography device shows promise as an alternative to standard colposcopy for detecting CIN2 + , particularly in low‐resource settings. While it enables remote evaluation by trained colposcopists, its diagnostic performance remains dependent on expert image interpretation. Standardizing biopsy protocols and integrating AI‐assisted analysis could further enhance its clinical utility. Trial Registration: TCTR20230907004; registered on September 7, 2023; https://thaiclinicaltrials.org .

The Burden of HPV Infections and HPV‐Related Diseases Among People With HIV: A Systematic Literature Review

ABSTRACT Human papillomavirus (HPV) is associated with a significant global burden of precancerous lesions and cancer. People with HIV (PWH) are at higher risk of HPV infection and HPV‐related diseases. This systematic review was conducted to synthesize data on the burden of HPV infection and HPV‐related diseases among PWH. Studies published between January 2018–June 2023 were sourced from databases and conferences. Included were 221 publications containing epidemiological data on HPV infections and the clinical burden of HPV‐related diseases among PWH. The burden varied by geographical region, age, sex, and sexual orientation. Compared to people without HIV (PWoH), PWH had higher prevalence and incidence of HPV infection and HPV‐related diseases. Among PWH, the prevalence of anal HPV infection ranged between 44% and 83%; men had a higher prevalence and incidence of anogenital warts than women. The incidence of anal HPV infection was over two‐fold greater among transgender women with HIV and men who have sex with men with HIV than among their respective counterparts without HIV. Incident HPV‐related anal cancer was up to two‐fold higher among PWH than PWoH, and incident cervical cancer was up to six times higher among women with HIV than those without. The most prevalent high‐risk (hr) HPV genotypes with HPV‐related disease were vaccine genotype HPV16/18/52/58. HPV35 was one of the most prevalent genotypes with anal or cervical HPV infection among PWH of African descent. PWH also have a higher burden of concurrent HPV infections and HPV‐related diseases. This study calls for strengthening appropriate HPV vaccine delivery and increasing vaccine uptake among this high‐risk group, potentially by integrating HPV vaccination with routine HIV care.

Accuracy of Liferiver HarmoniaHPV and VenusHPV Assays on Urine and Vaginal Self‐Samples

ABSTRACT In this report, the clinical performance of Liferiver HarmoniaHPV and Liferiver VenusHPV was evaluated under the VALHUDES framework. Five hundred and twenty‐three women collected first‐void urine (FVU) with Colli‐Pee and vaginal samples with Evalyn Brush or Qvintip. Cervical samples were taken with the Cervex Brush by a clinician. Both vaginal and cervical samples were resuspended in 20 mL ThinPrep. Triplet samples from 499 women were tested with HarmoniaHPV and VenusHPV tests. The clinical accuracy of HarmoniaHPV did not differ in FVU and vaginal self‐samples versus cervical samples. The relative sensitivity for CIN2+ on FVU and vaginal samples was 0.95 [95% CI 0.89–1.02] and 0.95 [95% CI 0.88–1.02], respectively. Relative specificity for < CIN2 was 0.95 [0.86–1.04] on FVU and 0.93 [0.86–1.01] on vaginal samples. VenusHPV demonstrated lower sensitivity on both self‐sample types, whereas the specificity was similar to cervical samples. Post‐hoc adjustment of the VenusHPV C t ‐values improved sensitivity (ratio FVU/cervical = 0.94 [95% CI 0.88–1.00]; ratio vaginal/cervical = 0.96 [95% CI 0.92–1.01]) without compromising specificity (ratio FVU/cervical = 1.00 [0.92–1.09]; ratio vaginal/cervical = 0.95 [95% CI 0.88–1.02]) on both self‐samples. In conclusion, HarmoniaHPV and VenusHPV tests demonstrated similar clinical accuracy on FVU and vaginal self‐ versus cervical samples, although VenusHPV test required cut‐off optimization.

Genome‐Wide Association Analyses of HPV16 and HPV18 Seropositivity Identify Susceptibility Loci for Cervical Cancer

ABSTRACTInfection by high‐risk human papillomavirus is known to exacerbate cervical cancer development. The host immune response is crucial in disease regression. Large‐scale genetic association studies for cervical cancer have identified few susceptibility variants, mainly at the human leukocyte antigen locus on chromosome 6. We hypothesized that the host immune response modifies cervical cancer risk and performed three genome‐wide association analyses for HPV16, HPV18 and HPV16/18 seropositivity in 7814, 7924, and 7924 samples from the UK Biobank, followed by validation genotyping in the German Cervigen case‐control series of cervical cancer and dysplasia. In GWAS analyses, we identified two loci associated with HPV16 seropositivity (6p21.32 and 15q26.2), two loci associated with HPV18 seropositivity (5q31.2 and 14q24.3), and one locus for HPV16 and/or HPV18 seropositivity (at 6p21.32). MAGMA gene‐based analysis identified HLA‐DQA1 and HLA‐DQB1 as genome‐wide significant (GWS) genes. In validation genotyping, the genome‐wide significant lead variant at 6p21.32, rs9272293 associated with overall cervical disease (OR = 0.86, p = 0.004, 95% CI = 0.78–0.95, n = 3710) and HPV16 positive invasive cancer (OR = 0.73, p = 0.005, 95% CI = 0.59–0.91, n = 1431). This variant was found to be a robust eQTL for HLA‐DRB1, HLA‐DQB1‐AS1, C4B, HLA‐DRB5, HLA‐DRB6, HLA‐DQB1, and HLA‐DPB1 in a series of cervical epithelial tissue samples. We additionally genotyped twenty‐four HPV seropositivity variants below the GWS threshold out of which eleven variants were found to be associated with cervical disease in our cohort, suggesting that further seropositivity variants may determine cervical disease outcome. Our study identifies novel genomic risk loci that associate with HPV type‐specific cervical cancer and dysplasia risk and provides evidence for candidate genes at one of the risk loci.

A 10‐year Retrospective Analysis on HPV Genotype Switching in a Tertiary Center in China: Infection Characterization and Clinical Outcome

ABSTRACTThis study aims to investigate the phenomenon of human papillomavirus (HPV) genotype switching (HGS), assess the potential influencing factors, and evaluate the clinical impact on the severity of cervical lesions. A total of 2569 HPV positive female patients with records of more than two follow‐up visits were included from the gynecology department at the Obstetrics and Gynecology Hospital of Fudan University, covering the period from May 2012 to September 2022. Patients' age, treatments, vaccination, HPV genotypes before and after HGS, and the final pathology results from colposcopy were recorded. Multifactorial analyses and correlation tests were performed. Single HPV infections accounted for 67% of the total population, while multiple HPV infections comprised 33%. The most prevalent genotypes in single HPV infections were HPV52 (18.6%), HPV16 (12.28%), HPV58 (11.72%), HPV53 (8.63%), and HPV81 (6.81%). Among cases of multiple infections, the most common genotype combinations were HPV52 + HPV53 (3.02%), HPV52 + HPV58 (3.13%), and HPV52 + HPV81 (3.02%). HGS was detected in 38.2% of the total cases (458/1200). The status of medication treatment was not found to correlate with the occurrence of HGS. However, age, surgical treatment status, vaccination status, and the genotype of HPV infection may be correlated with HGS. HPV52, HPV58, HPV53, HPV56, and HPV81 showed a positive association with the occurrence of HGS transitioning from multiple infections to a single infection (HGS‐MS) (p < 0.05). In contrast, HPV52, HPV16, HPV58, HPV39, HPV56, and HPV18 significantly influenced the occurrence of HGS from one single infection to another (HGS‐SS) (p < 0.05), albeit negatively. Notably, only one type of HGS, HGS‐MS, demonstrated a positive correlation with the severity of cervical lesions. Our findings suggest that HPV genotype switching from multiple infections to single infections is associated with cervical intraepithelial neoplasia (CIN). Different patterns of HGS could result from specific HPV genotype infections, particularly HPV16. HGS‐MS is revealed to plays a catalytic role in the progression of cervical lesions.

Clinical Performance of OncoPredict HPV Screening Assay on Self‐Collected Vaginal and Urine Specimens Within the VALHUDES Framework

ABSTRACT The introduction of self‐sampling in cervical cancer screening has raised the importance of HPV test validation on self‐collected samples. This study aimed to evaluate the clinical accuracy of the OncoPredict HPV Screening (SCR) assay on self‐collected vaginal and first‐void urine (FVU) samples, relative to cervical specimens, using the VALHUDES Framework. FVU and vaginal self‐samples followed by a clinician‐collected cervical brushing were collected from 500 women referred to colposcopy and tested using OncoPredict HPV SCR assay. The assay demonstrated clinical sensitivity to detect cervical intraepithelial neoplasia grade 2 or worse (≥ CIN2) similar to cervical samples in FVU (ratio: 0.95, [95% CI: 0.88–1.02]) and vaginal self‐samples (ratio: 0.96 [95% CI: 0.90–1.02]). The clinical specificity for < CIN2 was lower in vaginal (ratio: 0.90 [95% CI: 0.84–0.96]) but not in FVU samples (ratio: 1.03 [95% CI: 0.96–1.12) when compared to cervical samples. However, the relative specificity improved following cut‐off optimization (ratio: 0.94, 95% CI: [0.88–1.01]). Moderate to excellent agreement in HPV detection between self‐collected and cervical samples was demonstrated (Kappa values: 0.53–1.00). To conclude, OncoPredict HPV SCR assay demonstrated similar accuracy on FVU and cervical samples. On vaginal compared to cervical samples sensitivity was similar with a lower specificity, which improved with cut‐off optimization.

Risk‐stratified management of cervical high‐grade squamous intraepithelial lesion based on machine learning

AbstractThe concordance rate between conization and colposcopy‐directed biopsy (CDB) proven cervical high‐grade squamous intraepithelial lesion (HSIL) were 64−85%. We aimed to identify the risk factors associated with pathological upgrading or downgrading after conization in patients with cervical HSIL and to provide risk‐stratified management based on a machine learning predictive model.This retrospective study included patients who visited the Obstetrics and Gynecology Hospital of Fudan University from January 1 to December 31, 2019, were diagnosed with cervical HSIL by CDB, and subsequently underwent conization. A wide variety of data were collected from the medical records, including demographic data, laboratory findings, colposcopy descriptions, and pathological results. The patients were categorized into three groups according to their postconization pathological results: low‐grade squamous intraepithelial lesion (LSIL) or below (downgrading group), HSIL (HSIL group), and cervical cancer (upgrading group). Univariate and multivariate analyses were performed to identify the independent risk factors for pathological changes in patients with cervical HSIL. Machine learning prediction models were established, evaluated, and subsequently verified using external testing data.In total, 1585 patients were included, of whom 65 (4.1%) were upgraded to cervical cancer after conization, 1147 (72.4%) remained having HSIL, and 373 (23.5%) were downgraded to LSIL or below. Multivariate analysis showed a 2% decrease in the incidence of pathological downgrade for each additional year of age and a 1% increase in lesion size. Patients with cytology > LSIL (odds ratio [OR] = 0.33; 95% confidence interval [CI], 0.21–0.52), human papillomavirus (HPV) infection (OR = 0.33; 95% CI, 0.14–0.81), HPV 33 infection (OR = 0.37; 95% CI, 0.18–0.78), coarse punctate vessels on colposcopy examination (OR = 0.14; 95% CI, 0.06–0.32), HSIL lesions in the endocervical canal (OR = 0.48; 95% CI, 0.30–0.76), and HSIL impression (OR = 0.02; 95% CI, 0.01–0.03) were less likely to experience pathological downgrading after conization than their counterparts. The independent risk factors for pathological upgrading to cervical cancer after conization included the following: age (OR = 1.08; 95% CI, 1.04–1.12), HPV 16 infection (OR = 4.07; 95% CI, 1.70–9.78), the presence of coarse punctate vessels during colposcopy examination (OR = 2.21; 95% CI, 1.08–4.50), atypical vessels (OR = 6.87; 95% CI, 2.81–16.83), and HSIL lesions in the endocervical canal (OR = 2.91; 95% CI, 1.46–5.77). Among the six machine learning prediction models, the back propagation (BP) neural network model demonstrated the highest and most uniform predictive performance in the downgrading, HSIL, and upgrading groups, with areas under the curve (AUCs) of 0.90, 0.84, and 0.69; sensitivities of 0.74, 0.84, and 0.42; specificities of 0.90, 0.71, and 0.95; and accuracies of 0.74, 0.84, and 0.95, respectively. In the external testing set, the BP neural network model showed a higher predictive performance than the logistic regression model, with an overall AUC of 0.91. Therefore, a web‐based prediction tool was developed in this study.BP neural network prediction model has excellent predictive performance and can be used for the risk stratification of patients with CDB‐diagnosed HSIL.

Criteria for second generation comparator tests in validation of novel HPV DNA tests for use in cervical cancer screening

Abstract While HC2 and GP5+/6+ PCR‐EIA were pivotal in test validation of new HPV assays, they represent the first generation of comparator tests based upon technologies that are not in widespread use anymore. In the current guideline, criteria for second‐generation comparator tests are presented that include more detailed resolution of HPV genotypes. Second‐generation comparator tests should preferentially target only the 12 genotypes classified as carcinogenic (IARC‐group I), and show consistent non‐inferior sensitivity for CIN2+ and CIN3+ and specificity for ≤CIN1 compared to one of the first‐generations comparators, in at least three validation studies using benchmarks of 0.95 for relative sensitivity and 0.98 for relative specificity. Validation should take into account used storage media and other sample handling procedures. Meta‐analyses were conducted to identify the assays that fulfill these stringent criteria. Four tests fulfilled the new criteria: (1) RealTime High‐Risk HPV Test (Abbott), (2) Cobas‐4800 HPV test (Roche Molecular System), (3) Onclarity HPV Assay (BD Diagnostics), and (4) Anyplex II HPV HR Detection (Seegene), each evaluated in three to six studies. Whereas the four assays target 14 carcinogenic genotypes, the first two identify separately HPV16 and 18, the third assay identifies five types separately and the fourth identifies all the types separately.

p16/Ki67 dual stain triage versus cytology in primary human papillomavirus‐based cervical cancer screening with limited genotyping

AbstractThe introduction of primary human papillomavirus (HPV) cervical cancer screening requires the implementation of an appropriate triage strategy that will be effective in detecting high‐grade cervical disease without losing diagnostic specificity. From the 30.066 screening tests results, a total of 1086 with available high‐risk human papillomavirus (HRHPV) with limited genotyping, cytology, and p16/Ki67 dual‐stain were selected. Two triage strategies for primary HPV screening were analyzed retrospectively based on the study group. Performance characteristics for p16/Ki67 and cytology triage in the detection of cervical intraepithelial neoplasia grade 2 or worse (CIN2+) and grade 3 or worse (CIN3+) were calculated, detected in colposcopic biopsy. In HPV16/18‐positive cases, primary HPV with p16/Ki67 triage was significantly more specific than cytology (53.1%/16.8% for CIN2+; p < 0.0001; 45.9%/17.0% for CIN3+; p < 0.0001), with yielded sensitivity (95.7%/84.8% for CIN2+; p = 0.0955; 100.0%/87.5% for CIN3+; p = 0.0832). In other HRHPV‐positive cases (N16/N18), p16/Ki67 triage was also significantly higher specific (51.3%/15.3% for CIN2+; p < 0.0001; 44.5%/16.5% for CIN3+; p < 0.0001), with sensitivity (92.3%/74.4% for CIN2+; p = 0.0522; 90.9%/81.8% for CIN3+; p = 0.5637). Diagnostic predictive values were significantly higher for p16/Ki67 triage with the highest PPV in HPV16/18‐positive cases for CIN2+ (45.4%; 95% confidence interval [CI]: 35.2–55.8; p < 0.0001) and very high NPV in all HPV‐positive cases regardless of detected genotype (96.3%–100.0%). The risk (1‐NPV) for CIN3+ in HRHPV16/18‐positive/p16/Ki67‐negative women was 0.0%. Superior diagnostic performance compared to cytology for detecting cervical cancer precursors indicates that p16/Ki67 dual‐immunostain may be a highly effective tool of triage in primary HPV screening with limited HPV 16/18 genotyping in secondary cervical cancer prevention.

Clinical and Analytical Evaluation of the Abbott Alinity m HR HPV Assay in a New Generation First‐Void Urine Collector

ABSTRACTUrine‐based self‐sampling approaches can simplify cervical screening programs whilst increasing response. This study reports on the performance of Abbott Alinity m HR HPV on urine, self‐collected at home using a new generation first‐void urination device that is suitable for postal delivery (Novosanis Colli‐Pee Small Volumes). First‐void urine and paired cervical samples from 297 females attending colposcopy (age 25–65, NCT04530201) were analysed for the presence of Human Papillomavirus (HPV) DNA. Cervical disease was confirmed by colposcopy and/or histology. HPV testing on first‐void urine was less sensitive for high‐grade cervical intraepithelial neoplasia (CIN2 +; ratio 0.91; 95% CI: 0.83–0.99), though equally specific (< CIN2; ratio 1.04; 95% CI: 0.92–1.19) compared to cervical samples at the manufacturer established cut‐off for cervical samples. Adjusting the cut‐off for first‐void urine improved sensitivity for CIN2+ (ratio 0.96; 95% CI: 0.90–1.03), whilst maintaining equal specificity compared to cervical samples (ratio 1.00; 95% CI: 0.88–1.14). Cohen's kappa agreements of HPV outcomes between sample pairs were good to excellent at both cut‐offs (range: 0.64–0.85). Using the HPV test's adjusted cutoff for first‐void urine, no difference in clinical sensitivity or specificity was observed between first‐void urine and cervical samples. These data highlight the importance of evaluating self‐sample‐specific cut‐offs for HPV assays, previously validated on cervical samples.

Impact of a carrageenan gel on viral load of genital human papillomavirus infections in sexually active women: Findings from the Carrageenan‐gel Against Transmission of Cervical Human papillomavirus (CATCH) trial

AbstractPrevious research has shown that women's use of a carrageenan gel reduces the risk of acquiring genital human papillomavirus (HPV) infections but does not help to clear existing ones. Although gel use may not result in complete clearance, it may decrease the viral load of HPV infections. We tested this hypothesis in the Carrageenan‐gel Against Transmission of Cervical Human papillomavirus (CATCH) randomized controlled trial. Participants of the CATCH study were selected for viral load testing if they had completed the first four study visits and tested positive for HPV42 or HPV51 in at least one of these visits. HPV42 and HPV51 were chosen as they were among the most abundant low‐ and high‐risk types, respectively, in the study sample. We measured viral load with a type‐specific real‐time polymerase chain reaction. Results were displayed using summary statistics. Of 461 enrolled participants, 39 were included in the HPV42 analysis set and 56 in the HPV51 analysis set. The median time between visits 1 and 4 was 3.7 months. The viral load (copies/cell) of HPV42 ranged from <0.001 to 13 434.1, and that of HPV51 from <0.001 to 967.1. The net median change in HPV42 viral load over all four visits was −1.04 copies/cell in the carrageenan and −147 copies/cell in the placebo arm (Wilcoxon rank sum test, p = 0.26). There was no net median change in HPV51 viral load over all four visits in either arm (p = 0.45). The use of a carrageenan‐based gel is unlikely to reduce the viral load of HPVs 42 or 51.

Improving Efficiency in Dutch Cervical Screening by Genotyping: An Analysis of Real World Program Data

ABSTRACT High‐risk human papillomavirus (hrHPV)‐genotype specific risk stratification may improve cervical screening efficiency. This study evaluates the risks of cervical intraepithelial neoplasia (CIN), cancer and unnecessary referrals by hrHPV‐genotype in cytology‐positive (ASCUS+) women, using data from the Dutch population‐based cervical screening program. Data from hrHPV+/ASCUS+ women screened between January 2017 and March 2018 were analyzed using the Dutch Screening and Pathology databases. Risks for CIN2+/3+, cancer, and unnecessary referral (i.e., without CIN2+) were evaluated by hrHPV‐genotype (HPV16, HPV18, hrHPV‐other (i.e., non‐16/18 hrHPV), or mixed HPV16/18) using logistic regression, adjusted for age, laboratory (as proxy for region), sampling method (self‐ vs. clinician sampling), and stratified by age (< 50/≥ 50 years). HPV16+ women had 3.7 (CI: 3.42–3.95) and 4.6 (CI: 4.24–4.99) times higher risks of CIN2+ and CIN3+, respectively, compared to hrHPV‐other. HPV18+ women had 1.6 (CI: 1.43–1.79) and 1.9 (CI: 1.68–2.18) times higher risks. The cervical cancer risk was tenfold higher for both HPV16 (OR: 9.85, CI: 6.50–14.95) and HPV18 (OR: 10.27, CI: 6.33–16.68). Women with HPV16 had 70% and HPV18 40% lower risks of unnecessary referral, compared to hrHPV‐other. All risk differences between HPV16 or HPV18 and hrHPV‐other were statistically significant in both age groups (< 50 and ≥ 50 years). Given the significantly higher risk of CIN2+/3+ and cancer associated with HPV16 and HPV18 and the reduced likelihood of unnecessary referrals compared to hrHPV other, these findings support the use of genotype‐based colposcopy referrals in cervical screening to enhance screening efficiency.

Nobel calling Harald zur Hausen

Characteristics of high‐risk HPV infection in women with vaginal intraepithelial neoplasia in Beijing, China

AbstractWe evaluated the characteristics of high‐risk human papillomavirus (Hr‐HPV) infection in different grades of vaginal intraepithelial neoplasia (VaIN). 7469 participants were involved in this study, of which 601 were diagnosed with VaIN, including single vaginal intraepithelial neoplasia (s‐VaIN, n = 369) and VaIN+CIN (n = 232), 3414 with single cervical intraepithelial neoplasia (s‐CIN), 3446 with cervicitis or vaginitis and 8 with vaginal cancer. We got those results. First, the most popular HPV genotypes in VaIN were HPV16, 52, 58, 51, and 56. Second, our study showed that higher parity and older age were risk factors for VaIN3 (p < 0.005). Third, the median Hr‐HPV load of VaIN+CIN (725) was higher than that of s‐CIN (258) (p = 0.027), and the median Hr‐HPV load increased with the grade of VaIN. In addition, the risk of VaIN3 was higher in women with single HPV16 infections (p = 0.01), but those with multiple HPV16 infections faced a higher risk of s‐VaIN (p = 0.003) or VaIN+CIN (p = 0.01). Our results suggested that women with higher gravidity and parity, higher Hr‐HPV load, multiple HPV16 infections, and perimenopause or menopause status faced a higher risk for VaIN, while those with higher parity, single HPV16 infections, and menopause status are more prone to VaIN3.

Discovery of a small‐molecule inhibitor targeting the ovarian tumor domain of a novel Tamdy orthonairoviruse associated with human febrile illness

AbstractTick‐borne orthonairoviruses have been characterized as a global health threat to humans and animals. Tacheng Tick virus 1 (TcTV‐1) from this family was provided as evidence that is associated with the febrile illness syndrome. Here, we first identify and demonstrate that the ovarian tumor (OTU) domain of TcTV‐1 has remarkable deubiquitinating activity both in vitro and in vivo. By solving the crystal structure of TcTV‐1 OTU (tcOTU) domain and comparing it to that of human deubiquitinating enzymes, we found that overall structures of tcOTU and human OTU family are similar, but the residues involved in the catalytic pocket vary widely. Based on the tcOTU domain we screened 5090 bioactive compounds and found mecobalamin had a good effect on suppressing the deubiquitinating activity. The structural model of tcOTU and mecobalamin suggests that mecobalamin occupies the site of the substrate Ub, by blocking the substrate binding to the enzyme. Thus, our results showed OTU domain of TcTV‐1 has a robust deubiquitinating activity and mecobalamin or its derivatives might be promising candidates for the treatment or prevention of disease caused by the TcTV‐1 virus.

Genetic variation of E6 and E7 genes of human papillomavirus 52 from Central China

AbstractCervical cancer is the fourth most common malignant tumor in women worldwide and is closely related to human papillomavirus (HPV). Women have the highest susceptibility to HPV‐52 type in Jingzhou, China. In this study, E6‐E7 sequences of 183 HPV‐52 positive samples were amplified by a polymerase chain reaction and sequenced. HPV‐52 E6‐E7 gene variations were analyzed. The phylogenetic tree was constructed using the Kimura 2‐parameter method. The secondary structure of the protein was analyzed. The selective pressure to E6‐E7 genes was estimated using PAML. In addition, the B cell epitopes of the E6‐E7 sequences in HPV‐52 were predicted by the ABCpred server. In E6 sequences, 15 single nucleotide variants were observed, including 6 nonsynonymous variants and 9 synonymous variants. In E7 sequences, 19 single nucleotide variants occurred, including 10 nonsynonymous variants and 9 synonymous variants. Six amino acid variants, including 3 nonconservative substitutions, were found in sequences encoding the alpha helix. Eight amino acid variants, including three nonconservative substitutions, occurred in sequences encoding the strand. Through phylogenetic analysis, the E6‐E7 sequences were mainly distributed in B lineage. In HPV‐52 E6‐E7 sequences, no positively selected site was found. The nonconservative substitutions, such as K93R, K93E in E6, T37I, and D38N in E7, affected multiple hypothetical epitopes in the B cell. This study provides information for the investigation of HPV epidemic characters. The discovery of new variants of HPV‐52 may lay the basis for the development of the virus diagnosis, further study of cervical cancer, and vaccine design in Central China.

The prevalence of VAIN, CIN, and related HPV genotypes in Japanese women with abnormal cytology

AbstractVaginal intraepithelial neoplasia (VAIN) is often found by chance. We investigated the prevalence of VAIN and related human papillomavirus (HPV) types in comparison with cervical intraepithelial neoplasia (CIN). This study enrolled 648 women who were referred to the outpatient clinic of Kanazawa Medical University Hospital for abnormal cytology from January 2009 to January 2019. HPV genotypes were determined using Genosearch‐31 + 4, which can detect 35 different HPV types. Colposcopy was performed at the first visit by an experienced gynecological oncologist. Among 611 subjects with squamous cell lesions, 107 (17.5%) VAIN cases were identified, and 67 (11.0%) women had both VAIN and CIN. Ultimately, 72 VAIN1, 15 VAIN2/3, 203 CIN1, 249 CIN2/3, 32 cervical squamous cell carcinomas (SCC), and one vaginal SCC (Vag‐SCC) were identified. The prevalences of VAIN1, VAIN2/3, and Vag‐SCC were 35.5%, 6.0%, and 3.1% of equivalent cervical lesions, respectively. The VAIN patients were older than the CIN patients (P = .002). About half of the VAIN cases were diagnosed during the follow‐up. Multiple HPV infections were found in 42.9% of the VAIN and CIN patients. HPV52, 16, 51, 53, and 56 were the most common types in VAIN, whereas HPV16, 52, 58, 51, and 31 predominated in CIN. HPV18 was rare in VAIN, HPV58 was more common in CIN than in VAIN, and HPV53 and HPV73 were more common in VAIN. In conclusion, VAIN1 was identified more frequently than we expected. Various HPV types were identified in the vagina, which is likely a reservoir for HPV.

Correlation between human papillomavirus infection and histopathological diagnosis of women in Northeast Brazil

AbstractCervical carcinoma is the fourth leading cause of death among women worldwide. Epidemiological studies claim that human papillomavirus (HPV) infection is a necessary condition for cervical cancer development. Knowledge of the geographic distribution of HPV is important in guiding the introduction of prophylactic vaccines. This study analyzed the prevalence of HPV infection in cervical samples obtained from women with abnormal cervical histopathological diagnosis in Northeast Brazil. The study included an analysis of 211 women whose diagnosis was confirmed for cervical intraepithelial neoplasia type 1 (CIN‐1), cervical intraepithelial neoplasia type 2 (CIN‐2), cervical intraepithelial neoplasia type 3 (CIN‐3), and cancer. The identification of the HPV genotypes was based on the polymerase chain reaction–restriction fragment length polymorphism technique. A total of 42.7% of the samples showed a single HPV infection, while 57.3% showed multiple infections. The most common genotypes detected were HPV‐16, HPV‐18, and HPV‐31. HPV‐16, HPV‐31, HPV‐35, and HPV‐18 were the most common types in CIN‐1 with a single infection. HPV‐16 and HPV‐18 were the most often found in CIN‐2 with a single infection. HPV‐16, HPV‐18, and HPV‐31 were the most detected in CIN‐3 with a single infection. HPV‐16 and HPV‐31 were the most frequent in cancer with a single infection. Multiple infection with HPV‐16 shows a 2.7 times greater risk of CIN‐3 (P = .04). Multiple infections for HPV with HPV‐16 and excluding the HPV18/31 types, were associated with CIN‐3 (P = .01). The results allowed the detection and genotyping of HPV types circulating in the population studied. These findings must be taken into account when devising vaccination strategies against HPV.

Lineage analysis of human papillomavirus type 18 based on E6 region in cervical samples of Iranian women

AbstractDistinct human papillomavirus (HPV) 18 variants are thought to differ in oncogenic potential and geographic distribution. As such, understanding the regional variants of HPV 18 would be of great importance for evolutionary, epidemiological, and biological analysis. In this regard, the sequence variations of E6 gene were investigated to characterize more common variants of HPV 18 in normal cells, premalignant, and malignant samples collected from the cervix. In total, 99 samples of HPV 18 were analyzed by polymerase chain reaction and sequencing. In overall, lineages A was identified in all study subjects, among which sublineage A4 was dominant although the difference observed was not statistically significant with regard to different stages of disease. Sublineage A4 comprised 90.9% of samples and the remaining were belonged to sublineages A1, A2, A3, and A5 at the frequency of 6.1%, 1%, 1%, and 1%, respectively. In conclusion, our findings clearly highlight the sublineage A4 of HPV 18 as the most dominant variant in Iran.

Development of a sensitive and specific nanoparticle‐assisted PCR assay for detecting HPV‐16 and HPV‐18 DNA

AbstractCarcinoma precursor lesion caused by persistent infection of human papillomavirus (HPV) types 16 and 18 is known as a principal inducer of cervical cancer. Therefore, rapid and effective detection of HPV‐16 and HPV‐18 infection at early stage is an important strategy for preventing such disease. In this study, a novel duplex nanoparticle‐assisted polymerase chain reaction (nanoPCR) assay was developed to detect both of the two genotypes simultaneously. Two pairs of primers for nanoPCR were designed based on the conserved region within the early 6 (E6) gene of HPV‐16 and HPV‐18, respectively. After optimizing reaction conditions, the nanoPCR assay displayed 10‐fold more sensitive than that of conventional PCR and showed high specificity. The detection limit of nanoPCR was 1.7 × 101 copies/μL for HPV‐16, 1.2 × 102 copies/μL for HPV‐18, and no cross‐reaction was detected after using other viruses or HPV subtypes as templates. Of 209 clinical samples collected from patients, as also confirmed by sequencing, the nanoPCR method gave consistent results with conventional PCR assay: 7 positives for HPV‐16, 4 positives for HPV‐18, and no co‐infection. Here is the first report to introduce a reproducible nanoPCR assay for detecting HPV DNA with high sensitivity and specificity, which may point out a useful diagnostic tool for potential clinical application.

Epidemiology and persistence of cervical human papillomavirus infection among outpatient women in Heilongjiang province: A retrospective cohort study

AbstractAs persistent carcinogenic human papillomavirus (HPV) infection is a prominent driver of cervical cancer, it is essential to explore HPV persistence and its associated factors for cancer screening and prevention. A retrospective cohort study was performed in outpatient women between March 2010 and 2019 in Heilongjiang, northeast China. HPV genotyping was performed by polymerase chain reaction‐membrane hybridization. An unconditional logistic regression model was used to analyze the association of factors with persistence. The overall prevalence of HPV at baseline was 27.1%, with a downward trend from 2010 to 2019 (P <  .0001). The most commonly observed high‐ and low‐risk HPVs were HPV16 (N = 1094, 5.9%) and HPV11 (N = 596, 3.2%), respectively. The probabilities of 6‐month persistence were high for women infected with HPV16 (P = .0001), HPV58 (P = .018), and HPV53 (P = .014), as well as for women with multiple infections (P = .009), and those who were 51 to 60 years old (P = .004) or more than  60 years old (P = .007). The probabilities of 12‐month persistence were high for women infected with HPV53 (P = .017) and 51‐ to 60‐year‐old women (P = .044). HPV16 is the dominant HPV type in Heilongjiang. An age in the range of 51 to 60 years and infection with HPV53 is associated with HPV infection persistence in the Heilongjiang population.

Koilocytic changes are not elicited by human papillomavirus genotypes with higher oncogenic potential

AbstractKoilocytes are considered a common cytopathological effect in patients with human papillomavirus (HPV) infection. Thus, we aimed to elucidate whether koilocytes are common to all HPV infections. Liquid‐based cytology samples from 651 patients with abnormal Papanicolaou (Pap) test results were used to analyze the presence of koilocytes and HPV genotype. HPV genotype was determined in complete liquid cytology samples and microdissected cell samples from Pap smear slides using the uniplex E6/E7 polymerase chain reaction method, which can detect 39 mucosal HPV genotypes. Koilocytes were found in 29.3% (191) of all patients. Logistical regression analysis of diverse HPV genotypes revealed that infections with low‐risk HPV types (HPV‐6b, HPV‐40, HPV‐42, HPV‐61, HPV‐74, HPV‐89, and HPV‐90), probably high‐risk HPV types (HPV‐53 and HPV‐66), and high‐risk types (HPV‐39 and HPV‐56) were significantly associated with the presence of koilocytes. However, HPV‐16, HPV‐18, and HPV‐52, which have higher oncogenic potential, were not found to be associated with koilocytes. These results were confirmed by HPV genotyping using microdissected koilocytes in 27 patients.Most common high‐risk types belonging to α‐9 and α‐7 genotypes appear to rarely induce koilocytic changes. Therefore, koilocytes may provide additional useful information for predicting the risk of progression to high‐grade lesions.

Prevalence and genotype distribution of human papillomavirus in women with cervical cancer or cervical intraepithelial neoplasia in Henan province, central China

AbstractTo evaluate the prevalence of human papillomavirus (HPV) infection and its genotype among women with cervical lesions in Henan Province, central China. A total of 1317     cervical scrapes from patients with cervical intraepithelial neoplasia 1 (CIN1) (n = 91), CIN2/3 (n = 466), and cervical cancer (CC; n = 760) were collected from 2013 to 2018, and then tested for HPV genotypes using polymerase chain reaction followed by flow‐through hybridization assay. The prevalence of HPV was 62.64% for patients with CIN1, 86.91% for patients with CIN2/3%, and 89.21% for patients with CC. In total, the HPV prevalence was 86.56%, and the most common HPV type was HPV16 (58.77%) followed by HPV58 (10.33%), 18 (7.67%), 52 (6.61%), and 33 (5.54%). In this study, the high‐risk HPV cumulative attribution rate of nine‐valent vaccine coverage was markedly higher than that of bivalent or quadrivalent vaccine coverage in each histopathological category or overall (P < .001). Single HPV infection was the main infection category in each histopathological diagnosis, and the total infection rate was 65.83% (867/1317; P < .001). The prevalence of HPV16 or single HPV infection increased with the severity of cervical lesions (P < .001). HPV16, 58, 18, 52, and 33 may be predominant high‐risk factors for cervical lesions in Henan Province. The nine‐valent prophylactic HPV vaccine is more effective than a bivalent or quadrivalent vaccine for protecting women from CC in the region.

Characterization of major capsid protein (L1) variants of Human papillomavirus type 16 by cervical neoplastic status in Indian women: Phylogenetic and functional analysis

AbstractThe etiological role of infection with Human papillomavirus type 16 (HPV16) in cervical cancer is well established. HPV16 variants, classified based on less than 10% nucleotide variations in the major capsid (L1 ORF) are known to contribute to persistent infection leading to cancer development. L1 protein forms the cornerstone of HPV structure and antigenicity. In the present study, HPV16 L1 variants were characterized by cervical lesion grade and variations in sequences were correlated to structure and function. The L1 gene was analyzed in 152 HPV16 positive cervical samples obtained from Indian women using polymerase chain reaction‐directed sequencing. Phylogenetic analysis was carried out for lineage typing. Sixty‐one SNPs were detected in L1 genes resulting in 20 nonsynonymous amino acid substitutions of which N56T, N92T, L158F, V178G, N181I, K236T, K443Q, K454T, and K475R are reported in Indian isolates for the first time. The substitutions N181T, T353P, and T389S were significantly associated with high‐grade cervical disease. The predominance of lineage A (A1‐A4, 84.96%) was observed among the isolates, while the D3 sublineage showed significant association with high‐grade cervical lesions. No evidence for recombination and the positive selection was obtained. These substitutions, when mapped on three‐dimensional structure, revealed that 11 and 4 substitutions are part of experimentally validated B‐ and T‐cell epitopes, of which T266A and N285T were common to both types of epitopes and may impact HPV vaccine efficacy. The variants identified through this study have the potential to serve as translational leads for designing diagnostic probes and vaccines.

The papillomavirus E5 gene does not affect EGFR transcription and overall survival in cervical cancer

AbstractIntroductionThe human papillomavirus (HPV) E5 gene encodes a small and highly hydrophobic oncoprotein that affects immune evasion, cell proliferation, loss of apoptotic capacity and angiogenesis in tumors. E5 shows an affinity for biological membranes and was associated with an increase of epidermal growth factor/epidermal growth factor receptor (EGF/EGFR) signaling through the accumulation of EGFR in cellular membranes. Due to the frequent integration of the HPV genome into the host cell genome, E5 is frequently not transcribed in cervical tumors.AimIn this study we looked forward to verifying whether the potential expression of E5 protein in human papillomavirus 16 positive (HPV16+) and human papillomavirus 18 positive (HPV18+) cervical tumors was associated with levels of EGFR and vascular endothelial growth factor A (VEGFA) transcription and with patients overall survival.ResultsAssociation between the presence of E5 transcripts and viral genome disruption was observed for HPV16+ and HPV18+ tumors. Association was not observed between tumors potentially capable of translating E5 and EGFR or VEGFA transcriptional levels. Similarly, the capability of translating E5 and overall survival in patients with HPV16+ squamous cell carcinoma tumors stage ≥ IB2 were not associated.ConclusionThe likely presence of E5 transcripts was neither associated to a higher activity of the EGFR‐VEGFA pathway nor to the overall survival of patients with HPV16+ squamous cell carcinoma in stages ≥ IB2.

Genetic variability and functional implication of HPV16 from cervical intraepithelial neoplasia in Shanghai women

AbstractHuman papillomavirus (HPV)16 gene mutation is usually associated with persistent HPV infection and cervical intraepithelial neoplasia (CIN). However, the functional implications of HPV16 mutations remain poorly understood.145 LCR/E6/E7 of the HPV16 isolates were amplified and sequenced, and HPV16 integration status was detected. In total, 89 SNPs (68 in the LCR, 13 in E6, 8 in E7) were discovered, 11 of which were nonsynonymous mutations (8 in E6, 3 in E7). The H85Y and E120D variants in E6 were significantly reduced in the high‐grade squamous intraepithelial lesion (HSIL) group compared to the <HSIL group (P = .046 and .005), conversely the N29S in E7(P = .01). Amino acid substitutions (D32N/E, E36Q, H85Y, and E120D in E6 and N29H/S and R77C in E7) were predicted to have an effect on conserved structural and functional residues, and five amino acid substitutions (H85Y, E36Q, I34L, and D32E in E6; R77C in E7) would potentially change the secondary structure. “6329G>T,” a potential binding site for TATA‐binding protein, is the most common in LCR variants. A4 (Asian) was associated with an increased risk of HSIL compared to A1–3(P = .009). The H85/E120 in E6 and N29 in HPV16 E7 might play a critical role in carcinogenesis by disrupting p53 and Rb degradation due to affecting their interaction, respectively. In a word, the findings in this study provide preventative and therapeutic interventions of HPV16 ‐related cervical lesions/cancer.

Comparison of the performance of paired urine and cervical samples for cervical cancer screening in screening population

AbstractThe main objective of this work is to determine the performance of urine for human papillomavirus (HPV) detection in cervical cancer screening in screening population. Paired urine and cervical samples were collected from 2038 women (careHPV group: 1002, cobas4800 group: 1036) in 2015. Urine was tested by a new urine‐based HPV test and cervical samples by careHPV or cobas4800 HPV test. Women were triaged based on cervical results and then referred to colposcopy with biopsy as clinically indicated. In 2017, women were followed up and screened with cotesting strategy, women with any positive would be referred and biopsied if necessary. In careHPV group, the HPV prevalence of urine was 14.1%, and 16.4% for cervical samples. In cobas4800 group, it was 19.1% and 20.4%, correspondingly. The concordance of urine samples compared with cervical samples was moderate (careHPV group: 86.6%; κ = 0.48; cobas4800 group: 83%; κ = 0.46). The baseline sensitivity and specificity for urine against CIN2+  detection were 85.7%, 86.8% in careHPV group, and 69.2%, 82.3% in cobas4800 group, respectively. Cervical samples were 100% sensitive for both tests (careHPV and cobas4800) and 85.2% specific in careHPV group and 81.9% specific in cobas4800 group, respectively. The corresponding cumulative sensitivity and specificity were 68.8% and 87.1%, 58.8% and 81.9%, 87.5% and 85.5%, and 94.1% and 81.4%. Urine demonstrated certain potential in cervical cancer screening and could be an alternative if no better screening strategies available.

Prevalence of high‐risk human papillomavirus and cervical lesion risk factors: A population‐based study in Zhejiang, China 2010–2019

AbstractThis study investigates the epidemiological characteristics of high‐risk human papillomavirus (hrHPV) and analyzes the risk of cervical lesions among women in Zhejiang province, China. HPV data were collected retrospectively from a cohort of 67 742 women who underwent routine cervical cancer screening from 2010 to 2019. Precancerous and cervical cancer cases (n = 980) were histologically diagnosed as a low‐grade squamous intraepithelial lesion (LSIL; n = 341) or a high‐grade squamous intraepithelial lesion (HSIL; n = 499) and invasive cervical cancer (ICC) (n = 140) groups. Disordered logistic regression analysis was used to test the relationship between different degrees of cervical lesions, HPV16/18 infection status, positive rate of p16INK4a (p16), Ki‐67 expression, and patient's age in SIL and ICC (270/980 cases) patients. HPV52 (4.7%) was the most prevalent HPV type, followed by HPV16 (3.3%) and HPV58 (2.6%). HPV16 was the most common HPV in SIL, peaking at the age of 30–39. The HPV16 infection rate was significantly higher in HSIL than in LSIL patients; moreover, HPV16, HPV18, and HPV51 infection rates were significantly higher in ICC patients than in HSIL (Bonferroni‐adjusted p < 0.0167). The presence of HPV16/18 was also associated with a higher risk of developing HSIL from LSIL (odds ratio [OR] = 9.198, 95% confidence interval [CI]: 2.76–127.49). The increased p16 expression and HPV16/18 were associated with the increased risk of cancer progression (OR = 1.092, 95% CI: 1.03–1.36; OR = 1.495, 95% CI: 1.23–2.19, respectively). The identified hrHPV genotypes in cervical lesions can serve as a baseline indicator for future vaccine assessment in Zhejiang, China.

Recent advances in treating female genital human papillomavirus related neoplasms with topical imiquimod

AbstractHuman papillomavirus (HPV) encompasses a group of viruses that infect the skin and mucous membranes. In the presence of certain factors, persistent infection with high‐risk HPVs can trigger a process of neoplastic transformation. Imiquimod is a topical agent that acts as a Toll‐like receptor 7/8 agonist, stimulating the innate and adaptive immune system to exert antitumor and antiviral effects. It has been approved for the treatment of various skin conditions, however, its efficacy and safety in the management of HPV‐related‐neoplasms of the lower genital tract, such as vulvar, vaginal, and cervical neoplasia, are still under investigation. This review summarizes the current evidence on the use of imiquimod for the treatment of HPV‐induced lesions of the female lower genital tract, focusing on its indications, mechanisms of action, outcomes, and predictors of response.

Prevalence and genotype distribution of high‐risk HPV infection among women in Beijing, China

AbstractCervical cancer (CC) is highly associated with high‐risk human papillomavirus (HPV) infection and genotype distribution of high‐risk HPV (HR‐HPV) infection varies greatly in different regions. Clinical specimens were collected from 46 365 patients at Beijing Friendship Hospital, Capital Medical University from January 2017 to December 2020. HPV DNA genotype testing was performed using real‐time PCR. The infection rates based on disease group were compared using the χ 2 test. The linear‐by‐linear association test and gamma value were used to assess the changes in HPV prevalence over calendar year and age group. A total of 10 514 women were infected with HR‐HPV, with an overall positive rate of 22.7%. The most prevalent HR‐HPV types were HPV52, 58, 16, 51, and 66, and HPV59 had a higher prevalence except for HPV16, 58, and 52 in the CC group. Single infection of HR‐HPV was dominant among different disease groups. The infection rate of HR‐HPV decreased first and then increased from below 20 years old to over 60 years old. There were significant differences in the HR‐HPV infection rates among the age and disease groups. Our findings demonstrate that the genotype distribution of HR‐HPV varied with age and diseases. The HR‐HPV genotypes prevalence was found to be directly useful for local governments to promote HPV targeted vaccination in the study region.

Respective prevalence of high‐risk HPVgenotypes in cervical neoplasia in Senegal

AbstractObjectivesWith the perspective of prophylactic vaccination against high‐risk human papillomavirus (HPV), we analyzed the viral epidemiology of cervical neoplasia in Senegal.MethodsAll patients were treated at the Institut Joliot Curie du Cancer in Dakar. HPV genotypes were characterized using a real‐time polymerase chain reaction‐based approach and sequencing.ResultsHistologically, there were 224 invasive carcinomas, 17 high‐grade intraepithelial neoplasia (CIN), and five undetermined histologies. Molecular analysis was conclusive in 241 cases. HPV DNA was found in 207/241 (85.9%) cases while 34/241 (14.1%) remained HPV negative. There was one single genotype in 127/207 (61.4%) cases and several in 80/207 (38.6%) corresponding to 308 genotypes identified. Viral genotyping found HPV16 in 175 (56.8%) cases, HPV18 in 45 (14.6%), HPV45 in 40 (13.0%), HPV58 in 35 (11.4%), HPV33 in 6 (2.0%), HPV35 in 3 (1.0%), HPV31 in 2 (0.6%), HPV39 and HPV56 in one (0.3% each).ConclusionOur analysis showed that 98.4% of the HPV‐positive cases were associated with viral genotypes covered by the 9‐valent HPV vaccine. However, 14.1% of cases remained HPV negative. Therefore, prophylactic vaccination using a 9‐valent vaccine should dramatically reduce the incidence of HPV‐associated neoplasia but the detection and treatment of CIN remain necessary for the optimal prevention of cervical cancer.

Prevalence and genotype distribution of high‐risk human papillomavirus in 34 420 cases in Yangzhou city, Jiangsu province, China

AbstractWe aimed to study the infection status and distribution of human papillomavirus (HPV) in Yangzhou City to provide precise guidance for the prevention and treatment of cervical cancer in this area. Reproductive tract secretions were collected from patients admitted at Subei People's Hospital over the past 3 years. Fifteen high‐risk HPV (HR‐HPV) genotypes were analyzed by fluorescent polymerase chain reaction. The positive rate of HR‐HPV in 34 420 subjects was 23.56%. There was no significant difference in the rate of overall infection between males and females (χ 2 = 0.04; p = 0.952 > 0.05). The five genotypes with high infection rates in the population were HPV52, HPV58, HPV16, HPV51, and HPV39. Single infection was found to be dominant, primarily with the HPV52 genotype. The infection rate was higher in patients less than 20 years old and more than 60 years old. Most patients with cervical intraepithelial neoplasms 2/3 and cervical cancer were infected by HPV16, followed by those infected by HPV52 and HPV58. There was a significant difference in the infection rate of HPV16 among patients with different cervical lesions (χ 2 = 31.660; p < 0.01), and the infection rate of HPV16 was higher in patients with cervical cancer than in healthy individuals. Single infection was dominant among the study patients with HPV infection in Yangzhou city. There was no significant difference in infection rate and genotype distribution between males and females. The infection rate in young and old women was higher, and the rate increased with age (>20 years).

Prevalence characteristics of cervical human papillomavirus genotypes in Nanning, China: A 10‐year survey of 77,756 women from one medical center

AbstractObjectivesThe prevalence of human papillomavirus (HPV) infection and HPV genotypes varies in different regions. However, there is little data on HPV prevalence and genotyping in Guangxi Province, South China. This study conducted a 10‐year survey in a health center, to estimate the prevalence characteristics of HPV genotypes.MethodsBy using polymerase chain reaction (PCR) amplification and nucleic acid molecular hybridization, the HPV genotypes were detected from 77,756 females who were patients of the Department of Obstetrics and Gynecology and those who visited the Health Management Center for a physical examination between August 2011 and November 2020. The prevalence, genotypes, age‐related HPV infections, as well as chronological change of HPV prevalence, and the HPV genotype distribution were analyzed.ResultsThe overall prevalence of HPV infection was 21.14% (16,439/77,756). The HPV infection rate differed significantly between the patients of the Department of Obstetrics and Gynecology and the women who underwent a physical examination (22.98% vs. 9.88%, p < 0.05). The prevalence rates of high‐risk HPV, low‐risk HPV, mixed HPV (mixed high‐risk, and low‐risk HPV infection), and multiple HPV infections were 18.96% (14,739/77,756), 4.09% (3178/77,756), 1.90% (1478/77,756), and 4.94% (3838/77,756), respectively. The most prevalent genotypes were HPV 52, 16, and 58. The age‐associated HPV prevalence showed bimodal curves, with the first peak at <25 years and the second peak at >56 years.ConclusionsThis study provides baseline data on the HPV prevalence in the general female population of Nanning, Guangxi Province. Women <25 and >56 years old faced the greatest threat of HPV infection, and HPV 52, 16, and 58 were the most common genotypes.

Epidemiology and analysis of potential risk factors of high‐risk human papillomavirus (HPV) in Shanghai China: A cross‐sectional one‐year study in non‐vaccinated women

AbstractData regarding human papillomavirus (HPV) prevalence, its associated risk factors, and women's knowledge about this disease before the HPV vaccine was approved are limited in Shanghai, China. Therefore, we investigated these questions among females in Shanghai and aimed to provide comprehensive data to guide HPV vaccination and present the biopsychosocial risk factors that impact high‐risk HPV infection, and evaluate the level of knowledge and awareness of this disease among women aged 21–65 years old. A total of 6619 (aged from 21 to 65) women from different communities volunteered to participate in the HPV screening and complete questionnaires from December 2016 to December 2017 in the Department of Obstetrics and Gynecology of nine hospitals in Shanghai. Data were analyzed using sample logistic regression to assess biopsychosocial risk factors that impact high‐risk HPV infection and knowledge of HPV infection. A total of 632 (9.5%) cases were positive for high‐risk HPV test, 22.6% of them were HPV 16/18 infection, 77.4% of them were non HPV 16/18 infection. 40 potential risk factors may be related to high‐risk HPV infection, and there were 19 factors' p value < 0.1 from single factor logistic analysis. Finally, multivariable regression revealed education level, type of vaginitis, history of hyperlipidemias, family history of cancer, number of pregnancies, number of sex partners were independent risk factors for high‐risk HPV infection (p < 0.05). When stratified by education level, women who finished graduate school had significantly greater knowledge of cervical cancer, cervical screening, and the relationship between HPV and cervical cancer than other groups (p < 0.05). The prevalence rate of high‐risk HPV was a little lower than other regions in China and other countries, which may be related to regions, races, living habits, and economy. A less reported finding is that the history of vaginitis and the history of hyperlipidemias in our study were related to HPV infection. The majority of the participants had poor knowledge regarding cervical cancer, cervical screening, and the relationship between HPV and cervical cancer. Hence, these results should be served as a wake‐up call for the government to increase knowledge and awareness via the media and doctors.

Genetic variation in the E6 and E7 genes of human papillomavirus type 16 in northeastern Argentina

AbstractThe province of Misiones is considered a region with a high mortality rate due to cervical cancer (CC). To gain insight into this problem, we explored the association between genetic variation in the E6 and E7 oncogenes of HPV16 and the risk of CC. We studied 160 women with cytological diagnoses of negative for intraepithelial lesion or malignity, low‐grade squamous intraepithelial lesion, and high‐grade squamous intraepithelial lesion/CC and a positive test for HPV16 infection. The genetic characterization of E6 and E7 genes was undertaken through PCR amplification and direct Sanger sequencing. Phylogenetic classification was conducted using Bayesian methods. To estimate the odds ratio (OR) for an association between genetic variants in the E6 and E7 genes and the risk of CC, we used ordinal logistic regression adjusted by age. The final data set comprised 112 samples. Diagnostic single‐nucleotide polymorphisms (SNPs) and phylogenetic trees confirmed the presence of Lineage A (95.5%) and D (4.5%) in the samples. For the E6 gene, we identified eleven different sequences, with the most common ones being Lineage A E6 350G (58.9%) and E6 350T (37.5%). The E6 350G was associated with progression to HSIL/CC, with an OR of 19.41 (4.95–76.10). The E7 gene was more conserved than E6, probably due to the functional constraints of this small protein. Our results confirmed the association of the E6 350G SNP with a higher risk of developing CC. These data will contribute to understanding the biological bases of CC incidence in this region.

Postvaccination prevalence of vaccine‐Human Papillomavirus (vHPV) genotypes among the target population: A systematic review and meta‐analysis

AbstractHuman Papillomavirus (HPV) vaccines are safe and are highly effective in reducing the prevalence of HPV infections and subsequent HPV associated diseases in the target population. A systematic review and meta‐analysis was carried out searching electronic databases for articles published between January 2007 and September 2020 reporting the prevalence estimates of vaccine HPV (vHPV) types in women who had received one or more doses of quadrivalent or bivalent vaccines. This systematic review was based on standard systematic review guidelines and the meta‐analysis was performed by pooling the HPV vaccine type prevalence data with 95% confidence interval (CI) among 16,929 young women who had received the prophylactic HPV vaccines before the age of 27 years. The overall pooled prevalence of vHPV types was.0.04 (95% CI: 0.02, 0.05). The meta‐analysis concludes that prophylactic HPV vaccination before the age of 27 years results in a decline of vHPV types in young women.

Lineage and sublineage analysis of human papillomavirus type 56 in cervical samples of Iranian women

AbstractUnderstanding the regional lineages and sublineages of human papillomavirus type 56 (HPV 56) would be of great importance for further evolutionary, epidemiological, and biological investigations. To identify the distribution of lineages and sublineages of HPV 56 in Iran, the sequence variations of the E6 gene were analyzed in normal, premalignant, and malignant samples obtained from the cervix. In total, 58 HPV 56‐positive samples were investigated by nested‐PCR and followed by bidirectional direct nucleotide sequencing analysis. Both lineages A and B were identified in the studied samples. Lineage B was dominant as it was detected in 88.4% of all samples and the remaining samples belonged to lineage A (11.6%). Sublineages A1 and A2 were detected in 3.3% and 8.3% of all samples, respectively. With regard to the pathological stages of cervical specimens, no statistically significant differences were found in the three studied groups (p > 0.05). In conclusion, our findings showed that lineage B of HPV 56 was prevalent in Iran. However, further studies with a larger sample size are warranted to estimate the pathogenicity risk of HPV 56 lineages/sublineages to the progression of cervical cancer among Iranian women.

Risks for cervical abnormalities in women with non‐16/18 high‐risk human papillomavirus infections in south Shanghai, China

AbstractThe study was aimed to analyze the prevalence characteristics of non‐16/18 high‐risk human papillomaviruses (HR‐HPV) and the related risks for cervical abnormalities in south Shanghai. A total of 2291 HPV women who had been referred for a colposcopy due to HPV infection from @@@@@2016.12 to 2019.6 were enrolled. Combined with liquid‐based thin‐layer cell test (TCT) and pathological results of cervical biopsy, the infection spectrum and pathogenic risk of non‐16/18 HR‐HPV in local population were investigated. The results showed that the single HR‐HPV infection rate was significantly higher than that of multiple infection, and the five most frequently detected types were HPV16, HPV52, HPV18, HPV53, HPV58 in the group. The total proportion of non‐16/18 HR‐HPV infection was 68.22%, more than twice of HPV16/18. In cases with high‐grade cervical intraepithelial lesions (HSIL) or cervical cancer, non‐16/18 HR‐HPV infections account for 50.84% (single infection: 28.57%, multiple infection: 22.27%). The risk of cervical abnormalities caused by single HPV infection was ranked as HPV16 > HPV52 > HPV18 = HPV58 > HPV51 > HPV53 = HPV56 > others. Notably, among non‐16/18 HR‐HPV infected patients with HSIL/cancer lesions, the omission diagnostic rate of TCT was 62.81%. The infection rate of non‐16/18 HR‐HPV in whole study population was much higher than that of 16/18 type, and the infection rate of the former was also slightly higher in patients with HSIL and cancer. Due to the high omission diagnostic rate of TCT, we suggest patients with persistent non‐16/18 HPV infection should undergo colposcopy biopsy to reduce missed detection of HSIL and cancers.

A meta‐analysis of human papillomavirus prevalence and types among Iranian women with normal cervical cytology, premalignant lesions, and cervical cancer

AbstractIn this study, all data from Iran on human papillomavirus (HPV) prevalence and types among women with normal cervical cytology, premalignant lesions, and cervical cancer were obtained and pooled. The overall HPV prevalence was found to be 9% in women with a normal cervix, 55% in atypical squamous cells of undetermined significance or atypia cases, 58% and 69% in women with low and high grade squamous intraepithelial lesions, respectively, and 81% among women with invasive cervical cancer. In all of the studied groups, HPV 16 was the most common HPV type, followed by HPV 18. In conclusion, this meta‐analysis revealed that it will be beneficial if current HPV vaccines are integrated into the national vaccination programs of Iran.

Comparison of Aptima and hybrid capture‐2 HPV tests and Pap test in the referral population in Japan

AbstractThe Aptima human papillomavirus (HPV) test (APTIMA) detects E6‐E7 mRNA in abnormal cells in the uterine cervix. To investigate the accuracy of APTIMA for cervical cancer screening in Japan, 423 subjects, mostly referrals with abnormal cytology or being followed up for cervical intraepithelial neoplasia (CIN)1, were screened using two HPV tests, hybrid capture 2 (HC2) and APTIMA, and by the Pap test. Colposcopy was conducted in all subjects with a positive result in either test type. HPV genotyping was performed by Genosearch‐31.A result of atypical squamous cells‐undetermined significance (ASC‐US) or worse on the HC2 test (ASC‐US‐HC2), and low‐grade squamous intraepithelial lesion (LSIL) or worse (LSIL+) on the Pap test, was regarded as positive. APTIMA (97.5%) was more sensitive than LSIL+ (85.1%) for detecting CIN2 or worse (CIN2+) (McNemar test; p = .0003), and more sensitive (98.6%) than ASC‐US‐HC2 (92.7%) for detecting CIN3+. APTIMA and HC2 had similar sensitivities. HPV genotyping revealed that CIN2/3 with high‐risk HPV (HR‐HPV) was overlooked in five cases by ASC‐US‐HC2, and in four cases by HC2, while no such lesions were missed by APTIMA. Thus, APTIMA might be superior to HC2 for primary HPV screening in Japan. One cancer case positive for HPV67 (potentially high risk, [pHR]) was overlooked by Pap test and both HPV tests, suggesting a need for a new HPV test able to detect pHR‐HPV types.

Human papillomavirus infection status of single cells isolated from cervical cytology specimens by simple manual microdissection

AbstractHuman papillomavirus (HPV) testing with cytology triage for cervical cancer screening has proven to be useful. It is considered that a significant percentage of HPV‐positive women followed by reflex cytology have had multiple‐type HPV infections rather than single‐type infections. However, the effects of multiple‐type infections on changes in the cytomorphology of exfoliated cervical cells have not been investigated. The aim of this study was to validate simple manual microdissection (MMD) maneuver and investigate the HPV infection status of single cells isolated from Papanicolaou (Pap) smears prepared from women with multiple‐type infections. Using cytology samples from 90 patients with abnormal Pap smear results, we evaluated the efficiency of the MMD procedure and determined the HPV infection status of single squamous intraepithelial lesion (SIL) cells microdissected from patients with multiple‐type infection. When validating the MMD procedure, the HPV‐positive rate was 81.5% using 119 MMD samples from the Pap smear in 61 cases with single‐type infection. This MMD procedure was able to efficiently collect single cells. Of 119 MMD samples from 29 cases with multiple‐type infection, the HPV‐positive rate was 42.9%, and most (96.1%) MMD samples exhibited only one genotype. Our MMD maneuver successfully identified HPV genotypes using single cells isolated from cytology specimens. A majority of single SIL cells prepared from multiple‐type infection cases turned out to contain only one genotype. In the future, the MMD method could be applied while studying the relationship between the morphological changes exhibited by SIL cells on Pap smear and the infected HPV genotype.

Anyplex II HPV test in detection and follow‐up after surgical treatment of CIN2+ lesions

AbstractHuman papillomavirus (HPV) tests differ for technology, targets, and information on the genotype and viral load. In this study, we evaluated the performance of the Seegene Anyplex II HPV HR (Anyplex) assay in the detection of cervical intraepithelial lesions (CIN) and as a test‐of‐cure in the follow‐up after surgical treatment. One hundred and sixty‐seven women referred to the European Institute of Oncology, Milan, for surgical treatment of CIN2+ were enrolled. A cervical sample was taken before treatment and at the first follow‐up visit: on these samples, Qiagen Hybrid Capture 2 (HC2), Roche Linear Array HPV Test (Linear Array), cytology and histology were performed at baseline, HC2, and cytology at follow‐up. Anyplex genotyping HPV test was performed on a post aliquot from liquid‐based cytology specimens when available. The concordance between Anyplex and HC2 was 93.6% at baseline and 76.7% at follow‐up (3–9 months after treatment), respectively. The concordance between Anyplex and Linear Array was evaluable only at baseline (92.9%). No recurrence occurred in women without the persistence of the same genotype at follow‐up. Seven women relapsed: six had persistence of the same genotypes (five HPV16, one HPV33, and one HPV39), while one tested negative not only with Anyplex but also with HC2 for the persistence of low‐risk genotype infection (HPV73 only detected by Linear Array). Anyplex test represents a valid option for HPV detection and genotyping in order to stratify women at risk of high‐grade lesions at baseline and to monitor patients treated for CIN2+ lesions during follow‐up.

CRISPR/Cas9‐HPV‐liposome enhances antitumor immunity and treatment of HPV infection‐associated cervical cancer

Abstract Increasing evidence shows that human papillomavirus (HPV) E6/E7 deletion in cervical cancer cells may be related to the immunosuppressive tumor microenvironment and adverse reactions or resistance to immune checkpoint blockade. Here, we demonstrate that liposome delivery of CRISPR/cas9 can effectively knock out HPV, which, in turn, induces autophagy and triggers cell death‐related immune activation by releasing damage‐related molecular patterns. The results of in vivo experiments showed that HPV‐targeting guide RNA–liposomes could promote CD8+ T cell infiltration in tumor tissues; enhance the expression of proinflammatory cytokines, such as interleukin‐12, tumor necrosis factor‐α, and interferon‐γ, and reduce regulatory T cells and myeloid suppressor cells. The combination of HPV‐targeting guide RNA–liposomes with immune checkpoint inhibitors and antiprogrammed death‐1 antibodies produced highly effective antitumor effects. In addition, combination therapy induced immune memory in the cervical cancer model.

Single‐cell RNA‐sequencing dissects cellular heterogeneity and identifies two tumor‐suppressing immune cell subclusters in HPV‐related cervical adenosquamous carcinoma

AbstractThe intratumor heterogeneity of human papillomavirus (HPV)‐related cervical cancer remains poorly defined. We performed single‐cell RNA sequencing on 18 046 individual cells derived from two HPV‐related cervical adenosquamous carcinoma samples to analyze the transcriptional heterogeneity of both epithelial and immune constituents, identifying seven epithelial (Epi1‐7) and 11 immune subclusters. Based on expression of known cervical cancer markers, Epi1‐2 primarily displayed features of adenocarcinoma, whereas Epi3‐6 were instead characterized by features of squamous carcinoma. Our analyses also revealed that hypoxia and Kirsten rat sarcoma viral oncogene signaling were highly represented within Epi1; metabolic pathways mediating glycolysis and oxidative phosphorylation were enriched in Epi2‐4; while Epi5 was enriched in p53 pathway components and features of epithelial–mesenchymal transition. Moreover, CD8+FGFBP2+ T cells and FGFBP2+ natural killer cells were found to display high levels of cytotoxic effectors (GZMA, GZMB, GNLY, and PRF1) and low levels of inhibitory markers (PDCD1, TIGIT, and CTLA4), such that tumor infiltration by these populations was positively associated with survival in a cohort of n = 165 patients with HPV‐related cervical cancer from The Cancer Genome Atlas database (p = 0.017 and 0.014, respectively). These results shed new light on the intratumor heterogeneity of HPV‐related cervical adenosquamous carcinoma, which will help to refine diagnostic and treatment approaches.

Concordance between the BD Onclarity and Roche cobas assays for detection of HPV DNA in a Chinese population

AbstractAs cervical cancer screening shifts from cytology to human papillomavirus (HPV) testing, a major issue involves validating more HPV tests. In recent years, some HPV tests are used for clinical performance verification in China. The purpose of this study was to explore whether the BD Onclarity (Becton, Dickinson and Company)HPV assay differs from the Roche cobas (Roche Molecular Systems)HPV assay, as determined using 944 cervical samples, including 588 with sequencing results. In the nucleic acid assay accuracy verification, the assays showed excellent concordance for detection of HPV16 (κ = 0.93, 95% confidence interval [CI]: 0.89–0.97) and HPV18 (κ = 0.90, 95% CI: 0.83–0.97), and very good concordance for the 12 other high‐risk types (HPV31/33/35/39/45/51/52/56/58/59/66/68, κ = 0.79, 95% CI: 0.75–0.83). The overall agreement for HPV DNA detection between Onclarity and cobas was very good (κ = 0.7755). No difference for ≥CIN2 sensitivity was observed between Onclarity and cobas (both 96.5%), whereas the ≥CIN2 specificity for detection of Onclarity (16.6%, 95% CI: 13.7–19.9) was higher than that of cobas (11.5%, 95% CI: 9.1–14.5). Onclarity exhibited comparable screening performance and triage efficiency compared to cobas in the detection of cervical disease in Chinese women.

Prevalence of multiple human papillomavirus infections and association with cervical lesions among outpatients in Fujian, China: A cross‐sectional study

AbstractMultiple human papillomavirus (HPV) infections are common, but their impact on cervical lesions remains controversial. A total of 6225 female patients who underwent colposcopies/conization following abnormal cervical cancer screening results were included in the study. The final pathological diagnosis was determined by the most severe pathological grade among the cervical biopsy, endocervical curettage, and conization. Univariate and multivariate logistic regression analyses were used to investigate the association between multiple HPV infections and cervical lesions, adjusting for age, HPV genotype, gravidity and parity. In total, 33.3% (n = 2076) of the study population was infected with multiple HPV genotypes. Multiple HPV infections were more prevalent in patients younger than 25 years and older than 55 years, with the rate of multiple HPV infections at 52.8% and 44.3%, respectively. HPV16\52\18\58 are the most common HPV genotypes and usually appear as a single infection. Compared to single HR‐HPV infection, multiple HR‐HPV infections do not increase the risk of HSIL+, while single HR‐HPV coinfected with LR‐HPV seems to reduce the risk of HSIL+ (odds ratio = 0.515, confidence interval: 0.370–0.719, p < 0.001). Multiple HR‐HPV infections cannot be risk‐stratified for triage of HR‐HPV‐positive women.

Prevotella as the hub of the cervicovaginal microbiota affects the occurrence of persistent human papillomavirus infection and cervical lesions in women of childbearing age via host NF‐κB/C‐myc

AbstractThere is evidence that coinfection of cervicovaginal high‐risk human papillomavirus (HR‐HPV) and bacteria is common in women of childbearing age. However, the relationship between bacterial vaginosis (BV) and persistent HR‐HPV infection in women of childbearing age and the underlying mechanisms remain unclear. In this study, we determined whether BV affects persistent HR‐HPV infection in women aged 20–45 years and explored the possible mechanisms of their interactions. From January 1 to April 30, 2020, we recruited women aged 20–45 years with and without BV at a ratio of 1:2 from Fujian Maternity and Child Health Hospital. All women were followed up at 0, 12, and 24 months. A BV assay, HR‐HPV genotyping and cervical cytology were performed at each follow‐up. At 0 months, additional vaginal secretions and cervical exfoliated cells were collected for 16S ribosomal RNA sequencing, bacterial metabolite determination, and POU5F1B, C‐myc, TLR4, NF‐κB, and hTERT quantification. A total of 920 women were included. The abundance of Prevotella (p = 0.016) and Gardnerella (p = 0.027) were higher, whereas the abundance of Lactobacillus was lower (p = 0.001) in women with persistent HR‐HPV infection and high‐grade squamous intraepithelial lesions (HSIL). The abundance of Prevotella (p = 0.025) and Gardnerella (p = 0.018) increased in the vaginas of women with persistent HPV16 infection, whereas only the abundance of Prevotella (p = 0.026) was increased in women with persistent HPV18 infection. The abundance of Prevotella in the vagina was significantly positively correlated with the expression levels of TLR4, NF‐κB, C‐myc, and hTERT in host cervical cells (p < 0.05). Our findings suggest that overgrowth of Prevotella in the vagina may influence the occurrence of persistent HR‐HPV infection‐related cervical lesions through host NF‐κB and C‐myc signaling.

Longitudinal follow‐up of HPV16 sequence after cervical infection: Low intrahost variation and no correlation with clinical evolution

AbstractHuman papillomavirus (HPV) 16 exhibits different variants that may differ in their carcinogenic risk. To identify some high‐risk variants, we sequenced and compared HPV16 whole genomes obtained from a longitudinal cohort of 34 HPV16‐infected women who had either spontaneously cleared their infection (clearance group or “C”), or developed cervical high‐grade lesions following a viral persistence (group persistence or “P”). Phylogenetic analysis of paired samples obtained at the beginning (C0 or P0) and at the end (C2 or P2) of the follow‐up (median intervals between C0–C2 and between P0–P2 were 16 and 36.5 months, respectively) revealed a low genetic variability within the host compared to the genetic interhost diversity. By comparing our HPV16 sequences to a reference sequence, we observed 301 different substitutions, more often transitions (60.9%) than transversions (39.1%), that occurred throughout the viral genome, but with a low frequency in E6 and E7 oncogenes (10 and 9 substitutions), suggesting a high conservation of these genes. Deletions and insertions were mostly observed in intergenic regions of the virus. The only significant substitution found between the subgroups C2 and P2 was observed in the L2 gene (L330F), with an unclear biological relevance. Our results suggest a low longitudinal intrahost evolution of HPV16 sequences and no correlation between genetic variations and clinical evolution.

The prevalence and distribution of human papillomavirus in 4267 Turkish women with or without cervical lesions: A hospital‐based study

AbstractIn the present study, it was aimed to screen the genotypes of human papillomavirus (HPV) retrospectively in women with gynecological symptoms who were admitted to a tertiary care university hospital in Ankara, Turkey. A total of 4267 cervical swab samples of women aged 18–79 years were sent to Medical Virology Laboratory from January 2017 to November 2020. Nucleic acid extraction and amplification of samples were done by an automated system. The test can detect 14 high‐risk HPV (HR‐HPV) types in a single analysis that performs a real‐time polymerase chain reaction, by providing individual results on the highest‐risk genotypes HPV 16 and HPV 18 and pooled results on other high‐risk genotypes (OHR‐HPV) (31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68). HPV DNA positivity was detected in 14.2% (605/4267) of the samples. HPV type 16 and type 18 were detected in 2.4% and 0.7% of the samples, respectively. OHR‐HPV types were found in 8.8% of the samples. Of the 1.9% and 0.4% samples had mixed types with type 16+ OHR‐HPV and type 18+ OHR‐HPV, respectively. The results of this study presented the rates of HR‐HPV genotypes of a university hospital in Ankara, over a 4‐year period. It was observed that the positivity rate of type 18 is decreasing and some OHR‐HPV types are increasing. HPV vaccination is not in the national immunization program in Turkey yet, however, HPV vaccines are available and the vaccination rates for women are increasing.

HPV E6/E7mRNA association with interleukin 10 (rs1800872) polymorphism in a group of Macedonian women

AbstractInterleukin 10 (IL‐10) is an immunosuppressive cytokine and its genetic variants could have an indirect impact on viral biology and human papillomavirus (HPV) E6/E7 messenger RNA (mRNA) expression as well. This study evaluates the association between IL‐10‐592 C/A (rs1800872) single‐nucleotide polymorphism and HPV E6/E7 mRNA expression in a group of women from the Republic of North Macedonia. Using a commercial test, 272 women's cervical samples were analyzed for HPV E6/E7 mRNA and HPV DNA presence. The cases were stratified into three groups: double‐positive (n = 108, positive for both tests), negative (n = 51, negative for HPV E6/E7 mRNA and HPV DNA positive), and the control group (n = 113, negative for both tests). The IL‐10‐592 C/A polymorphism was analyzed using polymerase chain reaction‐restriction fragment length polymorphism. The results showed the CC genotype and the C allele frequencies of IL‐10‐592C/A were significantly higher in double‐positive (59.3% and 78.2%) compared to negative group (39.2% and 65.7%), (p = 0.018, confidence interval [CI] = 2.25; 1.14–4.45 and p = 0.016, CI = 1.88; 1.11–3.16, respectively). The CC genotype and C allele of rs1800872 polymorphism were shown to be associated with HPV E6/E7 mRNA but not with HPV DNA positivity, which implies a possible role of this polymorphism in the course of the infection only after HPV onset, and lack of association with the susceptibility to HPV.

Human Papillomavirus E6/E7 oncogene transcripts as biomarkers for the early detection of cervical cancer

AbstractCancer of the cervix uteri is the fourth most common cancer worldwide with a high mortality rate. Due to limitations of the existing methods, alternative methods for triage are needed for early detection of cervical cancer precursors before progression to high‐grade disease. The aim of this study was to evaluate human papillomavirus (HPV) E6/E7 oncogene expression as markers for early identification of cervical cancer risk in women with minor cytological abnormalities and in those with negative cytology. The detection of HPV was done using PCR and confirmed by southern hybridization. The high‐risk (HR) and low‐risk HPV types were identified by HPV typing. HPV DNA‐positive patients were further tested for markers of oncogene expression by real‐time PCR. Out of the women screened, 54/512 (10.54%) women tested positive for HPV infection. HR HPV DNA was found in 32/485 (6.60%) women with normal cytology (Pap negative) and 22/27 (81.5%) atypical squamous cells of undetermined significance/low‐grade intraepithelial lesion cases. HR HPV E6/E7 oncogene transcripts were detected in 36/512 (7.03%) patients. The positivity rate of E6/E7 messenger RNA (mRNA) was 2.48% (12/485) in normal cervical cytology group and 88.9% (24/27) in abnormal cervical cytology group. The HPV E6/E7 mRNA test sensitivity was found to be 88.89% and specificity was 97.53%. In comparison, the sensitivity of the HPV DNA test was found to be 81.48% and specificity was 93.40%. In conclusion, E6 and E7 transcripts could provide a sensitive, early predictor of cervical cancer risk in women with normal cytology and minor cytological alterations.

Genetic variation of E6, E7, and L1 genes of human papillomavirus 51 from central China

AbstractThe incidence of cervical cancer is closely related to high‐risk human papillomavirus (HR‐HPV). Women in Jingzhou had relatively high susceptibility to HPV‐51, whose ratio was 9.61% (456/4743) among HR‐HPV‐positive samples and ranked fifth in all analyzed HR‐HPV types. In this study, variations and phylogenetic trees of HPV‐51 E6−E7 and L1 sequences were analyzed by MEGA‐X. The selective pressure was estimated using PAML. The B‐cell epitope of L1 amino acid sequences and T‐cell epitope of E6 and E7 amino acid sequences were further predicted by ABCpred server and IEDB website, respectively. In the E6−E7 sequences 14 single nucleotide variants occurred, among which 4 were nonsynonymous variants and 10 were synonymous variants. A total of 41 single nucleotide variants were identified in the L1 sequences, including 10 nonsynonymous variants and 31 synonymous variants. All the isolates of both E6−E7 and L1 were classified into the A variant lineage. In HPV‐51 E6−E7 and L1 sequences, no positively selected site was found. Two nonconservative substitutions, H119Y and N176S in L1, affected multiple hypothetical B‐cell epitopes. Three nonconservative substitutions, T86P, S100L in E6, and F29L in E7, affected multiple hypothetical T‐cell epitopes. Elucidation of the HR‐HPV prevalence characteristics and genetic variations of HPV‐51 in central China may contribute to future investigations of diagnostic probes, therapeutic or preventative vaccines with wider coverage.

Study on the Clinical Characteristics, Persistent Infection Ability and Viral Load of Human Papillomavirus Type 73 Single Infection

ABSTRACTHuman papillomavirus (HPV) infection is a main cause of cervical cancer. Different HPV types exhibit significant differences in pathogenicity. Clinical data on HPV73 single infection is limited, this study aimed to analyze the clinical characteristics of HPV73 single infections. We collected cervical exfoliated cells to examine the infection rate and the clinical characteristics of patients with single HPV73 infection, and to investigate the association of viral load, persistent infection, and pathogenicity. The positive rate of HPV73 among women tested was approximately 0.14% (609/435 072). Among 214 HPV73 single infection cases, the number of patients with normal cytology, cervical intraepithelial neoplasia (CIN)1, CIN2, CIN3, and cervical cancer were 182, 24, 5, 1, and 1, respectively. Follow‐up data showed that 14.29% (3/21) of CIN1 patients developed to higher‐grade lesions, with 3.73% (5/134) of normal patients progressed to CIN. The viral load in the CIN group was significantly higher than that in the normal group (p = 0.001), and the viral load in the persistent infection group was significantly higher than that of the viral‐clearance group (p < 0.001). HPV73 single infection can lead to cervical cancer, with relatively low pathogenicity. And high viral load is a crucial factor in its persistent infection and pathogenicity.

Characteristics of Human Papillomavirus Prevalence and Infection Patterns Among Women Aged 35–65 in Fujian Province, China: A Nine‐Year Retrospective Observational Study

ABSTRACTThe assessment of human papillomavirus (HPV) genotype distribution could inform targeted cervical cancer prevention strategies. The epidemiology of HPV genotypes in terms of age and cervical lesions in Fujian Province, China has not been well described. This 9‐year retrospective study aimed to delineate the prevalence pattern and trend of HPV genotypes among a large‐scale community‐based population. Deidentified data were retrieved from the national cervical cancer screening program in China. We included eligible women aged 35–65 years who underwent cervical cancer screening between 2014 and 2022 in Fujian Province. The HPV prevalence within distinct subpopulations was calculated, and trends in HPV prevalence over the years and across age groups were examined using the Cochran‐Armitage trend test. A total of 551 604 women (median age 49 years [42, 54]; 0.10% with cervical cancer) were included in this study. The overall HPV prevalence was 11.72% (95% CI: 11.63%–11.80%), with HR‐HPV (high‐risk HPV) and HPV 16/18 prevalence at 10.02% (9.94%–10.10%) and 1.74% (1.71%–1.78%), respectively. HPV‐52, 58, 16, 39, 51, and 68 were the most predominant genotypes in the general population. Nearly all genotypes, except for HPV‐39 and 66, showed a decreasing trend in prevalence over the years, while a relatively high prevalence of HR‐HPV was observed across all age groups. As lesion severity increased, HR‐HPV and 9v‐HPV prevalence also increased. Our study underscores the importance of ongoing surveillance of HPV prevalence in China. While the overall decline in HPV infections over the years is encouraging, the relatively high prevalence of HR‐HPV warrants continued attention. Strengthening public health strategies—including prioritizing and promoting the current 9‐valent vaccination, extending HPV testing and cervical cancer screening to older women where feasible, and developing future vaccines targeting more HR‐HPV genotypes—will be crucial in eliminating cervical cancer and HPV‐related disease in China and beyond.

Mock Samples That Mimic Human Cervicovaginal Samples to Accelerate the Development and Evaluation of Assays for High‐Risk HPV for Cervical Cancer Screening

ABSTRACT Nearly all cervical cancer cases are caused by high‐risk human papillomavirus (hrHPV) infections. The World Health Organization recommends screening for hrHPV using nucleic acid amplification tests (NAATs) that detect hrHPV DNA or mRNA. Lack of access to affordable, point‐of‐care screening tests in resource‐limited settings leads to women presenting with advanced‐stage cervical cancer, with many dying of the disease. There is a significant need to develop point‐of‐care NAATs to improve screening and early detection. Because access to real clinical samples is limited, initial evaluation of NAATs is often performed using contrived samples created using some combination of extracted hrHPV DNA and/or cultured hrHPV‐positive cells. When mock samples do not adequately recapitulate the contents of clinical cervicovaginal samples, it can delay clinical translation of potentially promising assays. To improve the value of contrived samples, we characterized the composition of 32 hrHPV DNA‐positive cervicovaginal clinical samples. We also describe a simple method to generate contrived samples that mimic the diversity of clinical cervicovaginal samples, and test them using the methods used to characterize the clinical samples. Results show that the hrHPV DNA content of cervicovaginal samples varies by approximately eight orders of magnitude and spans from 100% linear integrated DNA to 100% circular non‐integrated DNA, that the concentration of hrHPV mRNA also varies by nearly nine orders of magnitude between patient samples, and that the concentration of potential inhibitors such as hemoglobin varies by more than three orders of magnitude. hrHPV DNA and mRNA extracted from contrived samples exhibited expected patterns of DNA quantity, DNA conformation, and mRNA quantity. Altogether, the protocols described here to generate mock samples can help NAAT developers optimize test performance prior to clinical evaluation, potentially improving test performance and reducing time to deployment.

What If Trojan Horse Nanoparticles Could Change the Game for HPV Gene‐Targeted Therapies?

ABSTRACT Human papillomavirus (HPV) is a common sexually transmitted infection linked to various cancers, particularly cervical cancer, primarily driven by high‐risk strains like HPV16 and HPV18. While vaccines are effective in preventing new infections, they do not address existing cases, highlighting the need for innovative therapies. Gene‐targeted approaches, such as CRISPR/Cas and siRNA, show promise in inhibiting HPV oncogenes. Recent advancements in Trojan horse nanoparticles (NPs) offer a strategy for delivering these therapies directly to HPV‐infected cells. These NPs improve stability and targeted delivery, enhancing the biodistribution of CRISPR/Cas systems and siRNAs while protecting them from degradation. However, challenges like immune responses and regulatory hurdles persist. Therefore, this review emphasizes the potential of Trojan horse NPs in treating HPV‐related cancers, identifies critical areas for future research, and provides updates on gene‐targeted therapy encapsulated NPs in preclinical and clinical trials.

Negative Xpert HPV Test (Cepheid) With Late Amplification Signals: Is There a Clinical Significance? (the PaPCR Study)

ABSTRACT Cervical cancer is the fourth most common cancer among women worldwide, with higher prevalence in low‐resource countries due to limited access to screening and HPV vaccination. Nucleic acid amplification tests (NAATs) such as Xpert HPV (Cepheid), enable rapid detection of high‐risk HPV (HR‐HPV) genotypes. However, the clinical significance of weak and late amplification signals (cycle threshold ≥ 38), interpreted as negative, remains unclear. In this study, a comparison with cytological results aimed to assess their significance. Out of 6846 cervical samples analyzed at Brest University Hospital (Jan. 2022–June 2024), 45 showed late amplification and were reported negative by Xpert. These samples were retested using an in‐house HPV pangenotypic PCR followed by genotyping (LiPA) and were assessed by cytology. Of the 44 available cytology results, 65.9% were normal, 15.9% showed low‐grade squamous intraepithelial lesions (LSIL), and 18.2% were atypical squamous cells of undetermined significance (ASC‐US). No high‐grade squamous intraepithelial lesions (HSIL) were found at initial cytology. Among the 42 samples available for in‐house PCR, 59.5% tested positive for HPV, and 11.9% were confirmed as HR‐HPV genotypes (types 52, 56, 35). Additionally, among patients with available HPV history, 73.3% had a previous positive test. In 91% of these cases, the genotype suspected during the late amplification was concordant with the previous infection. During the 1‐year follow‐up, one patient initially classified as LSIL developed a histologically confirmed HSIL (CIN2) and needed treatment by conization, whereas 3 others were found to have low‐grade CIN1 cell lesions. Overall, for 15 out of 45 patients with weak HPV PCR signal and abnormal cytology (33.3%), clinicians chose to recommend a closer 1‐year follow‐up and not at 5 years as recommended for HPV negative patients. In conclusion, cytological triage and a review of patient history remain crucial to avoid missing at‐risk patients, especially in settings where HPV‐negative women are re‐screened only 5 years later. A multicenter study using Xpert HPV and other automated platforms is recommended to strengthen these findings and further explore potential correlations between viral load and clinical severity.

Beyond HPV‐16 and 18: Clinical Significance of HPV‐58 and 59 in Cervical Abnormalities in Northeast India

ABSTRACT Cervical cancer exerts a significant public health burden globally and in India. While HPV‐16 and HPV‐18 are well‐known etiological agents, emerging evidence highlights the regional significance of other high‐risk genotypes like HPV‐58 and HPV‐59. This study aimed to evaluate the prevalence and cytological associations of HPV genotypes, particularly 58 and 59, in Northeast Indian women. A total of 429 cervical swab specimens were collected from women attending the routine outpatient department at NEIGRIHMS, Shillong, between 2021 and 2025, following informed consent and institutional ethical approval. Cytological analysis and demographic data collection were performed. HPV genotyping was carried out at the reference lab at Pune using the Xpert HPV assay and qPCR Panel. Among 429 participants, 74 (17.25%) tested positive for HPV. Among the HPV positive cases, the genotype distribution was: HPV‐16 (52.70%), HPV‐18 (8.10%), and HPV‐58(5.40%) and HPV‐59 (13.51%). Notably, HPV‐58 positive women exhibited abnormal cytology, while HPV‐59 positive patients ( n  = 10), Pap smears showed NILM (6), ASC‐US (1), Bacterial vaginosis (2), and One case of Poorly Differentiated Malignancy. This study underscores the emerging clinical relevance of HPV‐58 and 59 in Northeast India. The higher cytological abnormality in HPV‐58 cases highlights the need for expanded HPV genotyping in screening programs.

Tumor Core‐Edge Divergence in T‐Cell Exhaustion and Clonal Expansion in HPV‐Related Cervical Squamous Cell Carcinoma Unveiled by Simultaneous Single‐Cell RNA and TCR Sequencing

ABSTRACT Tumor spatial architecture significantly influences immune responses, but the impact of regional variations on T cell exhaustion and clonality in human papillomavirus (HPV)‐related cervical squamous cell carcinoma (CESC) remains poorly understood. Using single‐cell RNA sequencing (scRNA‐seq, n  = 13) and TCR sequencing (scTCR‐seq, n  = 9), this study profiled T cells from paired tumor core and edge samples of patients with CESC. This was supplemented by bulk RNA‐seq data ( n  = 41), TCGA datasets from three cancer types (CESC, head and neck squamous cell carcinoma, and lung squamous cell carcinoma), and scRNA‐seq data from colorectal cancer for broader validation. We found that CD8 + T cells, natural killer T (NKT) cells, and γδ T cells in the tumor core exhibited higher inhibitory and cytotoxicity scores, while CD4 + T cells showed increased regulatory T (Treg) and cytotoxic features in the tumor core. Bulk RNA‐seq data confirmed elevated inhibitory and cytotoxic scores in the tumor core relative to the edge. Additionally, four subsets of exhausted CD8 + T cells (CD8 + Tex) were identified, including a stress‐associated subset characterized by heat shock protein (HSP) family gene expression, which was validated by immunofluorescence and inversely correlated with survival in patients with CESC undergoing radiotherapy. TCR analysis revealed clonal expansion and reduced clonal diversity in the tumor core. Notably, CD8 + Teff‐CD160 cells displayed substantial clonal sharing across regions and were associated with a favorable prognosis. Overall, our findings highlight distinct T cell states between the tumor core and edge in HPV‐related CESC, offering insights into prognostic biomarkers and potential therapeutic targets.

Human CLIC5 as a Recurrent Hotspot of HPV 16 Integration in Cervical Cancer

ABSTRACT Despite extensive global efforts to eradicate cervical cancer through improved screening and widespread HPV vaccination, the disease continues to claim over 350 000 lives annually. Persistent infection with high‐risk HPV genotypes, primarily HPV‐16 and HPV‐18, is the principal etiological driver, with viral DNA integration into the host genome representing a critical event in malignant progression. In this study, HPV integration was investigated using 25 RNA‐seq datasets from HPV‐positive cervical cancer cell lines and 2 datasets from the HPV‐negative C‐33A cell line. Analysis revealed a recurrent HPV‐16 integration hotspot on Chromosome 6 within the CLIC5 gene. This integration was validated by qRT‐PCR and Sanger sequencing. Subsequent gene expression analysis demonstrated significant CLIC5 upregulation in HPV‐16‐positive cell lines, with a 21‐fold increase in CaSki and a 4‐fold increase in SiHa compared to HPV‐negative and HPV‐18‐positive controls. These findings delineate a distinct HPV‐16 integration pattern and its associated transcriptional impact. The identification and validation of this strain‐specific integration site nominates HPV‐16 integration at the CLIC5 locus as a potential biomarker for HPV‐driven cervical cancer.

Targeting HSP70‐E7 Interaction With SHetA2: A Novel Therapeutic Strategy for Cervical Cancer

ABSTRACT Cervical cancer is predominantly driven by persistent infections with high‐risk human papillomavirus and the continuous activity of its E6 and E7 oncoproteins. This study explored the role of heat shock proteins 70 kDa (HSP70s) in enhancing the function of these oncoproteins and examined the impact of SHetA2, an investigational new drug, on this interaction. We found that HSP70 specifically binds to E7, but not E6, protein and that SHetA2 disrupts this binding. This disruption led to a significant reduction in E6 and E7 mRNA and E7 protein levels, while effects on E6 protein levels were minimal. SHetA2 treatment also resulted in altered levels of cell cycle regulatory proteins, reduced cell cycle progression, and decreased metabolic viability in cervical cancer cell lines and xenograft models. These findings support the potential of SHetA2 to impair cervical cancer progression by targeting HSP70/E7 interactions, highlighting its promise as a therapeutic strategy for treating cervical cancer.

The Impact of HPV/HBV Coinfection on Cervical Cancer Risk: Potential Mediating Role of Ki67

ABSTRACTPersistent high‐risk human papillomavirus (HPV) infection is the primary cause of cervical cancer, with co‐factors such as hepatitis B virus (HBV) playing a role in oncogenic progression. This study investigates the interaction of HPV and HBV coinfection with cervical cancer risk. A total of 4488 women undergoing cervical cancer screening and HPV and HBV testing were included in this study, employing multinomial regression to analyze associations between viral infections and cervical intraepithelial neoplasia and cancer, while interaction analysis assessed synergistic effects on cervical carcinogenesis and examined the mediating role of ki67 in co‐infections. Co‐infection significantly increases the risk of all levels of cervical lesions compared to the reference group, with odds ratios for LSIL, HSIL, and cervical cancer demonstrating substantial elevation (OR = 3.13, 95% CI: 2.25–4.35; OR = 3.5, 95% CI: 2.55–4.81; and OR = 11.49, 95% CI: 5.85–22.57; respectively). On the additive scale, significant positive interactions were observed between HPV and HBV. Specifically, for cervical cancer, there was an attributable proportion (AP) due to interaction of 0.43 (p = 0.01), and a synergy index (SI) of 1.88 (p < 0.001). Similarly, for HSIL, significant positive interactions were noted with AP = 0.28 (p = 0.01) and SI = 1.65 (p < 0.001). Mediation analysis revealed that Ki67+ accounted for 49.39%, 70.44%, and 78.81% of the total effect of HPV and HBV co‐infection on cervical cancer, HSIL, and LSIL, respectively (all p < 0.05). HPV and HBV coinfection significantly impacts cervical lesions, enhancing cervical oncogenesis synergistically. Ki67 may be involved in mediating the process of cancer progression caused by viral co‐infection, stressing comprehensive screening and molecular interaction research.

Human Papillomavirus (HPV) Type Distribution in Cervical Carcinoma, Low‐Grade, and High‐Grade Squamous Intraepithelial Lesions in Women Under 25 Years Old From the Largest Women Hospital in China

ABSTRACT This study aimed to profile high‐risk HPV genotypes across the spectrum of cervical lesions (low‐grade squamous intraepithelial lesion [LSIL], HSIL, carcinoma) in young patients under 25. Patients under 25 with histologically confirmed LSIL ( n  = 200), HSIL ( n  = 200), or cervical cancer (CC) ( n  = 32) between January 2018 and December 2021 underwent real‐time PCR‐based HPV genotyping (21 types) to assess type‐specific prevalence. The overall HPV detection rates were 91% in LSIL and 100% in both HSIL and CC. HR‐HPV and LR‐HPV was observed in 90.5%/14.5% of LSIL cases, 87.5%/5.5% of HSIL cases, and 93.8%/12.5% of CC cases. HPV16 was the most prevalent genotype (25.5%), followed by HPV52 and HPV58 in LSIL. The most frequent HR‐HPV genotype in HSIL were HPV16 (63.0%), followed by HPV33, HPV52, HPV58, and HPV18. HPV16 (43.8%) and HPV18 (34.4%) were the most common genotypes in CC tissues, followed by HPV68, HPV53, HPV6, HPV31, HPV52, and HPV59. In squamous cell carcinoma, HPV16 was predominant (70.0%), followed by HPV6, HPV59, and HPV68. HPV18 prevalence was significantly higher in adenocarcinoma, adenosquamous carcinoma, and neuroendocrine carcinoma (100.0%, 71.4%, and 100.0%, respectively) than in squamous cell carcinoma. Theoretically, the protective rates of 2‐valent, 4‐valent, and 9‐valent HPV vaccine were 75.0%, 84.4%, and 87.5%, respectively for CC in Chinese women under 25 years old. The results of this study provide baseline information on the HPV genotypes distribution in women under 25 years of age, which may contribute to promoting the implementation of CC prevention and control programs for this demographic.

The Dual Role of DNA Methylation Test Can Optimize the Clinical Application of High‐Risk Human Papillomavirus Testing in Detecting Cervical Intraepithelial Neoplasia Grade 3 or Worse

ABSTRACT With the rapid development and maturation of high‐risk human papillomavirus (hrHPV) genotyping kits, it is crucial to understand the specific patterns of methylation in different hrHPV statuses, rather than simply categorizing them as hrHPV positive or negative. To achieve this goal, we first assessed the prevalence and CIN3+ risk of specific hrHPV genotypes in 184 cervical scrapings and then evaluated the performance of five host genes methylation to distinguish CIN3+ from <CIN3 not only among hrHPV‐positive and hrHPV‐negative women, but also among women positive for HPV16, HPV18, HPV52, and HPV58. The results showed that women with HPV16 infections had a higher risk of developing CIN3+ than women with other hrHPV infections. More importantly, we found that hrHPV testing caused a high false‐positive rate (43.54%) and leading to over‐referral of <CIN3 cases. Triage of patients with HPV 52/58 positive using ZIC1 m could accurately identify all CIN3+ cases to avoid over‐referral of false‐positive cases. In addition, implementing primary hrHPV testing resulted in 5.7% of CIN3+ patients being missed in our study. Our exploratory analysis found that DNA methylation of specific genes, including ZIC1 m , ZNF582 m , PAX1 m , and MIR129‐2 m , could lower the high false‐positive rate and find 80% (4/5) missed CIN3+ cases caused by hrHPV testing. This study substantiates the dual role of DNA methylation detection in cervical cancer screening. By incorporating methylation detection, the existing HPV‐based screening strategy can be further optimized to achieve precise risk stratification and facilitate the shift from cervical cancer screening to personalized and precise management.

Reproducibility of HPV Testing With Two Versions of the Papilloplex High‐Risk HPV Assay

ABSTRACT Validation of HPV tests usable in cervical cancer screening require demonstration of noninferior clinical accuracy and sufficient reproducibility. The Papilloplex HR‐HPV assay [version1] (Genefirst, UK) has been validated only for clinical accuracy. Here, we assessed its reproducibility, aiming to complete the validation process. A panel of 550 cryopreserved cervical cell samples collected from women attending the cervical cancer screening program in Belgium was used to assess the intra‐ and inter‐laboratory reproducibility of Papilloplex HR‐HPV [version1], a full genotyping assay that identifies separately 14 high‐risk HPV (hrHPV) types using multiple probe amplification technology. We assessed whether the reproducibility fulfils validation criteria (lower 95% confidence interval [CI] bound ≥ 87% and κ ≥ 0.50). Subsequently, we compared the concordance between version1 and a new version6 of the assay and between three analysis methods for PCR curve interpretation. Papilloplex HR‐HPV version1 assay showed an excellent reproducibility for hrHPV (97.5% [CI: 95.8%–98.7%], κ = 0.94 for intra‐ and 93.5% [CI: 91.0%–95.4%], κ = 0.85 for inter‐laboratory reproducibility). Concordance analyses exhibited an excellent agreement between two assay versions and between three PCR curve analysis methods. Papilloplex HR‐HPV version1 assay exhibited excellent reproducibility, completing the international validation criteria. Papilloplex HR‐HPV version6 showed excellent concordance with version1 but still lacks clinical validation.

HPV Genomic Status in Early Cervical Cancer and Its Prognosis Association

ABSTRACT Identify specific Human Papilloma Virus (HPV) genomic status by HPV capture technique coupled with next‐generation sequencing (NGS) in patients with early cervical cancer (ECC) regarding 2018 FIGO stages and correlate these statuses with prognosis. HPV sequencing by Capture technique coupled with NGS was performed on 34 tumors samples of ECC patients without histologically sentinel lymph node (SLN) metastasis in a retrospective study. NGS data were analyzed in our in‐house VIROCAPT2 pipeline to determine precise HPV genomic status such as the complete sequence of HPV genome, its integration or not in host genome and correlate this viral identity card with patient's clinical prognosis. As expected, HPV16 remains the most common genotype (60%, n  = 21/35) in ECC. HPV integration was identified in 40% ( n  = 14/35) of samples. We found that in ECC, integration events were related with HPV genotype ( p  = 0.0005). Among the 40% of integrated HPV, 8 were found integrated within human genes, including 2 into oncogenes. Finally, women with integrated HPV had a poorest outcome with a strong correlation between progression‐free survival and HPV integration ( p  = 0.0035) and interestingly, the 2 HPV integrated into oncogenes were observed in patients who died from cancer. HPV genomic status seems to be a key prognostic factor in early stage cervical cancer, with HPV integration strongly linked to poor outcomes. Larger studies are needed to confirm the clinical relevance of HPV ID card for risk stratification and treatment decisions.

Association of Vaginal Microbiota and Sociodemographic Factors With Cervical Human Papillomavirus in the Eastern Region of India

ABSTRACTProlonged exposure to the High‐risk Human Papillomavirus (HPV) leads to the development of cervical carcinoma. Numerous factors aid in this virus's acquisition, persistence, and clearance. This study aims to determine the association of vaginal microbiota and socio‐demographic factors with oncogenic HPV infections among women from Eastern region of India. Cervical scrapes and vaginal swab samples were collected from women (n = 300) having some gynecological complaints with informed consent. Cervical cytology and HPV types were screened among them. A subset of samples (n = 12) were subjected for next generation sequencing based 16S rRNA profiling to determine the vaginal bacterial diversity among the HPV−positive and HPV−negative women with normal cervical cytology. The taxonomic profiling, diversity and relative abundance of bacterial species were determined subsequently. With appropriate statistical tests, vaginal bacterial diversity along with socio‐demographic risk factors was correlated with HPV infections. A PCR‐based approach further detected the bacterial taxa that were exclusively present among the groups in the whole data set. HPV infection was identified among 11.60% of individuals, with the dominance of HPV18 (80%) among carcinogenic HPV. 16S rRNA profiling revealed that the HPV‐positive group had lower abundance of Firmicutes phylum and higher abundance of Proteobacteria and Actinobacteria phyla than the HPV‐negative group. A positive correlation between Genus Acinetobacter and HPV positivity was established, presenting higher susceptibility for oncogenic HPV16/18 (p = 0.016; relative mean abundance = 19.67). PCR‐based detection of this genus in the whole data set (n = 300), a statistically significant association with oncogenic HPV16/18 infection (p ≤ 0.01, odds ratio (OR)= 22.48 [95% CI = 5.23–96.63]) was found. Among sociodemographic factors, Tobacco users (16.67%, 4/24; OR = 6.70 [95% CI: 1.86–24.18]; p = 0.001) and field‐workers (15.79%, 3/19; OR = 5.67 [95% CI: 1.40–22.99]; p = 0.007) were associated with a higher risk of HPV18 infections. Assessment of geography‐specific vaginal microbiota and other lifestyle induced risk factors might contribute to acquire chronic infection of oncogenic HPV and progression to cervical cancer.

Human Papillomavirus 42: A Novel Sublineage Identification, and Comparative Analysis of HPV42 Genetic Variability Present in Cervical Cell and Digital Papillary Adenocarcinoma

ABSTRACT Although HPV 42 is classified as a low‐risk HPV, it has been identified as an oncogenic driver of digital papillary adenocarcinoma (DPA) and is associated with high‐grade squamous intraepithelial lesion (HSIL) in the mucosal genital region. This study aim to identify novel HPV42 sublineage, characterize its genetic variability, and explore the differences or associations of genetic variation between HPV42 variants derived from cervical and DPA samples. We successfully amplified 17 complete HPV42 genomes from cervical cells, including 5 A1, 11 A2, and 1 novel A4 sublineages. The A4 sublineage (SDWF08) was identified based on the phylogenetic analysis and pairwise distance of whole genome sequences, which differed by 0.57–0.70%, 0.50–0.74%, and 0.74% from A1, A2, and A3 sublineages, respectively. 13 A2 variants from China clustered into a minor branch, separate from other A2 sublineage. The A2 sublineage had the largest number of variation sites, with 71 single‐nucleotide polymorphisms (SNPs) and 11 nonsynonymous amino acid mutations in E6, E7, E2, E4, L2, and L1. Three amino acids (P, S, I) deletions in E2 of SDWF08 overlaps with the deletion in E2 that originated from DPA. These HPV42 genomic sequences obtained in this study expand the number of publicly available, and provide an updated phylogenetic analysis to support further functional and evolutionary research on HPV42 genetic diversity. The observed deletions in E2 and the aspartate residue (D) upstream of the LxCxE motif in E7 may suggest a possible role for HPV42 in carcinogenesis, further functional studies are needed to validate this potential association.

Clinical Validation of the Venus HPV Full‐Genotyping Assay for Cervical Cancer Screening in the VALGENT‐4 Framework

ABSTRACTThe Venus HPV assay (VenusHPV) is a real‐time PCR‐based human papillomavirus (HPV) test that is widely used in China but lacks extensive clinical validation. The VALidation of HPV GENotyping Tests (VALGENT) framework is an established protocol for evaluating HPV genotyping assays against a standard comparator test. This study aimed to assess the clinical accuracy and reproducibility of the VenusHPV assay following international validation criteria. The clinical performance of VenusHPV was evaluated against the GP5+/6+ PCR‐based enzyme immunoassay (GP‐EIA) using the VALGENT‐4 panel, which included 998 consecutive routine screening samples enriched with 297 samples with abnormal cytology from the Danish cervical cancer screening program. Cases were defined as women diagnosed with histologically confirmed cervical intraepithelial neoplasia 2 or more (CIN2+), while two consecutive negative cytology results served as a proxy for nondisease. Intra‐ and interlaboratory reproducibility was assessed on 500 samples. Using the manufacturer‐recommended cutoff, VenusHPV demonstrated noninferior sensitivity for detection of CIN2+, but its specificity for ≤ CIN1 was inferior to that of GP‐EIA. Applying an optimized a posteriori cutoff improved the specificity, yielding relative specificity of 1.02 (95% CI [CI], 1.00–1.03; p noninferiority [pn.inf] < 0.0001), while maintaining a noninferior sensitivity (of 1.02; CI, 1.00–1.08; pn.inf < 0.0001). The intra‐ and interlaboratory reproducibility was excellent (95.2%, CI, 93.3–97.1%, Kappa [κ] = 0.87 and 94.0%, CI, 92.0%–96.0%, κ = 0.85, respectively). Notably, the reproducibility criteria were met consistently, regardless of whether the unadjusted or optimized cutoff was applied. The VenusHPV was as sensitive as the GP‐EIA for detecting cervical precancer using the unadjusted cutoff but less specific. However, after cutoff optimization, VenusHPV met the international accuracy criteria for cervical cancer screening. Additionally, the assay demonstrated excellent reproducibility.

Human Papillomavirus Genotype Distribution and Viral Load in Relation to Cervical Disease Severity: A Retrospective Study of Outpatient Data in Beijing, China

ABSTRACTPersistent human papillomavirus (HPV) infection drives cervical carcinogenesis, yet regional variations in genotype distribution and viral load dynamics remain understudied in Beijing, China. This study investigated HPV prevalence, genotype‐specific patterns, and viral load correlations with cervical disease severity among women outpatient clinics from Peking University First Hospital in Beijing. In a retrospective study of 25 197 women undergoing cervical ThinPrep cytology (TCT) and HPV testing from October 2023 to October 2024, colposcopy results from 714 cases were also analyzed. HPV genotyping and quantitative for HPV types (16, 18, 33, 52, 58) were performed. Overall HPV positivity was 16.50% (4157/25 197), peaking in women aged 31~40 years (30.05%). Dominant genotypes were HPV 52 (18.89%), 16 (18.55%), and 58 (12.10%). HR‐HPV prevalence and viral load escalated with disease severity (p < 0.001), notably HPV16 and HPV33. HPV11, 43, 45, and 68 were absent in severe lesions. Single infections (76.35%) predominated, but multi‐infections showed genotype synergism (e.g., HPV51 + 42). The study highlights specific HPV epidemiology and genotype dominance in China, indicating gaps in vaccine coverage. Viral load is a key biomarker for risk assessment, suggesting the need for tailored vaccination programs and viral load monitoring in clinical practice.

Improving Detection of CIN2+ and CIN3+ Lesions: Evaluation of E6/E7 mRNA, P16, and Ki‐67, Individually and in Combination

ABSTRACTCytology screening has lower cervical precancer sensitivity than HPV DNA testing, which lacks specificity. To overcome this limitation, we assessed the diagnostic performance of E6/E7 mRNA, P16, and Ki‐67, both alone and in combination, in detecting cervical intraepithelial neoplasia grade 2 or higher (CIN2+). A total of 403 cervical samples were collected from the First People's Hospital of Nanning between January 2019 and January 2024, and each biomarker was evaluated for its diagnostic accuracy. E6/E7 mRNA showed moderate performance, with 68.2% sensitivity and 61.8% specificity in detecting CIN2+ lesions. P16 exhibited superior performance, achieving 82.2% sensitivity and 90.1% specificity. Although Ki‐67 showed the highest sensitivity at 95%, it had the lowest specificity at 27.2%. The combination of P16 and Ki‐67 yielded the best diagnostic results, with 90% sensitivity and 79.8% specificity for CIN2+, representing a significant enhancement over individual biomarkers. These findings highlight the superior accuracy of P16, especially when combined with Ki‐67, in detecting both CIN2+ and CIN3+ lesions. This approach improves high‐risk lesion detection by reducing false negatives. Incorporating P16/Ki‐67 biomarkers enhances screening sensitivity/specificity and clinical outcomes.

Electrochemical Duplex Detection of E2 and E6 Genes of Human Papillomavirus Type 16 and Determination of Physical Status in High‐Risk Cervical Carcinoma

ABSTRACT Human papillomavirus type 16 (HPV‐16) is a key driver in the development of cervical carcinoma, with the integration of its genome into the host DNA marking a critical step in disease progression. Monitoring the physical state of HPV‐16, particularly the transition from episomal to integrated forms, is essential for evaluating the risk of malignancy development in cervix. This study presents the development of a duplex electrochemical biosensor for the simultaneous detection of the E2 and E6 genes of HPV‐16. Using a one‐step sandwich hybridization assay, the biosensor was able to detect HPV‐16 E2 and E6 genes with a sensitivity of 8 copies/mL and 12 copies/mL respectively and distinguish between the episomal and integrated forms based on the E2/E6 ratio (cut‐off 0.77, 100% sensitivity/specificity). The sensor was validated with 30 clinical cervical tissue samples, providing results comparable to qPCR method. This novel biosensor offers a rapid and efficient platform for the detection and monitoring of HPV‐16, with potential applications in cervical cancer screening and prognosis.

Declining Prevalence of Human Papillomavirus Co‐Infections Among Young Japanese Women With Cervical Cancer and Its Precursors

ABSTRACTCo‐infections with human papillomavirus (HPV) of multiple genotypes mainly occur due to increased sexual activity. To address the prevalence and trend of HPV co‐infections in Japan, HPV‐type‐specific data from Japanese women (n = 8128) aged < 40 years and newly diagnosed with cervical abnormalities at 24 hospitals between 2012 and 2023 were analyzed. These included cervical intraepithelial neoplasia grade 1/2 (CIN1/2, n = 2745), CIN3/adenocarcinoma in situ (AIS) (n = 3953), and invasive cervical cancer (ICC, n = 1430). For women enrolled in this study since 2019, information on sexual behaviors was collected via a self‐administered questionnaire. Time‐trend analyses by disease category showed significant declines in the prevalence of multiple HPV infections in CIN1/2 (49.1%−38.3%, ptrend = 0.0004), CIN3/AIS (44.7%–31.5%, ptrend = 0.0002), and ICC (26.7%–10.5%, ptrend < 0.0001) during the last decade. When these data were analyzed separately for women aged 20–29 and 30–39 years, similar declining trends were observed in each disease category. Using data from 2111 women for whom information on sexual history was available, the number of sexual partners was strongly associated with increased multiple HPV infections (p < 0.0001). In conclusion, the declining prevalence of HPV co‐infections in cervical cancer and its precursors may reflect a decrease in sexual activity among Japanese women of reproductive age.

Assessment of 5A's of Human Papilloma Virus Vaccination: Awareness, Attitude, Apprehension, Action Expected and Acceptability Amongst Health Care Providers, Medical Students, Para‐Medical and Administration Staff in AIIMS Rajkot

ABSTRACTThe study aims to assess awareness, attitude, apprehension, action expected from policy makers, and acceptability towards HPV vaccination amongst health care providers (HCP), medical students, paramedical staff and administrative personnel. A cross‐sectional study was conducted with 501 participants, including HCP, medical students, paramedical and administrative staff. Their awareness, attitudes, apprehensions, actions expected from policymakers, and acceptability towards HPV vaccine were assessed through a Google Form survey. Of the 501 participants, 92.4% were aware of HPV vaccines, 24.6% had a thorough understanding of HPV, but only 5.8% had received the vaccine. Most participants held a positive attitude toward cervical cancer prevention and HPV vaccination, although 46.7% expressed apprehensions. Before viewing educational video, 278 participants (55.8%) were willing to receive the HPV vaccine, while 116 (44.2%) were not. After viewing the educational video, willingness increased to 394 participants (78.6%), and those unwilling decreased to 107 (21.4%). Factors such as age, gender, marital status, education, and professional role did not significantly predict knowledge or apprehension regarding HPV and the HPV vaccine. However, professional role and younger adult (20–39 years) groups were statistically significant predictors of a positive attitude as well as stronger expectations for action from policymakers towards HPV vaccine. The study reveals significant gaps in HPV vaccine awareness and uptake among medical professionals, paramedics, and administrative staff. It highlights the need for broad educational programs to address these knowledge gaps, emphasizing HPV's health impacts and preventive measures. Multimedia educational interventions effectively boost vaccine acceptance among the groups and the general public.

Vacuolated Parabasal Cells in Papanicolaou Smears Are Cellular Changes Caused by Human Papillomavirus 16 Infection

ABSTRACTIn cervical cancer screening, cytology is used as a triage test to refer high‐risk human papillomavirus (HR‐HPV)‐positive women for colposcopy, but its accuracy is inadequate. The present study aimed to demonstrate that the presence of atypical cells with large vacuoles in the cytoplasm of parabasal cells, referred to as vacuolated parabasal cells (VPCs), which are observed in the Pap smears of HPV‐positive women, is associated with specific HPV genotypes. Among 2175 patients, 310 with a single HR‐HPV infection and cytological diagnosis of high‐grade squamous intraepithelial lesions (HSIL) or atypical squamous cells not excluding HSIL (ASC‐H) were included, of which 86 were infected with HPV16. Biopsy results revealed that 69 (80.2%) patients had cervical intraepithelial neoplasia Grade 2 or higher (CIN2+). VPCs were found in 47 (54.7%) of HPV16‐infected cases, indicating a significant increase of VPCs in HPV16‐infected cases (p < 0.01). Episomal HPV16 load was quantified in 142 VPCs and 156 HSIL (ASC‐H) cells using liquid‐based cytology samples from five patients, with a median of 987 copies in VPCs, significantly higher than those in HSIL (ASC‐H) cells (176 copies; p < 0.001). VPCs in Pap specimens were identified not only as cells altered by HPV16 infection but also as CIN2+‐derived cells and HPV16‐producing cells.

Single‐Cell RNA Sequencing Reveals the Cellular Origin and Evolution of Small‐Cell Neuroendocrine Carcinoma of the Cervix

ABSTRACTSmall‐cell neuroendocrine cancer (SCNEC) of the uterine cervix is an exceedingly rare, highly aggressive tumor with an extremely poor prognosis. The cellular heterogeneity, origin, and tumorigenesis trajectories of SCNEC of the cervix remain largely unclear. We performed single‐cell RNA sequencing and whole‐exome sequencing on tumor tissues and adjacent normal cervical tissues from two patients diagnosed with SCNEC of the cervix. Here, we provide the first comprehensive insights into the cellular composition, HPV infection‐related features, and gene expression profiles of SCNEC of the cervix at single‐cell resolution. Correlation analyses suggested that SCNEC of the cervix may originate from squamous epithelial cells, and this observation was validated with bulk RNA‐seq data from external cervical neuroendocrine cancer. Furthermore, sex‐determining region Y‐box 2 (SOX2), a key transcription factor that functions in direct neural differentiation, was located in the copy number gain region and highly expressed in neuroendocrine tumor cells from both patients. Notable, the distributions of the HPV‐infected epithelium and SOX2 highly expressed epithelium were consistent with each other. Therefore, we supposed that high‐risk HPV infection and amplification of SOX2 in the squamous epithelium may contribute to the progression of small‐cell neuroendocrine tumorigenesis in the cervix.

The Distribution of HR‐HPV E6/E7 DNA and mRNA by Histological Grade and the Clinical Performance for Detection of Cervical Cancer and Precancer

ABSTRACT High‐risk human papillomavirus (HR‐HPV) testing, utilizing both DNA and RNA methods, offers enhanced sensitivity compared to cytology for detecting high‐grade cervical intraepithelial neoplasia (CIN2+). Meanwhile, HR‐HPV E6 and E7 mRNAs are more likely to differentiate the transient infection from the persistent than DNA. Aptima HPV can not only detect HPV mRNA but also HPV DNA though it is much more efficient at detecting HPV RNA than DNA. Currently, there are few studies on the distribution of HPV E6 and E7 mRNA and DNA in the same individual. It is interesting to compare the clinical performance of the Onclarity and Aptima HPV assays and to assess variations in viral load across different histological grades at both DNA and mRNA levels. The analysis included 1607 women (902 from a cervical cancer screening population and 705 inpatients and outpatients), with cervical cytological samples tested using the Aptima and Onclarity HPV assays. Both assays demonstrated high agreement for HPV types 18/45 and 16. Signal‐to‐cutoff (S/CO) values and Ct values for various HR‐HPV types increased with histological severity from normal tissue to cancer. Notably, HPV18 Ct values exceeded those for HPV16 and 45 in women with ≥ CIN1 lesions but decreased sharply in cancer cases. Across the screening population, both assays showed similar sensitivity and predictive values for detecting CIN2+ lesions. The area under the curve (AUC) for CIN2+ and CIN3+ detection in the study population was robust (CIN2+: 0.880, 0.863; CIN3+: 0.881, 0.863). The DNA level for various HR‐HPV types increased with histological severity from normal tissue to cancer, which might impact the performance of Aptima HPV assay. Both assays showed similar sensitivity and predictive values for detecting CIN2+ lesions.

A Narrative Review of the Putative Etiologic Role and Diagnostic Utility of the Cervicovaginal Microbiome in Human Papillomavirus‐Associated Cervical Carcinogenesis

ABSTRACT The cervicovaginal microbiome (CVM) may contribute to human papillomavirus (HPV)‐associated cervical carcinogenesis. We summarized the literature on the CVM in cervical carcinogenesis by searching Medline, Web of Science, and Embase for articles that sequenced the CVM using metagenomics. Additionally, we identified studies assessing the diagnostic role of the CVM in cervical carcinogenesis by searching PubMed. We performed an environmental scan of Google and Google Scholar to review common CVM characterization techniques. Twenty‐eight records presented or summarized associations between the CVM and HPV acquisition, prevalence, persistence, clearance, and cervical lesions or cancer, while three studies identified bacterial taxa detecting high‐risk HPV prevalence or cervical lesions. The area under the curve ranged from 0.802 to 0.952. 16S ribosomal RNA gene sequencing and whole metagenome sequencing have sufficient resolution to study the CVM bacteriome. Bacterial communities may have important implications in cervical cancer; however, there is a need for methodological standardization for CVM characterization.

The Sansure® Human Papillomavirus DNA Diagnostic Kit offers excellent reproducibility performance for the detection of high‐risk HPV

AbstractCervical cancer screening is a cornerstone of cervical cancer elimination. Detection of high‐risk human papillomavirus (hrHPV) is recommended as the first step in screening provided that the assay used has been adequately validated. The Sansure® Human Papillomavirus DNA Diagnostic Kit is a new assay designed to detect HPV16, HPV18 and 13 other HPV in aggregate. The study aimed to evaluate the intra‐ and interlaboratory reproducibility of the assay according to international guidelines. Five hundred and fifty cervical residual cell samples from women attending cervical cancer screening were selected from the biobank of the HPV National Reference Centre in Belgium and used in this study. After DNA extraction, HPV was tested using the Sansure® Human Papillomavirus DNA Diagnostic Kit. The lower 95% confidence limit around the general reproducibility of this assay should be greater than or equal to 87%, with κ ≥ 0.50. Five hundred and thirty‐three samples had valid results. The Sansure® Human Papillomavirus DNA Diagnostic Kit demonstrated an excellent intra‐laboratory reproducibility of 93.8% (95% confidence interval [CI]: 91.4–95.7, κ = 0.85). The interlaboratory reproducibility was 93.4 (95% CI: 91.0–95.4, κ = 0.84). Intra and interlaboratory reproducibility were also excellent at the genotype level. Excluding HPV53 single infection samples from the analyses also resulted in excellent agreement. These data show that the Sansure® Human Papillomavirus DNA Diagnostic Kit is highly reproducible.

Eliminating cervical cancer: Don't forget HPV test and vaccination in men

The level of expression of HPV16 early transcripts is not associated with the natural history of cervical lesions

Abstract The natural history of cervical cancer is closely linked to that of high‐risk human papillomaviruses (HPV) infection. It is recognized that upon HPV DNA integration, partial or complete loss of the E2 open reading frame precludes expression of the corresponding protein, resulting in upregulation of the E6 and E7 viral oncoproteins. To better characterize HPV16 infection at the cervical level, viral load, viral DNA integration, and viral early transcript expression (E2, E5, and E6) were analyzed in a series of 158 cervical specimens representative of the full spectrum of cervical disease. Overall, the frequency of early transcript detection varied from 45% to 90% and tended to increase with lesion severity. In addition, the levels of E2, E5, and E6 transcript expression were slightly higher in high‐grade lesions than in cervical specimens without abnormalities. Notably, early transcript expression was clearly associated with viral load, and no inverse correlation was found between the expression of E2 and E6 transcripts. No clear association was found between early transcript expression and HPV16 DNA integration, with the exception that samples with a fully integrated HPV16 genome did not harbor E2 or E5 transcripts. In conclusion, early HPV16 transcript expression appears to be associated with viral load rather than lesion grade. From a practical point of view, quantification of HPV16 early transcripts is difficult to translate into a relevant biomarker for cervical cancer screening.

HPV genotyping in clinical samples using long‐read single‐molecule real‐time sequencing

AbstractHuman papillomavirus (HPV) genotyping is widely used, particularly in combination with high‐risk (HR) HPV tests for cervical cancer screening. We developed a genotyping method using sequences of approximately 800 bp in the E6/E7 region obtained by PacBio single molecule real‐time sequencing (SMRT) and evaluated its performance against MY09‐11 L1 sequencing and after the APTIMA HPV genotyping assay. The levels of concordance of PacBio E6/E7 SMRT sequencing with MY09‐11 L1 sequencing and APTIMA HPV genotyping were 100% and 90.8%, respectively. The sensitivity of PacBio E6/EA7 SMRT was slightly greater than that of L1 sequencing and, as expected, lower than that of HR‐HPV tests. In the context of cervical cancer screening, PacBio E6/E7 SMRT is then best used after a positive HPV test. PacBio E6/E7 SMRT genotyping is an attractive alternative for HR and LR‐HPV genotyping of clinical samples. PacBio SMRT sequencing provides unbiased genotyping and can detect multiple HPV infections and haplotypes within a genotype.

Distribution and diagnostic value of single and multiple high‐risk HPV infections in detection of cervical intraepithelial neoplasia: A retrospective multicenter study in China

AbstractThe risk associated with single and multiple human papillomavirus (HPV) infections in cervical intraepithelial neoplasia (CIN) remains uncertain. This study aims to explore the distribution and diagnostic significance of the number of high‐risk HPV (hr‐HPV) infections in detecting CIN, addressing a crucial gap in our understanding. This comprehensive multicenter, retrospective study meticulously analyzed the distribution of single and multiple hr‐HPV, the risk of CIN2+, the relationship with CIN, and the impact on the diagnostic performance of colposcopy using demographic information, clinical histories, and tissue samples. The composition of a single infection was predominantly HPV16, 52, 58, 18, and 51, while HPV16 and 33 were identified as the primary causes of CIN2+. The primary instances of dual infection were mainly observed in combinations such as HPV16/18, HPV16/52, and HPV16/58, while HPV16/33 was identified as the primary cause of CIN2+. The incidence of hr‐HPV infections shows a dose–response relationship with the risk of CIN (p for trend <0.001). Compared to single hr‐HPV, multiple hr‐HPV infections were associated with increased risks of CIN1 (1.44, 95% confidence interval [CI]: 1.20–1.72), CIN2 (1.70, 95% CI: 1.38–2.09), and CIN3 (1.08, 95% CI: 0.86–1.37). The colposcopy‐based specificity of single hr‐HPV (93.4, 95% CI: 92.4–94.4) and multiple hr‐HPV (92.9, 95% CI: 90.8–94.6) was significantly lower than negative (97.9, 95% CI: 97.0–98.5) in detecting high‐grade squamous intraepithelial lesion or worse (HSIL+). However, the sensitivity of single hr‐HPV (73.5, 95% CI: 70.8–76.0) and multiple hr‐HPV (71.8, 95% CI: 67.0–76.2) was higher than negative (62.0, 95% CI: 51.0–71.9) in detecting HSIL+. We found that multiple hr‐HPV infections increase the risk of developing CIN lesions compared to a single infection. Colposcopy for HSIL+ detection showed high sensitivity and low specificity for hr‐HPV infection. Apart from HPV16, this study also found that HPV33 is a major pathogenic genotype.

Analysis of the diagnostic performance of PAX1/SOX1 gene methylation in cervical precancerous lesions and its role in triage diagnosis

AbstractMethylation panels, tools for investigating epigenetic changes associated with diseases like cancer, can identify DNA methylation patterns indicative of disease, providing diagnostic or prognostic insights. However, the application of methylation panels focusing on the sex‐determining region Y‐box 1 (SOX1) and paired box gene 1 (PAX1) genes for diagnosing cervical lesions is under‐researched. This study aims to examine the diagnostic performance of PAX1/SOX1 gene methylation as a marker for cervical precancerous lesions and its potential application in triage diagnosis. From September 2022 to April 2023, 181 patients with abnormal HPV‐DNA tests or cytological exam results requiring colposcopy were studied at Hubei Maternal and Child Health Hospital, China. Data were collected from colposcopy, cytology, HPV‐DNA tests, and PAX1/SOX1 methylation detection. Patients were categorized as control, cervical intraepithelial neoplasia Grade 1 (CIN1), Grade 2 (CIN2), Grade 3 (CIN3), and cervical cancer (CC) groups based on histopathology. We performed HPV testing, liquid‐based cytology, and PAX1/SOX1 gene methylation testing. We evaluated the diagnostic value of methylation detection in cervical cancer using DNA methylation positivity rate, sensitivity, specificity, and area under the curve (AUC), and explored its potential for triage diagnosis. PAX1/SOX1 methylation positivity rates were: control 17.1%, CIN1 22.5%, CIN2 100.0%, CIN3 90.0%, and CC 100.0%. The AUC values for PAX1 gene methylation detection in diagnosing CIN1+, CIN2+, and CIN3+ were 0.52 (95% confidence interval [CI]: 0.43–0.62), 0.88 (95% CI: 0.80–0.97), and 0.88 (95% CI: 0.75–1.00), respectively. Corresponding AUC values for SOX1 gene methylation detection were 0.47 (95% CI: 0.40–0.58), 0.80 (95% CI: 0.68–0.93), and 0.92 (95% CI: 0.811–1.00), respectively. In HPV16/18‐negative patients, methylation detection showed sensitivity of 32.4% and specificity of 83.7% for CIN1+. For CIN2+ and CIN3+, sensitivity was all 100%, with specificities of 83.0% and 81.1%. Among the patients who underwent colposcopy examination, 166 cases had cytological examination results ≤ASCUS, of which 37 cases were positive for methylation, and the colposcopy referral rate was 22.29%. PAX1/SOX1 gene methylation detection exhibits strong diagnostic efficacy for cervical precancerous lesions and holds significant value in triage diagnosis.

Identification of transcriptionally‐active human papillomavirus integrants through nanopore sequencing reveals viable targets for gene therapy against cervical cancer

Abstract Integration of the human papillomavirus (HPV) genome into the cellular genome is a key event that leads to constitutive expression of viral oncoprotein E6/E7 and drives the progression of cervical cancer. However, HPV integration patterns differ on a case‐by‐case basis among related malignancies. Next‐generation sequencing technologies still face challenges for interrogating HPV integration sites. In this study, utilizing Nanopore long‐read sequencing, we identified 452 and 108 potential integration sites from the cervical cancer cell lines (CaSki and HeLa) and five tissue samples, respectively. Based on long Nanopore chimeric reads, we were able to analyze the methylation status of the HPV long control region (LCR), which controls oncogene E6/E7 expression, and to identify transcriptionally‐active integrants among the numerous integrants. As a proof of concept, we identified an active HPV integrant in between RUNX2 and CLIC5 on chromosome 6 in the CaSki cell line, which was supported by ATAC‐seq, H3K27Ac ChIP‐seq, and RNA‐seq analysis. Knockout of the active HPV integrant, by the CRISPR/Cas9 system, dramatically crippled cell proliferation and induced cell senescence. In conclusion, identifying transcriptionally‐active HPV integrants with Nanopore sequencing can provide viable targets for gene therapy against HPV‐associated cancers.

Advanced technologies towards improved HPV diagnostics

Abstract Persistent infection with high‐risk types of human papillomaviruses (HPV) is a major cause of cervical cancer, and an important factor in other malignancies, for example, head and neck cancer. Despite recent progress in screening and vaccination, the incidence and mortality are still relatively high, especially in low‐income countries. The mortality and financial burden associated with the treatment could be decreased if a simple, rapid, and inexpensive technology for HPV testing becomes available, targeting individuals for further monitoring with increased risk of developing cancer. Commercial HPV tests available in the market are often relatively expensive, time‐consuming, and require sophisticated instrumentation, which limits their more widespread utilization. To address these challenges, novel technologies are being implemented also for HPV diagnostics that include for example, isothermal amplification techniques, lateral flow assays, CRISPR‐Cas‐based systems, as well as microfluidics, paperfluidics and lab‐on‐a‐chip devices, ideal for point‐of‐care testing in decentralized settings. In this review, we first evaluate current commercial HPV tests, followed by a description of advanced technologies, explanation of their principles, critical evaluation of their strengths and weaknesses, and suggestions for their possible implementation into medical diagnostics.

Effectiveness of prophylactic HPV vaccines against cervical abnormalities and HPV infection in Japan: The J‐HERS 2021 multicenter study

Abstract This study investigated the efficacy of the prophylactic human papillomavirus (HPV) vaccine, which was initiated between 2009 and 2013 in Japan. The study involved 1529 eligible women aged 16–39 years who visited 11 outpatient clinics in Japan for various reasons. These patients underwent HPV genotype analysis and a Pap test of cervical cell samples. A total of 299 women (19.6%) had received the prophylactic HPV vaccine (bivalent:quadrivalent vaccine ratio = 2:1). Of the 5062 participants in the Japanese Human Papillomavirus Disease Education and Research Survey (J‐HERS 2011), which was conducted in the pre‐vaccination era, 3236 eligible participants were included as controls. In this study (J‐HERS 2021), the highest rate of HPV vaccination (53%) was observed in patients aged 22–27 years. Vaccinated individuals exhibited a 49% rate of protection against low‐grade intraepithelial lesions (LSILs) and atypical squamous cells, not excluding high‐grade squamous intraepithelial lesions (ASCH) or worse (LSIL/ASCH+), and a 100% rate of protection against high‐grade squamous intraepithelial lesions (HSILs) or worse (HSIL+). Significant reductions in HPV16 (95%) and HPV18 (100%) infections were noted, but no differences were observed in HPV6 and HPV11 infections. The prevalences of HPV51 and HPV59 increased with vaccination, although these changes were not confirmed in the comparative study with J‐HERS 2011. Comparing the prevaccination (J‐HERS 2011) and postvaccination (J‐HERS 2021) periods, 43%, 51%, 88%, and 62% reductions in HPV16, HPV18, HPV16/18, and HPV31/58 infection rates were observed, respectively. Similarly, 62% and 71% reductions in LSIL/ASCH+ and HSIL+ rates were noted, respectively. There were 88% and 87% reductions in LSIL/ASCH+ and HSIL+ rates in 16–21‐ and 28–33‐year‐old patients, respectively. Bivalent or quadrivalent vaccines provided 100% protection against high‐grade squamous cell lesions (suggestive of CIN2 or CIN3) in young women aged <39 years at 9–12 years after initiation of Japan's first nationwide HPV vaccination program. Cross‐protection against HPV31 and HPV58 is likely to occur, although some HPV‐type replacements are inconsistent across vaccination regimens. This demonstrates the effectiveness of the HPV vaccine. However, continuous monitoring of cervical cancer and precancer is necessary in younger generations (born 1997–2007), who were rarely vaccinated due to the prolonged suspension of the vaccine recommendations in Japan.

The International Human Papillomavirus Reference Center: Standardization, collaboration, and quality assurance in HPV research and diagnostics

AbstractThe International Human Papillomavirus (HPV) Reference Center (IHRC) confirms and assigns type numbers to novel HPV types, maintains a reference clone repository, and issues international proficiency panels for HPV screening and genotyping. Furthermore, the Center coordinates the Global HPV Reference Laboratory Network that promotes collaboration and international exchange of experiences among national HPV reference laboratories, to further international standardization and quality assurance in the HPV field. The established HPV types (n = 225) belong to 5 different genera: alpha (n = 65), beta (n = 54), gamma (n = 102), mu (n = 3) and nu (n = 1). Since the last published IHRC overview in 2018, 6 novel types have been established, with 5/6 belonging to the gamma genus and 1/6 to beta genus. Also, 474 reference clones have been provided to 55 different research laboratories and the global proficiency program for HPV genotyping has seen an increasing proficiency (despite a decrease seen in 2019), from 68% proficiency in 2017 to 77.3% in 2022. The first proficiency study for HPV screening found an international proficiency of up to 77%. In summary, increasing complexity of the HPVs and demands on quality assurance in the era of cervical cancer elimination requires international efforts to support proficiency and recognized quality and order among HPV types.

A paradigm for post‐embryonic Oct4 re‐expression: E7‐induced hydroxymethylation regulates Oct4 expression in cervical cancer

Abstract The Octamer‐binding transcription factor‐4 (Oct4) is upregulated in different malignancies, yet a paradigm for mechanisms of Oct4 post‐embryonic re‐expression is inadequately understood. In cervical cancer, Oct4 expression is higher in human papillomavirus (HPV)‐related than HPV‐unrelated cervical cancers and this upregulation correlates with the expression of the E7 oncogene. We have reported that E7 affects the Oct4‐transcriptional output and Oct4‐related phenotypes in cervical cancer, however, the underlying mechanism remains elusive. Here, we characterize the Oct4‐protein interactions in cervical cancer cells via computational analyses and Mass Spectrometry and reveal that Methyl‐binding proteins (MBD2 and MBD3), are determinants of Oct4‐driven transcription. E7 triggers MBD2 downregulation and TET1 upregulation, thereby disrupting the methylation status of the O ct4 gene. This coincides with an increase in the total DNA hydroxymethylation leading to the re‐expression of Oct4 in cervical cancer and likely affecting broader transcriptional patterns. Our findings reveal a previously unreported mechanism by which the E7 oncogene can regulate Oct4 re‐expression and global transcriptional patterns by increasing DNA hydroxymethylation and lowering the barrier to cellular plasticity during carcinogenesis.

The impact of cycling hypoxia on the phenotype of HPV‐positive cervical cancer cells

AbstractCycling hypoxia (cycH) is a prevalent form of tumor hypoxia that is characterized by exposure of tumor cells to recurrent phases of hypoxia and reoxygenation. CycH has been associated with a particularly aggressive cellular phenotype of tumor cells and increased therapy resistance. By performing comparative analyses under normoxia, physoxia, chronic hypoxia, and cycH, we here uncover distinct effects of cycH on the phenotype of human papillomavirus (HPV)‐positive cervical cancer cells. We show that—other than under chronic hypoxia—viral E6/E7 oncogene expression is largely maintained under cycH as is the E6/E7‐dependent regulation of p53 and retinoblastoma protein. Further, cycH enables HPV‐positive cancer cells to evade prosenescent chemotherapy, similar to chronic hypoxia. Moreover, cells under cycH exhibit a particularly pronounced resistance to the proapoptotic effects of Cisplatin. Quantitative proteome analyses reveal that cycH induces a unique proteomic signature in cervical cancer cells, which includes a significant downregulation of luminal lysosomal proteins. These encompass the potentially proapoptotic cathepsins B and cathepsin L, which, however, appear not to affect the response to Cisplatin under any of the O2 conditions tested. Rather, we show that the proapoptotic Caspase 8/BH3‐interacting domain death agonist (BID) cascade plays a pivotal role for the efficiency of Cisplatin‐induced apoptosis in HPV‐positive cancer cells under all investigated O2 conditions. In addition, we provide evidence that BID activation by Cisplatin is impaired under cycH, which could contribute to the high resistance to the proapoptotic effects of Cisplatin. Collectively, this study provides the first insights into the profound phenotypic alterations induced by cycH in HPV‐positive cancer cells, with implications for their therapeutic susceptibility.

The potential of PCNA inhibition as a therapeutic strategy in cervical cancer

AbstractCervical cancers are the fourth most common and most deadly cancer in women worldwide. Despite being a tremendous public health burden, few novel approaches to improve care for these malignancies have been introduced. We discuss the potential for proliferating cell nuclear antigen (PCNA) inhibition to address this need as well as the advantages and disadvantages for compounds that can therapeutically inhibit PCNA with a specific focus on cervical cancer.

Revisiting the role of endogenous STAT3 in HPV‐positive cervical cancer cells

AbstractNovel treatment options for human papillomavirus (HPV)‐induced cancers are urgently required. The oncogenic transcription factor signal transducer and activator of transcription 3 (STAT3) is considered to be constitutively active in HPV‐positive cervical cancer cells and essential for their proliferation. Moreover, STAT3 was reported to undergo mutually stimulatory interactions with the HPV E6/E7 oncogenes. Thus, inhibiting STAT3 in HPV‐positive cancer cells is under discussion to provide a powerful novel therapeutic strategy. We here show that the antifungal drug ciclopirox destabilizes the STAT3 protein by acting as an iron chelator. However, by exploring the functional consequences of STAT3 inhibition in HPV‐positive cancer cells, we obtained several unexpected results. Chemical STAT3 inhibitors heterogeneously affect cervical cancer cell proliferation and those which act antiproliferative also block the growth of STAT3 knockout cells, indicating induction of off‐target effects. In contrast to several chemical inhibitors, genetic inhibition of STAT3 expression by either RNA interference or the CRISPR/Cas9 method does not appreciably affect cervical cancer cell proliferation. Transcriptome analyses indicate that blocking STAT3 expression in HPV‐positive cancer cells has very limited effects on putative STAT3 target genes. Although the targeted inhibition of specific growth‐promoting signaling pathways leads to a feedback activation of STAT3 in cervical cancer cells via Janus kinase 1/2, this does not lead to treatment resistance. Moreover, we did not obtain experimental evidence for a STAT3‐linked activation of HPV E6/E7 oncogene expression or, vice versa, an E6/E7‐dependent activation of STAT3, at endogenous conditions in cervical cancer cells. Collectively, these findings question the essential role of STAT3 in cervical cancer cell proliferation and the strategy to inhibit STAT3 in these cells for therapeutic purposes.

The impact of the COVID‐19 pandemic on the prevalence and genotype distribution of HPV infection in Beijing, China

AbstractHuman papillomavirus (HPV) is one of the most common sexually transmitted infections nationwide. The COVID‐19 pandemic has greatly influenced on the HPV prevention project. The objective of this study was to examine the influence of the pandemic on HPV prevalence and genotype distribution in Beijing, China. A total of 44 401 genital swabs were obtained from outpatients at Peking Union Medical College Hospital during two distinct periods: the prepandemic stage from January 2017 to December 2019 and the pandemic stage from January 2020 to December 2022. During the prepandemic and pandemic stages, a total of 33 531 and 10 870 swabs were respectively collected. Fifteen high‐risk HPV (HR‐HPV) DNA type and a combination of two low‐risk (LR‐HPV) types (6/11) of genital swabs were detected to compare the HPV infection rates and genotype distributions in two stages. The results showed that the pandemic period witnessed a decrease in the overall HPV infection rate from 33.43% (11 245/33 531) to 29.43% (5527/18 780) compared to the prepandemic. There were statistically significant differences in infection rates between females and males (p < 0.05). Single infection was the predominant type while multiple infection was more prevalent in males than females in both prepandemic and pandemic periods. HR‐HPV infection constituted the majority of infections and cannot be disregarded. The distribution of HR‐HPV genotypes exhibited little variation before and after the outbreak, but there were some differences between females and males. HPV 16, 52, 58, 56, and 66 were the most commonly detected genotypes in females, whereas HPV 16, 52, 51, 58, and 18 were frequently detected in males. Additionally, HPV 6/11 exhibited a higher prevalence in males than in females. Notably, the age group of 31−40 years old exhibited the highest prevalence of HPV and the lowest infection rate was detected among individuals aged ≤20 years (p < 0.05), which remained relatively consistent before and during the pandemic. These findings underscore the importance of monitoring the trend of HPV epidemic and offer valuable insights for the prevention, treatment, and scientific investigation of HPV in the post‐COVID‐19 era.

Intra‐ and interlaboratory reproducibility of the RIATOL qPCR HPV genotyping assay

AbstractThe implementation of cervical screening based on human papillomavirus (HPV) continues to progress rapidly across countries. Evidence has shown that assays detecting high‐risk human papillomavirus (hrHPV) deoxyribonucleic acid (DNA) are more effective than cytology‐based screening. Validation of new hrHPV DNA assays requires both noninferior clinical accuracy compared to a standard comparator for cervical precancer and good reproducibility. This study builds upon previous diagnostic accuracy assessments of the RIATOL HPV genotyping qPCR assay and aims to evaluate the international validation criteria for reproducibility. The intra‐ and interreproducibility of the RIATOL‐qPCR assay were assessed using 550 remnant cervical cell material from the cytology archive of the National Reference Center for HPV in Belgium. Specimens were collected in the context of cervical cancer screening and tested in two different laboratories. The international reproducibility criteria include the lower bound of 95% confidence interval of the intra‐ and interlaboratory agreement regarding the detection of hrHPV DNA exceeding 87% with kappa ≥0.50. The RIATOL‐qPCR assay demonstrated excellent intralaboratory reproducibility, achieving an overall agreement of 98.2 (95% CI 96.6–99.1%) and a kappa of 0.96. Interlaboratory testing showed an overall agreement of 98.5 (95% CI 97.1–99.4%) with a kappa of 0.97. The RIATOL‐qPCR assay fulfills the third criterion for HPV test reproducibility requirement for use in cervical cancer screening.

Identification of HPV oncogene and host cell differentiation associated cellular heterogeneity in cervical cancer via single‐cell transcriptomic analysis

Abstract Human Papillomaviruses (HPVs) are associated with around 5%–10% of human cancer, notably nearly 99% of cervical cancer. The mechanisms HPV interacts with stratified epithelium (differentiated layers) during the viral life cycle, and oncogenesis remain unclear. In this study, we used single‐cell transcriptome analysis to study viral gene and host cell differentiation‐associated heterogeneity of HPV‐positive cervical cancer tissue. We examined the HPV16 genes—E1, E6, and E7, and found they expressed differently across nine epithelial clusters. We found that three epithelial clusters had the highest proportion of HPV‐positive cells (33.6%, 37.5%, and 32.4%, respectively), while two exhibited the lowest proportions (7.21% and 5.63%, respectively). Notably, the cluster with the most HPV‐positive cells deviated significantly from normal epithelial layer markers, exhibiting functional heterogeneity and altered epithelial structuring, indicating that significant molecular heterogeneity existed in cancer tissues and that these cells exhibited unique/different gene signatures compared with normal epithelial cells. These HPV‐positive cells, compared to HPV‐negative, showed different gene expressions related to the extracellular matrix, cell adhesion, proliferation, and apoptosis. Further, the viral oncogenes E6 and E7 appeared to modify epithelial function via distinct pathways, thus contributing to cervical cancer progression. We investigated the HPV and host transcripts from a novel viewpoint focusing on layer heterogeneity. Our results indicated varied HPV expression across epithelial clusters and epithelial heterogeneity associated with viral oncogenes, contributing biological insights to this critical field of study.

CREB1 activation promotes human papillomavirus oncogene expression and cervical cancer cell transformation

AbstractHuman papillomaviruses (HPVs) infect the oral and anogenital mucosa and can cause cancer. The high‐risk (HR)‐HPV oncoproteins, E6 and E7, hijack cellular factors to promote cell proliferation, delay differentiation and induce genomic instability, thus predisposing infected cells to malignant transformation. cAMP response element (CRE)‐binding protein 1 (CREB1) is a master transcription factor that can function as a proto‐oncogene, the abnormal activity of which is associated with multiple cancers. However, little is known about the interplay between HPV and CREB1 activity in cervical cancer or the productive HPV lifecycle. We show that CREB is activated in productively infected primary keratinocytes and that CREB1 expression and phosphorylation is associated with the progression of HPV+ cervical disease. The depletion of CREB1 or inhibition of CREB1 activity results in decreased cell proliferation and reduced expression of markers of epithelial to mesenchymal transition, coupled with reduced migration in HPV+ cervical cancer cell lines. CREB1 expression is negatively regulated by the tumor suppressor microRNA, miR‐203a, and CREB1 phosphorylation is controlled through the MAPK/MSK pathway. Crucially, CREB1 directly binds the viral promoter to upregulate transcription of the E6/E7 oncogenes, establishing a positive feedback loop between the HPV oncoproteins and CREB1. Our findings demonstrate the oncogenic function of CREB1 in HPV+ cervical cancer and its relationship with the HPV oncogenes.

Risk‐Based Triage Strategy by Extended HPV Genotyping for Women With LSIL Cytology: A Real‐World Study

AbstractTo evaluate the immediate risk of (pre)cancer for cytology low‐grade squamous intraepithelial lesion (LSIL) women infected with or without specific HPV genotype and develop a risk‐based management strategy. A total of 4567 LSIL women with extended HPV genotyping and colposcopy results were enrolled according to the inclusive and exclusive criteria. The distribution and immediate cervical intraepithelial neoplasia grade 2 or worse and 3+ or worse (CIN2+/3+) risks of specific HPV genotypes were assessed using Minimum Estimate, Any Type Estimate, and Hierarchical Attribution Estimate. A risk‐based strategy was further established and evaluated. CIN2+/3+ were 729/328 cases, including 691/317 in 3398 HPV‐positive and 38/11 in 1169 HPV‐negative women. HPV16, 52, 58, and 18 were the most prevalent genotypes in both HPV‐positive and CIN2+/3+ cases. HPV16, 73, and 33 carried the highest immediate CIN2+/3+ risk. A risk‐based strategy was established, which suggested Group A (HPV 16, 33, 45, 31, 18, 58, 52, 35, 73, 82; with immediate CIN3+ risk of 4.08%–22.12%) for immediate colposcopy, Group B (HPV 59, 66, 56, 53) for 6‐month follow‐up or p16/Ki‐67 dual stain or DNA methylation triage, while Group C (HPV 51, 68, 39, 26) for 1‐year HPV repeat testing. Compared with conventional strategy, this new strategy showed significantly higher specificity (CIN2+: 52.16% vs. 29.47%, χ2 = 409.136, p < 0.001; CIN3+: 48.45% vs. 27.32%, χ2 = 402.395, p < 0.001) but similar sensitivity, which could reduce immediate colposcopy referrals by 19.82%. A risk‐based triage strategy for LSIL women with extended HPV genotyping could effectively reduce unnecessary colposcopies and maintain high efficacy for CIN2+/3+ detection.

Advances in HPV‐associated tumor management: Therapeutic strategies and emerging insights

AbstractWith the rapid increase in the incidence of cervical cancer, anal cancer and other cancers, human papillomavirus (HPV) infection has become a growing concern. Persistent infection with high‐risk HPV is a major cause of malignant tumors. In addition, microbiota and viruses such as human immunodeficiency virus, herpes simplex virus, and Epstein–Barr virus are closely associated with HPV infection. The limited effectiveness of existing treatments for HPV‐associated tumors and the high rates of recurrence and metastasis in patients create an urgent need for novel and effective approaches. In recent years, HPV vaccine coverage has increased and can reduce the incidence of serious adverse events. Overall, this article provides a comprehensive overview of HPV biology, microbiome, and other viral interactions in cancer development, highlighting the need for a more comprehensive approach to cancer prevention and treatment. Current and emerging HPV‐related cancer control and treatment strategies are also further explored.

NFX1‐123: A potential therapeutic target in cervical cancer

AbstractNFX1‐123 is a splice variant isoform of the NFX1 gene. It is highly expressed in cervical cancers caused by HPV, and NFX1‐123 is a protein partner with the HPV oncoprotein E6. Together, NFX1‐123 and E6 affect cellular growth, longevity, and differentiation. The expression status of NFX1‐123 in cancers beyond cervical and head and neck cancers, and its potential as therapeutic target, have not been investigated. TSVdb of TCGA was used to quantify NFX1‐123 expression in 24 cancers compared with normal tissues. The NFX1‐123 protein structure was predicted and then submitted to retrieve suitable drug molecules. The top four compounds, found to bind in silico to NFX1‐123, were tested experimentally to determine their effects on NFX1‐123‐related cellular growth, survival, and migration. 46% of cancers (11 of 24 had significant differences in NFX1‐123 expression, with nine having had greater NFX1‐123 expression, when compared with adjacent normal tissues. Bioinformatics and proteomic predictive analysis modeled the three‐dimensional structure of NFX1‐123, and drug libraries were screened for high‐binding affinity compounds using this modeled structure. Seventeen drugs with binding energies ranging from −1.3 to −10 Kcal/mol were identified. The top four compounds were used to treat HPV‐ and HPV+ cervical cancer cell lines, three of which (Ropitoin, R428 and Ketoconazole) reduced NFX1‐123 protein levels, inhibited cellular growth, survival, and migration, and enhanced the cytotoxicity of Cisplatin. These findings highlight cancers expressing high levels of NFX1‐123, and drugs that target it, may reduce cellular growth, survival, and migration, making NFX1‐123 a potential novel therapeutic target.

25‐hydroxycholesterol inhibits human papillomavirus infection in cervical epithelial cells by perturbing cytoskeletal remodeling

AbstractPersistent high‐risk human papilloma virus (HR‐HPV) infection is the main risk factor for cervical cancer, threatening women's health. Despite growing prophylactic vaccination, annual cervical cancer cases are still increasing and show a trend of younger onset age. However, therapeutic approaches towards HPV infection are still limited. 25‐hydrocholesterol (25HC) has a wide‐spectrum inhibitory effect on a variety of viruses. To explore efficient interventions to restrict HPV infection at an early time, we applied different pseudoviruses (PsV) to evaluate anti‐HPV efficacy of 25HC. We tested PsV inhibition by 25HC in cervical epithelial‐derived HeLa and C‐33A cells, using high‐risk (HPV16, HPV18, HPV59), possibly carcinogenic (HPV73), and low‐risk (HPV6) HPV PsVs. Then we established murine genital HPV PsV infection models and applied IVIS to evaluate anti‐HPV efficacy of 25HC in vivo. Next, with the help of confocal imaging, we targeted 25HC activity at filopodia upon HPV exposure. After that, we used RNA‐seq and Western blot analysis to investigate (1) how 25HC disturbs actin cytoskeleton remodeling during HPV infection and (2) how prenylation regulates the cytoskeletal remodeling signaling pathway. Our findings suggest that 25HC perturbs F‐actin rearrangement by reducing small GTPase prenylation. In this way, the phenomenon of HPV virion surfing was restricted, leading to failed infection.

Detection of human papillomavirus in fresh and dried urine through an automated system for cervical cancer screening in low‐ and middle‐income countries

AbstractThe majority of cervical cancer cases and associated deaths occur in low‐ and middle‐income countries (LMICs), where sociocultural barriers, poor access to prevention and care, and technical and practical difficulties hinder screening coverage improvement. Using urine specimens for human papillomaviruses (HPV) molecular screening through automated testing platforms can help to overcome these problems. We evaluated the high‐risk (HR) HPV detection performance of the Xpert® HPV test on GeneXpert® System (Cepheid), on fresh and dried urine (Dried Urine Spot [DUS]) samples as compared to an in‐house polymerase chain reaction (PCR) genotyping assay. Forty‐five concentrated urine samples collected from women with known cytological and HPV infection status, determined through in‐house PCR and genotyping assays, were tested “as is” and as DUS with the Xpert® HPV test. This system detected HR‐HPV in 86.4% of fresh and in 77.3% of dried urine samples collected from HPV+ women, correctly identifying HR‐HPV infection in 100% of women with low‐ and high‐grade lesions. High concordance (91.4%, k = 0.82) was found between PCR test and Xpert® HPV Test from urine. Urine‐based Xpert® HPV test seems to be a suitable screening test for detection of HR‐HPV infections associated with low‐ and high‐grade lesions requiring follow‐up monitoring or treatment. This methodology, relying on noninvasively collected samples and on available rapid testing platforms, could facilitate large, at‐scale screening programs, particularly in LMICs and rural areas, thus reducing adverse outcomes of HPV infection and facilitating achievement of the WHO cervical cancer elimination goal.

Chimeric oncolytic Ad5/3 virus replicates and lyses ovarian cancer cells through desmoglein‐2 cell entry receptor

AbstractDespite new therapies, the estimated 229 875 women living with ovarian cancer have a 5‐year survival rate of 47.6%. This cavity‐localized cancer lends itself to local administration of modalities, such as the oncolytic adenovirus (Ad) Ad5/3‐D24‐granulocyte‐macrophage colony‐stimulating factor virus (ONCOS‐102). Its repeated administration to a patient with chemotherapy‐refractory ovarian cancer induced CD8+ antitumor immune responses with the overall survival reaching 40 months. Here we probe the dominant receptor used by ONCOS‐102 in four established epithelial ovarian cancer cell lines. Ad3 can use the desmoglein‐2 (DSG2) and CD46 receptors on susceptible cells. DSG2 was nearly absent in A2780 cells but was expressed in more than 90% of OAW42, OVCAR3, and OV‐90 cells. After 96 hours, ONCOS‐102 treatment showed significant oncolytic activity (≧50%) in OAW42, OVCAR3, and OV‐90 cells, but minimal activity in A2780 cells, suggesting DSG2 as the dominant receptor for ONCOS‐102. Furthermore, retrospective analyses of phase I clinical trial of ONCOS‐102 treatment of 12 patients with varied tumors indicated a correlation between viral genomes in blood and DSG2 RNA expression. These data support the role of DSG2 expression on cancer cells in virus infectivity and the continued development of ONCOS‐102 for ovarian cancer treatment.

Laurencia johnstonii extract reverses early lesions in the K14E7HPV16 murine cervical carcinogenesis model

AbstractPersistent infection with high‐risk human papillomavirus (HR‐HPV) is a well‐established risk factor to the development of cervical intraepithelial neoplasia (CIN), a condition that can progress to cervical cancer (CC) a major health problem worldwide. Recently, there has been growing interest in exploring alternative therapies utilizing natural products, among which is the algae species Laurencia johnstonii Setchell & Gardner, 1924 (L. johnstonii), proposed for the management of precancerous lesions. The aim of this work was to determine the effect of an organic extract from L. johnstonii (ELj) in early cervical lesions (CIN 1). These CIN 1 lesions were generated in a murine model expressing the HR‐HPV16 E7 oncoprotein (K14E7HPV transgenic mice) with a single exogenous hormonal stimulus using 17β‐estradiol. The histopathological studies, the determination of cell proliferation and of the apoptotic levels in cervical tissue, showed that, seven doses of ELj (30 mg/kg weight per day diluted in a DMSO‐saline solution [1:7]) lead to recovery the architecture of cervical epithelium. Accordingly, in the transgenic mice it was observed a statistically significant decrease of the PCNA expression levels, a marker of cell proliferation, and a statistically significant increase in the apoptosis levels using Caspase 3 as a marker. In addition, we determined the expression levels of the tumor suppressor miR‐218 and the oncomiRNA miR‐21. Interestingly, our results may suggest that ELj treatment tended to restore the normal expression of both miRNAs as compared with controls being more evident in the non‐transgenic induced mice. Differences of p < 0.05 were considered statistically significant through the whole study. Based on these results, we propose that the use of ELj could be an alternative for the treatment of cervical early lesions.

Reflections Regarding Validation of New HPV Tests With Reduced HPV Genotypes: Report From an IARC Expert Consultation

ABSTRACT Of the 12 HPV genotypes classified as carcinogenic to humans (Group 1), over 95% of HPV‐positive cervical cancers are linked to eight genotypes (HPV16/18/31/33/35/45/52/58). Screening programmes may consider HPV tests incorporating only these genotypes to improve screening efficiency and reduce programmatic costs. Validation of such tests requires fine‐tuning of existing criteria. An expert group convened by the International Agency for Research on Cancer discussed how existing criteria by Meijer et al. for HPV screening clinical validation should be adapted to evaluate new reduced‐valency HPV tests. Experts identified four key criteria: (1) Clinical performance criteria should meet WHO HPV test Target Product Profiles (TPP) minimal standards with high relative sensitivity ( ≥ 0.90 for CIN2+ and ≥ 0.95 for CIN3+) and relative specificity ( ≥ 0.98 for ≤ CIN1) to detect CIN2/3+ lesions associated with types targeted by the test, as established by a comparator test providing information on the presence of the targeted genotypes; (2) Comparator tests should be clinically validated according to Meijer criteria principles for comparator tests, and should offer HPV genotyping to detect at least the types included in the reduced‐valency test; (3) Cervical samples should be representative of a population‐based screening programme; (4) Intra‐ and inter‐laboratory reproducibility should adhere to Meijer criteria and, preferentially also the more stringent TPP. As the global HPV type distribution in cervical cancer is well known, a future evaluation strategy may consider including both virological and simplified clinical standards. The consultation highlights essential criteria building on existing clinical accuracy standards, enriched with analytical standards. These criteria will be instrumental in ensuring both accuracy and reliability of new reduced‐valency HPV tests for cervical cancer screening highly needed to assure 70% coverage aim of cervical cancer elimination.

Significance of Genotype‐Specific High‐Risk Human Papillomavirus Testing in Cervical Cancer Screening: A Hospital‐Based Study

ABSTRACTThis study explored histopathological outcomes among women who tested positive for high‐risk human papillomavirus (hrHPV), examined the significance of extended HPV genotyping, and identified predictors of cervical intraepithelial neoplasia grade 2 or worse (CIN2+). This retrospective review assessed medical records of women who screened positive for hrHPV between January 1, 2020, and December 31, 2023. Genotyping results, diagnostic procedures, and histopathological findings were collected. Data were analyzed using SPSS, with p <  0.05 considered statistically significant. Among 1981 women, the median age was 40 years (IQR 32.0‒49.0), and the median parity was 1 (IQR 0‒2). Overall, 1223 women (61.7%) had prior screening, 1215 women (61.3%) had previous cytology, and 107 women (5.4%) had prior hrHPV testing. Single‐genotype infection occurred in 1408 women (74.7%), with HPV52, HPV16, and HPV58 identified in 23.7%, 15.6%, and 15.4% of cases, respectively. CIN2+ was detected in 152 women (7.7%), including 130 with CIN2/CIN3/AIS and 22 with cancer. Detection of HPV16 significantly increased the risk of CIN2+ (odds ratio [OR] 4.534, 95% CI: 3.197‒6.430), as did multiparity (OR 1.497, 95% CI: 1.070‒2.094). The immediate risk of CIN2+ for HPV31, HPV39, HPV56, HPV66, and HPV68 was below 4%. Among hrHPV‐positive women, 7.7% had CIN2+. Extended hrHPV genotyping may refine risk stratification by highlighting HPV16 and multiparity as significant predictors of CIN2+ lesions.

High‐Throughput Targeted Sequencing Identifies an HPV Methylation Panel for Detecting Cervical Lesion Progression

ABSTRACT High‐risk human papillomavirus (hrHPV) infection is the primary cause of cervical cancer. However, hrHPV testing lacks specificity in detecting neoplastic changes. This study explored the utility of quantitative methylated HPV DNA markers for precise detection of cervical lesions. Using hybridization capture‐based bisulfite sequencing, we analyzed genome‐wide HPV methylation patterns. The study included a training cohort of 60 cervical exfoliated cell samples and a validation cohort of 29 samples. Analysis of 112 CpG sites across the HPV genome revealed that genome‐wide HPV16 methylation levels correlated with disease progression. Squamous cell carcinoma (SCC) showed 1.4‐fold higher methylation levels compared to normal tissue ( p  = 0.0032). Progressive methylation increases in the E5‐α and L2 genes were observed across the spectrum of cervical lesion severity, from normal tissue through high‐grade squamous intraepithelial lesion (HSIL) to SCC. Intersection analysis of differentially methylated CpG sites between HSIL vs Normal and SCC vs Normal identified 16 consistently hypermethylated CpG sites in the E5‐α, E7, L2, and L1 genes, distinguishing both HSIL and SCC from normal tissue. This pilot study identifies a five‐CpG methylation panel (E5‐α_3887, E5‐α_3941, E7_701, L2_4441, and L2_5128) as promising triage biomarkers for HPV16‐positive women, achieving high discriminatory performance (AUC = 0.919 in a validation cohort) for detecting cervical lesions. This genome‐wide capture sequencing identified novel HPV16 methylation markers that distinguish cervical lesions from normal tissue, supporting the feasibility of HPV methylation‐based triage for HPV‐positive women in cervical cancer screening.

Effective vaccination strategies for human papillomavirus (HPV) infection and cervical cancer based on the mathematical model with a stochastic process

AbstractHuman papillomavirus (HPV) infection poses a significant risk to women's health by causing cervical cancer. In addition to HPV, cervical cancer incidence rates can be influenced by various factors, including human immunodeficiency virus and herpes, as well as screening policy. In this study, a mathematical model with stochastic processes was developed to analyze HPV transmission between genders and its subsequent impact on cervical cancer incidence. The model simulations suggest that both‐gender vaccination is far more effective than female‐only vaccination in preventing an increase in cervical cancer incidence. With increasing stochasticity, the difference between the number of patients in the vaccinated group and the number in the nonvaccinated group diminishes. To distinguish the patient population distribution of the vaccinated from the nonvaccinated, we calculated effect size (Cohen's distance) in addition to Student's t‐test. The model analysis suggests a threshold vaccination rate for both genders for a clear reduction of cancer incidence when significant stochastic factors are present.

Genetic variability analysis of human papillomavirus 58: Novel sublineage identification and persistent infection association

AbstractThis study aims to characterize the genetic variability of HPV58, identify novel lineages and sublineages, and explore the association between persistent/multiple HPV58 infections and genetic variation. In this study, samples from 124 women with HPV58 infection in Eastern China were collected and 81 isolates of E6 and L1 full‐length genes were successfully amplified from 55 samples. We evaluated the diversity of genetic variants and performed correlation analyses between genetic variability and pathology, vaccination, multiple infections, and persistent infections. Among the E6 and L1 gene sequences collected, the dominant prevailing sublineages were A1 (46.2%) and A2 (23.1%). In addition, we found two potential novel sublineages denoted as the A4 and A5 sublineage. A total of 50 nucleotide substitutions, including 28 synonymous substitutions and 22 nonsynonymous substitutions, were observed in the E6 and L1 genes. Among them, variants with A388C/K93N substitutions in the E6 gene correlated with persistent infection (≥1 and ≥2 years) (p < 0.005), and C307T/C66C was associated with persistent infection (≥2 years) (p < 0.005). Notably, two mutations above were detected in the isolate from the patient with breakthrough vaccine infection. Our study found two novel sublineages and sites of genetic variability in multiple and persistent infection variants. In addition, we identified two mutational sites associated with persistent infection. This study provides new insight into the clinical characteristics of HPV 58 genetic variations and offers new ideas for research on next‐generation vaccines in Eastern China.

Nobel laureate Harald zur Hausen passed. He was the first to discover the HPV‐cervical cancer link

Intra‐ and interlaboratory reproducibility evaluation toward international validation status of the AmpFire assay

Abstract To meet the screening goal of WHO's 90‐70‐90 strategy aimed at eliminating cervical cancer (CC) by 2030, clinical validation of human papillomavirus (HPV) assays is essential to provide accurate and valid results through fulfilling three criteria of the international validation guidelines (IVGs). Previously, the clinical accuracy of the AmpFire® HPV Screening 16/18/HR assay (AmpFire assay) was reported but reproducibility data are lacking. Here, we aim to evaluate the intra‐ and inter‐laboratory reproducibility of the AmpFire assay. The reproducibility of the isothermal AmpFire assay was assessed using 556 cervical cell samples collected from women attending CC screening and biobanked in a Belgian HPV national reference center. This assay detects HPV16, HPV18, and 12 other high‐risk HPV (hrHPV) types (31/33/35/39/45/51/52/56/58/59/66/68) in aggregate. Lower 95% confidence interval bound around the assay's reproducibility should exceed 87%, with κ  ≥ 0.50. Additionally, a literature review of the assay's clinical performance was performed. The AmpFire assay showed an excellent intralaboratory (96.4%, 95% CI:94.5–97.8%, κ  = 0.920) and interlaboratory (95.3%, 95% CI:93.2–96.9%, κ  = 0.897) reproducibility. One study demonstrated noninferior sensitivity of a prototype AmpFire assay targeting 15 hrHPV types (including HPV53) to detect CIN2+. However, clinical specificity became similar to the comparator after removing HPV53 from analyses. The low‐cost and easy‐to‐use AmpFire assay presents excellent reproducibility and—after removing HPV53 from the targeted types—fulfills also clinical accuracy requirements. Inclusion of HPV53, which is not recognized as carcinogenic, comprises clinical specificity of screening assays.

Species‐level characterization of the cervicovaginal microbiota and its role in human papillomavirus‐associated cervical carcinogenesis

Abstract The cervicovaginal microbiome may contribute to human papillomavirus (HPV)‐associated cervical carcinogenesis, but studies have been limited by low‐resolution analysis methods. Using a high‐resolution bioinformatics pipeline, we evaluated the relationship of the cervicovaginal microbiome with HPV and cervical intraepithelial neoplasia (CIN). The cervicovaginal microbiome of 186 women was characterized by sequencing 16S rRNA regions (V3–V4 and V5–V6) and annotated with the high‐resolution ANCHOR pipeline. Samples were genotyped for HPV using the Roche‐Cobas 4800 assay. We fitted logistic regression models using stepwise forward selection to select species (presence/absence) as correlates of CIN1+ and constructed a linear microbiome‐based score using the regression coefficients. An HPV‐based score was calculated from a separate logistic regression model to detect CIN1+ . Receiver operating characteristic curve analyses were performed; the area under the curve (AUC) and 95% confidence intervals (CI) were compared between scores. Overall, 66.7% of participants were HPV‐positive. 77 unique species were identified: 8 using V3–V4, 48 using V5–V6, and 21 shared. Twelve species were retained via stepwise selection. The AUCs for the microbiome‐, and HPV‐based scores were 0.7656 (95% CI 0.6885–0.8426), and 0.7529 (95% CI 0.6855–0.8204), respectively. Bacterial species may be involved in cervical carcinogenesis as the microbiome‐ and HPV‐based scores performed similarly for CIN1+ detection.

Changes in intrahost genetic diversity according to lesion severity in longitudinal HPV16 samples

AbstractHuman papillomavirus type 16 (HPV16) is the most common cause of cervical cancer, but most infections are transient with lesions not progressing to cancer. There is a lack of specific biomarkers for early cancer risk stratification. This study aimed to explore the intrahost HPV16 genomic variation in longitudinal samples from HPV16‐infected women with different cervical lesion severity (normal, low‐grade, and high‐grade). The TaME‐seq deep sequencing protocol was used to generate whole genome HPV16 sequences of 102 samples collected over time from 40 individuals. Single nucleotide variants (SNVs) and intrahost SNVs (iSNVs) were identified in the viral genomes. A majority of individuals had a unique set of SNVs and these SNVs were stable over time. Overall, the number of iSNVs and APOBEC3‐induced iSNVs were significantly lower in high‐grade relative to normal and low‐grade samples. A significant increase in the number of APOBEC3‐induced iSNVs over time was observed for normal samples when compared to high‐grade. Our results indicates that the lower incidence of iSNVs and APOBEC3‐induced iSNVs in high‐grade lesions may have implications for novel biomarkers discoveries, potentially aiding early stratification of HPV‐induced cervical precancerous lesions.

Targeted therapies for HPV‐associated cervical cancer: Harnessing the potential of exosome‐based chipsets in combating leukemia and HPV‐mediated cervical cancer

AbstractExosomes play a crucial role in intercellular communication and have emerged as significant vehicles for transporting disease‐specific biomarkers. This feature provides profound insights into the progression of diseases and the responses of patients to treatments. For example, in leukemia, exosomes convey critical information through the carriage of specific proteins and nucleic acids. In the case of human papillomavirus (HPV)‐mediated cervical cancer, exosomes are particularly useful for noninvasive detection as they transport high‐risk HPV DNA and specific biomolecules, which can be indicators of the disease. Despite their vast potential, there are several challenges associated with the use of exosomes in medical diagnostics. These include their inherent heterogeneity, the need for enhanced sensitivity in detection methods, the establishment of standardization protocols, and the requirement for cost‐effective scalability in their application. Addressing these challenges is crucial for the effective implementation of exosome‐based diagnostics. Future research and development are geared towards overcoming these obstacles. Efforts are concentrated on refining the processes of biomarker discovery, establishing comprehensive regulatory frameworks, developing convenient point‐of‐care devices, exploring methods for multimodal detection, and conducting extensive clinical trials. The ultimate goal of these efforts is to inaugurate a new era of precision diagnostics within healthcare. This would significantly improve patient outcomes and reduce the burden of diseases such as leukemia and HPV‐mediated cervical cancer. The integration of exosomes with cutting‐edge technology holds the promise of significantly reinforcing the foundations of healthcare, leading to enhanced diagnostic accuracy, better disease monitoring, and more personalized therapeutic approaches.

Genotype, cervical intraepithelial neoplasia, and type‐specific cervical intraepithelial neoplasia distributions in hrHPV+ cases referred to colposcopy: A multicenter study of Chinese mainland women

AbstractTo investigate age and type‐specific prevalences of high‐risk human papillomavirus (hrHPV) and cervical intraepithelial neoplasia (CIN) in hrHPV+ women referred to colposcopy. This is a retrospective, multicenter study. Participants were women referred to one of seven colposcopy clinics in China after testing positive for hrHPV. Patient characteristics, hrHPV genotyping, colposcopic impressions, and histological diagnoses were abstracted from electronic records. Main outcomes were age‐related type‐specific prevalences associated with hrHPV and CIN, and colposcopic accuracy. Among 4419 hrHPV+ women referred to colposcopy, HPV 16, 52, and 58 were the most common genotypes. HPV 16 prevalence was 39.96%, decreasing from 42.57% in the youngest group to 30.81% in the eldest group. CIN3+ prevalence was 15.00% and increased with age. As lesion severity increases, HPV16 prevalence increased while the prevalence of HPV 52 and 58 decreased. No age‐based trend was identified with HPV16 prevalence among CIN2+, and HPV16‐related CIN2+ was less common in women aged 60 and above (44.26%) compared to those younger than 60 years (59.61%). Colposcopy was 0.73 sensitive at detecting CIN2+ (95% confidence interval[CI]: 0.71, 0.75), with higher sensitivity (0.77) observed in HPV16+ women (95% CI: 0.74, 0.80) compared to HPV16− women (0.68, 95% CI: 0.64, 0.71). Distributions of hrHPV genotypes, CIN, and type‐specific CIN in Chinese mainland hrHPV+ women referred to colposcopy were investigated for the first time. Distributions were found to be age‐dependent and colposcopic performance appears related to HPV genotypes. These findings could be used to improve the management of women referred to colposcopy.

Analytical evaluation of the automated genotyping system (GenPlex) compared to a traditional real‐time PCR assay for the detection of high‐risk human papillomaviruses

AbstractThe detection of high‐risk human papillomaviruses (HPVs) is crucial for early screening and preventing cervical cancer. However, the substantial workload in high‐level hospitals or the limited resources in primary‐level hospitals hinder widespread testing. To address this issue, we explored a sample‐to‐answer genotyping system and assessed its performance by comparing it with the traditional real‐time polymerase chain reaction (PCR) method conducted manually. Samples randomly selected from those undergoing routine real‐time PCR detection were re‐analyzed using the fully automatic GenPlex® system. This system identifies 24 types of HPV through a combination of ordinary PCR and microarray‐based reverse hybridization. Inconsistent results were confirmed by repeated testing with both methods, and the κ concordance test was employed to evaluate differences between the two methods. A total of 365 samples were randomly selected from 7259 women. According to real‐time PCR results, 76 were high‐risk HPV negative, and 289 were positive. The GenPlex® system achieved a κ value greater than 0.9 (ranging from 0.920 to 1.000, p < 0.0001) for 14 types of high‐risk HPV, except HPV 51 (κ = 0.697, p < 0.0001). However, the inconsistent results in high‐risk HPV 51 were revealed to be false positive in real‐time PCR by other method. When counting by samples without discriminating the high‐risk HPV type, the results of both methods were entirely consistent (κ = 1.000, p < 0.0001). Notably, the GenPlex® system identified more positive cases, with 73 having an HPV type not covered by real‐time PCR, and 20 potentially due to low DNA concentration undetectable by the latter. Compared with the routinely used real‐time PCR assay, the GenPlex® system demonstrated high consistency. Importantly, the system's advantages in automatic operation and a sealed lab‐on‐chip format respectively reduce manual work and prevent aerosol pollution. For widespread use of GenPlex® system, formal clinical validation following international criteria should be warranted.

Eliminating cervical cancer in China: Opportunities come and challenges remain

Human papillomavirus genotyping in high‐grade vaginal intraepithelial neoplasia: A multicentric Italian study

Abstract This study aimed to analyze the human papillomavirus (HPV) genotype distribution in a large cohort of high‐grade vaginal intraepithelial neoplasia (VaIN) (vaginal HSIL, VaIN2/3) patients from two Italian referral centers. We included all patients with histologically confirmed VaIN2/3 from the Department of Surgical Sciences, Sant'Anna Hospital, University of Torino, Torino, Italy, and Ospedale Maggiore della Carità, Novara, Italy, between 2003 and 2022. After the histological evaluation of formalin‐fixed paraffin‐embedded samples, we performed HPV genotyping with VisionArray HPV Chip 1.0. We detected HPV DNA in 94.4% of VaIN2/3 (168/178), with HPV 16 as the most prevalent genotype, accounting for 51.8% of all infections, 41.2% of VaIN2 and 77.6% of VaIN3 cases. Other frequent genotypes were HPV 58 (8.3%, 10.9% of VaIN2 and 2.0% of VaIN3), HPV 73 (5.4%, 5.0% of VaIN2 and 6.1% of VaIN3), and HPV 31 (5.4%, 6.7% of VaIN2 and 2.0% of VaIN3). 73.2% of VaIN2/3 had a single HPV genotype infection and 26.8% a multiple infection (20.8% a double infection, 4.8% a triple infection, and 1.2% a quadruple infection). Single infection was more frequently present in VaIN3 than VaIN2 (81.6% vs. 69.8%). 69.1% of single infections and 73.3% of multiple infections had one or more genotypes covered by nine‐valent HPV vaccine. HPV vaccination is expected to have a large impact on reducing the incidence of vaginal intraepithelial neoplasia.

Prevalence and significance of HPV DNA detection below the clinical threshold of the commercial kit Alinity m HR‐HPV assay (Abbott)

Abstract The positive clinical threshold of human papillomavirus (HPV) tests validated for primary cervical cancer screening (CCS) is designed to offer an optimal balance between clinical sensitivity and specificity. However, there may be a gap between the analytical sensitivity of the test and the positive clinical threshold, referred to here as the “gray‐zone.” This study aims to determine the prevalence and significance of HPV results obtained in the gray‐zone in routine practice. Cervical samples obtained in our institution for CCS over a 22‐month‐period were tested with the Alinity m HR‐HPV Assay (Abbott). Clinical and biological data, including cytological results and patients' HPV history were collected. Of the 6101 samples collected, 1.7% had an HPV result in the gray‐zone (102 patients). The proportion of gray‐zone results varied according to HPV genotype, reaching 11.8% of samples with detectable HPV DNA in the case of HPV31/33/52/58 genotypes. Reflex cytologies showed no abnormalities or Atypical Squamous Cells of Undetermined Significance results in 74.6% and 17.9% of cases, respectively. A previous or subsequent HPV‐positive result with a (possibly) identical genotype was observed in 58% and 38% of cases, respectively. Two women with a history of persistent HPV detection had a CIN2+ lesion 1 year after the gray‐zone result. In conclusion, the proportion of HPV results in the gray‐zone varies according to genotype. No cytological abnormality is observed in the majority of cases, but a few rare patients with a history of persistent HPV infection should be closely monitored even if the HPV result is transiently located in the gray‐zone.

Cervicovaginal microbiota long‐term dynamics and prediction of different outcomes in persistent human papillomavirus infection

Abstract Persistent human papillomavirus (HPV) infection can lead to cervical intraepithelial neoplasia (CIN) and cervical cancer, posing serious threats to the health of women. Although the cervicovaginal microbiota is strongly associated with CIN, the dynamics of the microbiota during CIN development are unknown. In this retrospective cohort study, we analyzed 3‐year longitudinal data from 72 patients diagnosed with a persistent HPV infection almost all caused by high‐risk HPV types. Patients were categorized into groups with HPV persistent infection ( n  = 37), progression to CIN ( n  = 16), and CIN regression ( n  = 19) based on infection outcome during the follow‐up period. Furthermore, 16S rRNA gene sequencing was performed on consecutively collected cervical samples to explore the composition and dynamics of the cervicovaginal microbiota during the development and regression of CIN. Our results showed that the composition of the cervicovaginal microbiota varied among women with different HPV infection outcomes and remained relatively stable during the follow‐up period. Notably, the serial follow‐up data showed that these microbial alterations were present for at least 1–2 years and occurred before pathologic changes. In addition, microbial markers that were highly discriminatory for CIN progression or regression were identified. This study provides evidence for a temporal relationship between changes in the cervicovaginal microbiota and the development of CIN, and our findings provide support for future microbial intervention strategies for CIN.

The effects of HPV oncoproteins on host communication networks: Therapeutic connotations

AbstractHuman papillomavirus (HPV) infections are a leading cause of viral‐induced malignancies worldwide, with a prominent association with cervical and head and neck cancers. The pivotal role of HPV oncoproteins, E5, E6, and E7, in manipulating cellular events, which contribute to viral pathogenesis in various ways, has been extensively documented. This article reviews the influence of HPV oncoproteins on cellular signaling pathways within the host cell, shedding light on the underlying molecular mechanisms. A comprehensive understanding of these molecular alterations is essential for the development of targeted therapies and strategies to combat HPV‐induced premalignancies and prevent their progress to cancer. Furthermore, this review underscores the intricate interplay between HPV oncoproteins and some of the most important cellular signaling pathways: Notch, Wnt/β‐catenin, MAPK, JAK/STAT, and PI3K AKT/mTOR. The treatment efficacies of the currently available inhibitors on these pathways in an HPV‐positive context are also discussed. This review also highlights the importance of continued research to advance our knowledge and enhance therapeutic interventions for HPV‐associated diseases.

mRNA‐HPV vaccine encoding E6 and E7 improves therapeutic potential for HPV‐mediated cancers via subcutaneous immunization

AbstractThe E6 and E7 proteins of specific subtypes of human papillomavirus (HPV), including HPV 16 and 18, are highly associated with cervical cancer as they modulate cell cycle regulation. The aim of this study was to investigate the potential antitumor effects of a messenger RNA‐HPV therapeutic vaccine (mHTV) containing nononcogenic E6 and E7 proteins. To achieve this, C57BL/6j mice were injected with the vaccine via both intramuscular and subcutaneous routes, and the resulting effects were evaluated. mHTV immunization markedly induced robust T cell‐mediated immune responses and significantly suppressed tumor growth in both subcutaneous and orthotopic tumor‐implanted mouse model, with a significant infiltration of immune cells into tumor tissues. Tumor retransplantation at day 62 postprimary vaccination completely halted progression in all mHTV‐treated mice. Furthermore, tumor expansion was significantly reduced upon TC‐1 transplantation 160 days after the last immunization. Immunization of rhesus monkeys with mHTV elicited promising immune responses. The immunogenicity of mHTV in nonhuman primates provides strong evidence for clinical application against HPV‐related cancers in humans. All data suggest that mHTV can be used as both a therapeutic and prophylactic vaccine.

Cytological features of human papillomavirus‐infected immature squamous metaplastic cells from cervical intraepithelial neoplasia grade 2

AbstractIn reflex cytology, the presence of prominent nucleoli in immature metaplastic squamous cells (IM) may be underdiagnosed due to variations in interpretation. The aim of this study is to identify human papillomaviruses (HPVs) that infect IM clusters in cervical intraepithelial neoplasia 2 (CIN2) on Papanicolaou (Pap) smears to determine the cytological features of lesion‐derived cells. Thirty‐two patients with a simultaneous diagnosis of CIN2 on biopsy and high‐grade squamous intraepithelial lesions (HSIL) on cytology as well as with IM clusters on HSIL smears were included. CIN2 tissues and HSIL and IM clusters on Pap smears were isolated by manual microdissection, and HPV types were identified by PCR‐based genotyping. The nuclear area within the IM clusters was also measured. The median nuclear area of HPV‐negative IM clusters was 48 μm2, with a coefficient of variation (CV) of 0.20; those of HPV‐positive clusters were 66 μm2 and 0.34, respectively. The cut‐off values of the nuclear area and CV for HPV positivity were 62 μm2 and 0.25, respectively. IM clusters composed of cells with a nuclear area of more than twice that of neutrophils or cells with a wide variation in nuclear sizes are likely to be neoplastic cells caused by HPV.

Human papillomavirus testing using HPV APTIMA® assay and an external cellularity control in self‐collected samples

AbstractIn cervical cancer screening programs, the detection of high‐risk human papillomavirus (HR‐HPV) is now widely implemented on physician‐collected samples and has expanded to include self‐collected samples. The use of a cellularity control (CC) is needed to reduce false‐negative HPV results. An external mRNA CC for the HPV APTIMA® assay was assessed for its analytical performance and the results were compared with both cervix cytobrush samples taken by physicians and self‐collected vaginal samples from 148 women. The performance of the CC was adjusted to control for the presence of cellular mRNA in the ThinPrep® and Multitest® transport media. This CC is user‐friendly but implies to perform two independent assays on PANTHER® automate. Self‐collected vaginal sampling gives a lower median CC results (13.2 vs. 16.9 min) but a higher risk of negative CC results (3.3 vs. 0%). The usefulness of the CC for the HR‐HPV assay may be optimized by the definition of a threshold for a minimum cell number to be tested to increase confidence in HPV‐negative results. The systematic use of an RNA CC increases confidence for HPV RNA assays on self‐collected vaginal samples.

Coinfections with additional oncoviruses in HPV+ individuals: Status, function and potential clinical implications

AbstractOncovirus infections account for an estimated 12%−20% of human cancers worldwide. High‐risk human papillomavirus (HPV) infection is the etiological agent of some malignancies such as cervical, oropharyngeal, anal, penile, vaginal, and vulvar cancers. However, HPV infection is not the only cause of these cancers or may not be sufficient to initiate cancer development. Actually, certain other risk factors including additional oncoviruses coinfections have been reported to increase the risk of patients exposed to HPV for developing different HPV‐related cancers. In the current review, we summarize recent findings about coinfections with different oncoviruses in HPV+ patients from both clinical and mechanistic studies. We believe such efforts may lead to an interesting direction for improving our understanding and developing new treatments for virus‐induced cancers.

Impact of cervicovaginal microbiome on the risk of cervical abnormalities development

AbstractThe vaginal microbiome has emerged as potentially influencing the natural history of Human Papillomavirus (HPV) infections and their clinical impact. We aimed to characterize the vaginal microbiome in samples from 807 high‐risk HPVs (Hr‐HPV) positive women with a mean age of 41.45 ± 10.79 years who participated in the Regional Cervical Cancer Screening Program from the Northern Region of Portugal. Microbiome analysis was performed with commercial kits for the detection of 21 microorganisms. The most frequent microorganisms were Ureaplasma parvum (52.5%), Gardnerella vaginalis (GV) (34.5%), Atopobium vaginae (AV) (32.6%), Lacto (30.7%), and Mycoplasma hominis (MH) (23.5%). The distribution according to age reveals that MH, Mega1, GV, BVab2, AV, and Mob were more prevalent in women older than 41 years of age (p < 0.050), while Lacto is significantly decreased in this group (23.5% vs. 39.4%, p < 0.001; RR = 0.47). The risk analysis showed that Hr‐HPV‐16/‐18 and Hr‐HPV‐9val genotypes are associated with an increased risk of developing cervical abnormalities, while Lacto (p < 0.001; odd ratio [OR] = 0.33), GV (p = 0.0111; OR = 0.41), AV (p = 0.033; OR = 0.53) and Mob (p = 0.022; OR = 0.29) are associated with protection. Similar results were found for the risk of development atypical squamous cells cannot exclude HSIL/high‐grade squamous intraepithelial lesion. Overall, the multivariate analysis confirmed that lactobacillus and bacteria associated with bacterial vaginosis (GV, AV, and Mob) are associated with protection against the development of cervical abnormalities. This study provides important data to be included in the future management of risk stratification for Hr‐HPV‐positive women.

The effect of being diagnosed with human papillomavirus infection on women's sexual lives

AbstractProblemObjective studies that use validated questionnaires are needed to evaluate the changes in the sexual functions of women diagnosed with human papillomavirus (HPV) infection.Method of StudyThe study comprised 80 sexually active women diagnosed with a high‐risk HPV infection. These patients were divided into four groups as follows: group 1, HPV 16/18‐positive and normal cytology; group 2, HPV 16/18‐positive and abnormal cytology; group 3, non‐16/18 HPV‐positive and abnormal cytology; and group 4, non‐16/18 HPV‐positive and normal cytology. The sexual functions and anxiety statuses of the patients were assessed via the Female Sexual Function Index (FSFI) and Beck anxiety inventory (BAI) questionnaires, respectively, at their first clinical visits and then 2 months later.ResultsThere was no statistically significant difference among the study groups in terms of the overall FSFI and domain sub‐scores at either of the visits. Women who tested positive for the high‐risk HPV 16/18 strains had a significantly less sexual desire after being informed about the test results. Those with HPV 16/18 and normal cytology had significantly higher anxiety levels at their second than first visits. The BAI scores of the HPV 16/18‐positive women (normal or abnormal cytology) at the second visit were significantly higher than those of non‐16/18 HPV‐positive women with normal cytology. There was no significant difference between the patients with normal and abnormal cytology results regarding the difference of BAI, overall and domain FSFI sub‐scores at the first and second visits. The desire and lubrication domain scores of the HPV 16/18‐positive patients significantly decreased after the first visit compared with those of the non‐16/18 HPV‐positive patients.ConclusionHPV 16/18 positivity decreases women's total FSFI and desire domain sub‐scores.

Consistency in human papillomavirus type detection between self‐collected vaginal specimens and physician‐sampled cervical specimens

AbstractWith the rising need for accessible cervical cancer screening, self‐sampling methods offer a promising alternative to traditional physician‐led sampling. This study aims to evaluate the efficacy of the HygeiaTouch Self Sampling Kit for Women in detecting human papillomavirus (HPV) types and predicting cervical lesions. We studied the concordance in identifying high‐risk HPV (hrHPV) types between samples collected by physicians and those self‐collected by women using a self‐sampling kit for validation. Women aged 21–65, fitting into specific categories based on their cervical health history were eligible. Cohen's kappa coefficient to gauge concordance between the two specimen types and relative accuracy metrics in identifying cervical intraepithelial neoplasia (CIN) were also calculated, with physician‐sampled specimens serving as a reference. A total of 1210 participants from three institutes were involved. The self‐sampling kit closely matched the physician‐led method in terms of collecting valid specimens (100% vs. 100%), identifying hrHPV types (kappa: 0.75, 95% confidence interval [95% CI]: 0.72–0.79; agreement: 87.7%, 95% CI: 85.8–89.6) and predicting CIN grade 2 or worse (CIN2+) (relative sensitivity: 0.949, relative accuracy: 0.959). Kappa values varied between 0.71 and 0.83 for different hrHPV types and combinations, with an overall value 0.75 (95% CI: 0.72–0.79) signifying robust compatibility between the two methods. Our study underscores the potential of the HygeiaTouch Self Sampling Kit as a reliable, efficient, and user‐friendly alternative to traditional sampling methods. This suggests that self‐sampling could be pivotal in expanding cervical cancer screening accessibility and enhancing detection rates.

Human papillomavirus prevalence and genotype distribution in Liaocheng men between 2016 and 2022

AbstractHuman papillomavirus (HPV) infection can lead to HPV‐related cancer in men, including the anus, penile, and oropharyngeal cancers and precancerous lesions. This study retrospectively investigated HPV prevalence and genotype distribution in Liaocheng men between 2016 and 2022. The total HPV positive rate was 64.87% (2388/3681, 95% confidence interval [CI]: 63.32%–66.40%), where high risk (HR)‐HPV and low risk (LR)‐HPV accounted for 42.49% (1564/3681, 95% CI: 40.90%–44.09%) and 69.71% (2566/3681, 95% CI: 68.20%–71.17%), respectively. The mixed HPV infection rate of two and more genotypes was 35.72%. The infection rate of HR‐HPV increased with the number of positive cases annually from 2016 (16.91%) to 2022 (46.59%). The most common HR‐HPV genotypes were HPV16 (11.60%), HPV52 (6.95%), and HPV59 (6.28%), whereas the least common HR‐HPV was HPV26. The most common LR‐HPV genotypes were HPV6 (56.99%), HPV11 (23.79%), and HPV43 (6.37%). The 9 v HPV vaccine preventable for LR‐HPV and HR‐HPV accounted for 80.78% and 30.40%, respectively, in this study. Most HPV‐positive patients aged 1–86 were in the 30–39 age group. This study confirmed that HPV prevalence in Liaocheng men was common and diverse. HPV16, HPV52, and HPV59 are widely distributed in Liaocheng men, and the male HR‐HPV infection rate remained high in this region. Regarding public health and cancer prevention, it is recommended and effective to include the HPV vaccination in the national vaccination program for men.

Methodological and analytical challenges in microbiome‐HPV association studies

Prevalence and genotype distribution of human papillomavirus in cervical adenocarcinoma (usual type and variants): A systematic review and meta‐analysis

AbstractCervical glandular neoplasms represent a heterogeneous group of tumors for which a comprehensive overview of the involvement of high‐risk human papillomaviruses (HPV) in pathogenesis is still lacking. We first searched MEDLINE (PubMed), Embase, and Scopus databases (until October 2022), and systematically reviewed available literature. We then quantitatively estimated both pooled and genotype‐specific prevalence of HPV DNA as well as the influence of various factors (e.g., geographical region, histological subtype, tissue/sample type) on computed effect size by means of random effects meta‐analysis. In total, 379 studies comprising 17 129 cases of cervical adenocarcinoma were identified. The pooled HPV prevalence was 78.4% (95% confidence interval [95% CI]: 76.2–80.3) with a significant between‐study heterogeneity (I2 = 79.4%, Q test p < 0.0001). Subgroup analyses indicated that the effect size differed substantially by geographical region (from 72.5% [95% CI: 68.7–76.1] in Asia to 86.8% [95% CI: 82.2–90.3] in Oceania) (p < 0.0001) and histological subtype of cancer (from 9.8% [95% CI: 5.5–17] in gastric‐type to 85% [95% CI: 79.6–89.2] in usual‐type cervical adenocarcinoma) (p < 0.0001). HPV16 and HPV18 were by far the most frequently detected viral strains with specific prevalence of 49.8% (95% CI: 46.9–52.6) and 45.3% (95% CI: 42.8–47.8), respectively. When stratified by continent or histologic variant, these genotype‐specific results varied in a relatively limited manner. Altogether, these findings support that all histological subtypes of cervical adenocarcinoma are etiologically linked to high‐risk HPV but to varying degrees. Therefore, a dual‐criteria classification taking into account accurately both morphological and virological aspects could be an interesting evolution of the current binary World Health Organization classification, better reflecting the pathogenic diversity of the disease.

HPV and HPV‐associated cancer

Global and regional estimates of cervical cancer burden associated with human immunodeficiency virus infection from 1990 to 2019

AbstractPrevious studies reported human immunodeficiency virus (HIV) could enhance human papillomavirus (HPV)‐induced cervical cancer. Therefore, the burden of cervical cancer associated with HIV across different regions and time periods need to be assessed. We aim to investigate the global burden of cervical cancer associated with HIV infection. Age standardized rates (ASRs) of cervical cancer disability‐adjusted life‐years (DALYs) in females (≥15 years old) were calculated by standardization, according the age‐specific DALYs numbers extracted from GBD data set 2019. Population attributable fractions was calculated by combining the published risk ratio, with the HIV prevalence (≥15 years old) from Joint United Nations Programme on HIV and AIDS (UNAIDS), and transferred to estimate the HIV‐associated cervical cancer burden. Expected annual percentage changes (EAPCs) was calculated to describe the temporal trend of ASR from 1990 to 2019. Pearson correlation analysis were conducted to assess the correlation between the ASR or EAPCs and the socio‐demographic index. The worldwide DALYs ASR caused by HIV‐associated cervical cancer rose from 3.78 (95% confidence interval [CI]: 2.19–5.56) in 1990 to 9.50 (95% CI: 5.66–13.79) in 2019 per 100k population. In 2019, the region with the greatest burden was Eastern and Southern Africa, with the highest DALYs of 273 900 (95% CI: 149 100–476 400) and ASR of 254.44 per 100k population (95% CI: 168.86–329.28). Notably, the Eastern Europe and Central Asia regions had the highest EAPC (14.07%) of HIV‐associated DALYs ASR. Women in Eastern and Southern Africa experience the greatest burden of HIV‐associated cervical cancer, while the Eastern Europe and Central Asia regions had witnessed the largest increase over the last 30 years. Prioritize the promotion of HPV vaccination and cervical cancer screening for women living with HIV were crucial in these regions.

Diverse intratumoral heterogeneity and immune microenvironment of two HPV‐related cervical cancer types revealed by single‐cell RNA sequencing

AbstractCervical squamous cell carcinoma (SCC) and adenocarcinoma (AD) are the main histological types of human papillomavirus‐related cervical cancer. However, there are few reports on cell type‐specific molecular differences between SCC and AD. Here, we used unbiased droplet‐based single‐cell RNA sequencing to elucidate the cellular differences between SCC and AD in tumor heterogeneity, and tumor microenvironment (TME). A total of 61 723 cells from three SCC and three AD patients, were collected and divided into nine cell types. Epithelial cells exhibited high intra‐ and interpatient heterogeneity and functional diversity. Signaling pathways, such as epithelial‐to‐mesenchymal‐transition (EMT), hypoxia and inflammatory response were upregulated in SCC, while cell cycle‐related signaling pathways were highly enriched in AD. SCC was associated with high infiltration of cytotoxicity CD8 T, effector memory CD8 T, proliferative natural killer (NK), and CD160+ NK cells as well as tumor‐associated macrophages (TAMs) with high major histocompatibility complex‐II genes. AD exhibited a high proportion of naive CD8 T, naive CD4 T, Treg CD4, central memory CD8, and TAMs with immunomodulatory functions. Additionally, we also observed that the majority of cancer‐associated fibroblasts (CAFs) were from AD, and participated in inflammation regulation, while SCC‐derived CAFs exhibited similar functions to tumor cells, such as EMT and hypoxia. This study revealed the widespread reprogramming of multiple cell populations in SCC and AD, dissected the cellular heterogeneity and characteristics in TME, and proposed potential therapeutic strategies for CC, such as targeted therapy and immunotherapy.

Multi‐omics data reveals novel impacts of human papillomavirus integration on the epigenomic and transcriptomic signatures of cervical tumorigenesis

AbstractIntegration of human papilloma virus (HPV) DNA into the human genome may progressively contribute to cervical carcinogenesis. To explore how HPV integration affects gene expression by altering DNA methylation during carcinogenesis, we analyzed a multiomics dataset for cervical cancer. We obtained multiomics data by HPV‐capture sequencing, RNA sequencing, and Whole Genome Bisulfite Sequencing from 50 patients with cervical cancer. We detected 985 and 485 HPV‐integration sites in matched tumor and adjacent paratumor tissues. Of these, LINC00486 (n = 19), LINC02425 (n = 11), LLPH (n = 11), PROS1 (n = 5), KLF5 (n = 4), LINC00392 (n = 3), MIR205HG (n = 3) and NRG1 (n = 3) were identified as high‐frequency HPV‐integrated genes, including five novel recurrent genes. Patients at clinical stage II had the highest number of HPV integrations. E6 and E7 genes of HPV16 but not HPV18 showed significantly fewer breakpoints than random distribution. HPV integrations occurring in exons were associated with altered gene expression in tumor tissues but not in paratumor tissues. A list of HPV‐integrated genes regulated at transcriptomic or epigenetic level was reported. We also carefully checked the candidate genes with regulation pattern correlated in both levels. HPV fragments integrated at MIR205HG mainly came from the L1 gene of HPV16. RNA expression of PROS1 was downregulated when HPV integrated in its upstream region. RNA expression of MIR205HG was elevated when HPV integrated into its enhancer. The promoter methylation levels of PROS1 and MIR205HG were all negatively correlated with their gene expressions. Further experimental validations proved that upregulation of MIR205HG could promote the proliferative and migrative abilities of cervical cancer cells. Our data provides a new atlas for epigenetic and transcriptomic regulations regarding HPV integrations in cervical cancer genome. We demonstrate that HPV integration may affect gene expression by altering methylation levels of MIR205HG and PROS1. Our study provides novel biological and clinical insights into HPV‐induced cervical cancer.

Distribution of cervical intraepithelial neoplasia is closely associated with HPV status and uterine position

AbstractAlthough cervical intraepithelial neoplasia (CIN) lesions are considered to be not randomly distributed across the cervix, but predominantly in the anterior wall, the clinicopathological etiology remains unknown. Herein, we aimed to elucidate the relationship between quantitatively measured area of CIN2/3 and cervical cancer associated factors by retrospective cohort study. We analyzed 235 consecutive therapeutic conization specimens dissected as a single intact section to determine CIN2/3 area and its correlation with both clinical risk factors including human papillomavirus (HPV) status (single or multiple infection) and uterine position defined by transvaginal ultrasound. Cervical wall was classified into three groups: anterior: (11, 12, 1, and 2 o'clock), posterior (5, 6, 7, and 8 o'clock) and lateral (3, 4, 9, and 10 o'clock). Multiple regression revealed that younger age and HPV16 status were significantly correlated with CIN2/3 area (p = 0.0224 and p = 0.0075, respectively). The Jonckheere‐Terpstra test showed a significant trend: CIN2/3 area was highest in the single HPV16 group, followed by the multiple HPV16 group and the non‐HPV16 group (p < 0.0001). CIN2/3 area in the anterior wall was statistically significantly larger than the posterior and lateral wall (p = 0.0059 and p = 0.0107, respectively). CIN2/3 area in the anterior wall was significantly greater with anteversion‐anteflexion than retroversion‐retroflexion (p = 0.0485), whereas CIN2/3 area in the posterior wall was significantly larger with retroversion‐retroflexion than anteversion‐anteflexion (p = 0.0394). In conclusion, the topographical distribution of CIN2/3 area is closely associated with patient age, high‐risk HPV status, especially single HPV16 infection and uterine position.

Impact of HPV molecular testing with partial genotyping as a feasibility study in cervical cancer community screening program in South India

AbstractCervical cancer can be eradicated by 2030 by the implementation of a global strategy involving the vaccination of young girls against human papillomavirus (HPV), screening 70% of women in 30–69 years of age and treating 90% of the women with precancerous lesions. For a country with a large population like India, all the three strategies can be a challenge. There is a need for implementation of a high throughput technology that can be scalable. Cobas 4800, a multiplexed assay based on quantitative polymerase chain reaction technology, identifies HPV 16 and HPV 18 along with the concurrent detection of 12 pooled other high‐risk HPV infections. This technology was used to test 10 375 women from the South Indian community for the first time as a feasibility program. Upon testing, high‐risk HPV was found in 595 (5.73%) women. A total of 127 women (1.2%) were found to be infected with HPV 16, 36 women (0.34%) with HPV 18 and 382 women (3.68%) with the 12 pooled high‐risk HPV and multiple mixed infections were found in 50 women (0.48%). It was observed that there was a high prevalence of high‐risk HPV in younger women, 30–40 years of age and a second peak was observed at 46–50 years of age. The second peak had higher mixed infections in the 46–50 years of age and this association was statistically significant. We found that 24/50 (48%) of the multiple mixed high‐risk HPV infections were in the age group 46–50 years. The current study is the first attempt from India, on a completely automated platform using Cobas 4800 HPV test in a community screening program. This study shows HPV 16 and HPV 18 infections, when differentiated, can be valuable for risk stratification in community screening program. Women in the perimenopausal age (46‐50yrs) showed a higher prevalence of multiple mixed infections, signifying a higher risk.

Infection pattern and immunological characteristics of Epstein‐Barr virus latent infection in cervical squamous cell carcinoma

AbstractPrevious studies reported the association between Epstein‐Barr virus (EBV) and cervical squamous cell carcinoma (CSCC), but its infection pattern and clinical significance unclear. This study aimed to comprehensively investigate the infection pattern, clinicopathology, outcomes, and immunology of this entity in central China. We evaluated a total of 104 untreated CSCC tumor tissue specimens using in situ hybridization for EBV‐encoded small RNAs (EBERs), and by employing flowcytometry fluorescence hybridization for human papillomavirus (HPV) genotyping. The expression of EBV latency proteins and immune biomarkers was evaluated and quantified by immunohistochemistry. EBERs transcripts were detected in 21 (20.2%) cases overall (in malignant epithelial cells of 13 cases and in lymphocytes of 8 cases). EBV belonged to latency type I infection in CSCC. The high‐risk (HR)‐HPV was detected in all of EBV‐positive CSCC, and the difference of detection rate of HR‐HPV was significant when compared with EBV‐negative CSCC (p = 0.001). The specific clinicopathology with increased frequency of advanced clinical stages, tumor‐positive lymph nodes, neural invasion, and increased infiltration depth (all p value < 0.05) were observed in cases with EBV. However, EBV infection was found to have no impact on prognosis of patients with CSCC. Increased densities of forkhead box P3 (FoxP3)+‐tumor infiltrating lymphocytes (TILs) (p = 0.005) and cytotoxic T‐lymphocyte‐associated protein 4 (CTLA‐4)+‐TILs (p = 0.017) and higher expression of programmed cell death‐1 (PD‐1) (p = 0.002) and programmed cell death‐1 ligand 1 (PD‐L1) (p = 0.040) were associated with EBV latent infection in CSCC, and these immunological changes were more likely to be associated with the infection in lymphocytes rather than tumor cells. Moreover, in patients with HPV‐positive CSCC, similar significant differences were still found. In conclusions, EBV‐positive CSCC may have specific infection pattern and clinicopathology and can exhibit an immunosuppressive microenvironment dominated by Treg cells aggregation and immune checkpoint activation.

Evidence of human cytomegalovirus infection and expression of 5‐lipoxygenase in borderline ovarian tumors

AbstractBorderline ovarian tumors (BOTs) belong to a group of tumors that are distinctly different from ovarian carcinomas. There is an increased risk of BOTs in patients with pelvic inflammatory disease. Human cytomegalovirus (HCMV) has been detected in ovarian cancer tissue specimens. This virus favors the inflammatory milieu by inducing expression of the potent inflammatory factor 5‐lipoxygenase (5LO), which stimulates cellular viability, cellular proliferation and activates antiapoptotic signaling pathways. Here, we aimed to examine presence of HCMV and 5LO in BOTs. Expression levels of HCMV proteins (IE and pp65) and 5LO were examined in paraffin embedded BOT tissue sections by immunohistochemistry staining and HCMV immunoglobulin M and immunoglobulin G (IgG) levels were determined by serology in blood samples obtained from 15 patients with BOTs identified in a prospective study at Karolinska University Hospital. Extensive expression of HCMV‐IE, pp65, and 5LO were detected in 87%, 40%, and 90% of examined BOT tissue sections, respectively. HCMV–IgG prevalence and antibody levels were significantly higher in patients with BOT compared to age matched healthy women (83.3% vs. 65,6%, respectively, p = .01). Whether HCMV can induce inflammation and affect the pathogenesis of BOTs should therefore be further investigated.

Lineage analysis of human papillomavirus types 31 and 45 in cervical samples of Iranian women

AbstractKnowing the regional lineages/sublineages of human papillomavirus 31 (HPV 31) and 45 would be of great importance for further evolutionary, epidemiological, and biological analysis. In this regard, to characterize more common lineages and sublineages of HPV 31 and 45, the sequence variations of E6 gene were investigated in normal, premalignant, and malignant samples collected from the cervix in Iran. In total, 54 HPV 31‐ and 24 HPV 45‐positive samples were analyzed by hemi‐nested polymerase chain reaction (PCR) and nested‐PCR, respectively. All PCR products were subjected to direct sequencing analysis. The results indicated that all three lineages A, B, and C were detected in HPV 31‐positive samples; among which HPV 31 lineage A was dominant as it was found in 66.7% of all samples. HPV 31 lineages B and C were identified in 5.5% and 27.8% of samples, respectively. In HPV 45‐infected samples, lineage B comprised of 62.5% of all samples and the remaining 37.5%  belonged to lineage A. In conclusion, our findings showed that lineage A of HPV 31 was predominant in Iran. Lineage B of HPV 45 was also dominant among Iranian women. However, further studies with larger sample size should be addressed to estimate the pathogenicity risk of HPV 31 or HPV 45 lineages/sublineages in the development of cervical cancer among Iranian women.

A retrospective study of cytology and HPV genotypes results of 3229 vaginal intraepithelial neoplasia patients

AbstractTo analyze the distribution of human papillomavirus (HPV) genotype, cytology, and the clinical characteristics of vaginal intraepithelial neoplasia (VaIN). All patients with histological‐proven VaIN at West China Second University Hospital, between January 1, 2014, and October 1, 2020, were retrospectively identified. The demographics, medical history, HPV genotype, viral load, and cytology results were retrieved. Standard statistical analyses were conducted. Of 3229 patients included, 42.3% were diagnosed with VaIN 1, 30.3% with VaIN 2% and 27.4% with VaIN 3. Patients with VaIN 3 were the oldest (p < 0.001). The leading HPV genotypes were HPV 16, 52, 58, 53, 56 and 81. The positive rate of HPV 16 was positively correlated with the grade of VaIN and infected most VaIN 3 patients (76.0%). The sensitivities of cytology for VaIN only, concomitant VaIN, and VaIN after hysterectomy were 75.6%, 78.8%, and 82.9%, respectively (p = 0.013), and the sensitivities of HPV were 91.1%, 93.5%, and 91.7%, respectively (p = 0.205). Cotesting improved the sensitivities, up to 96.9%, 97.1%, and 98.1%, respectively. VaIN can occur alone or be concomitant with cervical or vulvar intraepithelial neoplasia. Most of those with VaIN 2/3 are infected with HPV 16. The sensitivity of cytology and HPV testing is non‐inferior to that of cervical intraepithelial neoplasia 2+. Therefore, these testings might be helpful in the early detection of VaIN.

The expression analysis of human endogenous retrovirus‐K Env, Np9, and Rec transcripts in cervical cancer

AbstractWhile infection with high‐risk human papillomavirus (HPV) types is necessary for cervical cancer (CC) development, it is not enough, and other risk factors are required. Several studies have reported the activation of HERV‐K in different cancers; however, the investigation of HERV‐K expression levels in CC is scarce. In this study, it was hypothesized that activation of HERV‐K could play an essential role in CC development. In this order, the expression levels of HERV‐K Env, Np9, and Rec transcripts were investigated on 147 normal to CC uterine cervical tissues using quantitative real‐time PCR. The significantly higher levels of HERV‐K Env and Np9 transcripts were found in patients with cervical intraepithelial neoplasia (CIN) II−III and CC groups compared to those in the normal/CIN I group. Expression of Rec transcript was also higher only in the CC group than normal/CIN I group. Among CC patients, meaningfully higher levels of HERV‐K Env and Np9 transcripts were found in patients with squamous cell carcinoma rather than in adenocarcinoma. When only the HPV 16 positive samples were investigated, it was found that the mean difference in Env and Np9 mRNA levels was meaningfully higher among precancer lesions and the cancer group in comparison with the normal group. However, the Rec mRNA level showed no significant differences. The association between the expression of HERV‐K genes was investigated, and a significant positive correlation of Env expression with Np9 transcript was found only in the group with precancer lesions (R = 0.6, p = 0.0037). Moreover, a significant positive correlation was found between Rec and Np9 transcripts in patients with normal cervix tissues (R = 0.26, p = 0.033). However, no correlations were observed between the expression of Env and Rec in the three groups. In conclusion, our results showed that HERV‐K transcripts, especially Env and Np9, upregulated during cervical lesion progression. These findings highlight the potential use of HERV‐K Env and Np9 as biomarkers for CC diagnosis and prognosis. Further investigation is needed to determine the clinical utility of these markers and whether targeting HERV‐K oncogenes could be a viable therapeutic strategy for CC.

The Impact of Specific Sexually Transmitted Pathogens on Cervix: A Prospective Study Based on Cervical Cancer Screening Cohort

ABSTRACT Previous studies showed the association between sexually transmitted infections (STIs) and cervical lesions remains ambiguous. This study was conducted among 8371 women from a screening cohort. Seven specific sexually transmitted pathogens (STPs), including one viral [high‐risk human papillomavirus (hrHPV), low‐risk HPV (lrHPV)], five bacterial [Ureaplasma parvum (UP), Mycoplasma hominis (MH), Ureaplasma urealyticum (UU), Chlamydia trachomatis (CT), and Mycoplasma genitalium (MG)], and one parasitic [Trichomonas vaginalis (TV)] pathogen, were tested by Next Generation Sequencing assay using well‐stored baseline samples. Odds ratios (ORs) for incident cervical lesions with different STPs were calculated by Logistic Regression analysis. Within 3‐year follow‐up, 133 and 72 participants were diagnosed with histopathological cervical intraepithelial neoplasia grade 1 (CIN1) and CIN2+, respectively. The adjusted ORs (aORs) of atypical squamous cells of undetermined significance or worse (ASC‐US+) for women with hrHPV, lrHPV, UP, MH, TV, CT, and MG infections were 2.62 (95% CI: 2.19–3.13), 1.94 (95% CI: 1.55–2.43), 1.48 (95% CI: 1.26–1.74), 1.47 (95% CI: 1.25–1.73), 1.65 (95% CI: 1.27–2.15), 1.26 (95% CI: 0.79–2.01) and 2.33 (95% CI: 1.41–3.85), respectively. The aORs of cytological high‐grade squamous intraepithelial lesions (HSIL) for women with hrHPV, TV, and MG infections were 13.01 (95% CI: 5.78–29.31), 3.48 (95% CI: 1.38–8.75), and 5.87 (95% CI: 1.58–21.77). The aORs of CIN1 for hrHPV, lrHPV, and MH were 6.88(95% CI: 4.79–9.90), 2.04(95% CI: 1.29–3.14), and 1.47(95% CI: 1.02–2.11). The aOR of CIN2+ for women with hrHPV infection was 17.56 (95% CI: 10.31–29.92), no significance was observed for CIN2+ with non‐hrHPV STIs. Specific STP infections were significantly associated with subsequent cervical cytological ASC‐US+ (hrHPV, lrHPV, UP, MH, TV, and MG) and HSIL (hrHPV, TV, and MG). Infection with lrHPV and MH could increase the CIN1 risk in future though no obvious CIN2+ risk elevation was observed.

LAMP‐based electrochemical platform for monitoring HPV genome integration at the mRNA level associated with higher risk of cervical cancer progression

AbstractHuman papillomaviruses (HPVs) represent a diverse group of double‐stranded DNA viruses associated with various types of cancers, notably cervical cancer. High‐risk types of HPVs exhibit their oncogenic potential through the integration of their DNA into the host genome. This integration event contributes significantly to genomic instability and the progression of malignancy. However, traditional detection methods, such as immunohistochemistry or PCR‐based assays, face inherent challenges, and thus alternative tools are being developed to fasten and simplify the analysis. Our study introduces an innovative biosensing platform that combines loop‐mediated amplification with electrochemical (EC) analysis for the specific detection of HPV16 integration. By targeting key elements like the E7 mRNA, a central player in HPV integration, and the E2 viral gene transcript lost upon integration, we show clear distinction between episomal and integrated forms of HPV16. Our EC data confirmed higher E7 expression in HPV16‐positive cell lines having integrated forms of viral genome, while E2 expression was diminished in cells with fully integrated genomes. Moreover, we revealed distinct expression patterns in cervical tissue of patients, correlating well with digital droplet PCR, qRT‐PCR, or immunohistochemical staining. Our platform thus offers insights into HPV integration in clinical samples and facilitates further advancements in cervical cancer research and diagnostics.

Human Papillomavirus‐Associated Risk Factors and Clinical Characteristics of Vaginal Intraepithelial Neoplasia: A Retrospective Study

ABSTRACT Vaginal intraepithelial neoplasia (VaIN) is a rare premalignant lesion with the potential to progress to invasive vaginal carcinoma, yet its clinical characteristics and associated risk factors remain incompletely defined. This retrospective study evaluated the prevalence, human papillomavirus (HPV) genotype distribution, and clinical risk factors of VaIN in women undergoing colposcopy. A total of 8206 patients who received colposcopic examination and vaginal biopsy due to abnormal ThinPrep cytologic test results and/or high‐risk HPV positivity between January 2021 and December 2023 were reviewed. Patients with cervical cancer, vulvar intraepithelial lesions, or condyloma acuminatum were excluded. Clinical characteristics, HPV genotypes, cytology findings, and histopathological results were analyzed. VaIN was diagnosed in 682 patients (8.22%). The most prevalent HPV genotypes were HPV16, HPV52, HPV58, and HPV53. High‐grade VaIN (VaIN3) occurred more frequently in older and postmenopausal women. Concurrent VaIN and cervical intraepithelial neoplasia was significantly associated with advanced age, postmenopausal status, multiple HPV infections, and a history of cervical intraepithelial neoplasia. Among women with prior hysterectomy, VaIN was more commonly observed after hysterectomy for cervical lesions, although 24.7% of cases followed hysterectomy for non‐cervical indications. In non‐hysterectomized patients, 98.2% of VaIN lesions were located in the upper third of the vagina. Multivariate analysis identified older age, postmenopausal status, and multiple HPV infections as independent risk factors for VaIN. These findings underscore the importance of vigilant vaginal surveillance, particularly in women with persistent or multiple HPV infections and those with a history of hysterectomy.

The influence of IL1RN VNTR polymorphism on HPV infection among some tribal communities

AbstractPersistent infection of human Papillomavirus is the main etiological factor for cervical cancer. Austro‐Asiatic tribes are early settlers in India and they have unique genetic variations compared to other people. The immunological response is crucial for the prevention of viral associated diseases. Interleukin‐1 receptor antagonist (IL‐1RN) is considered being an important regulator of host immune surveillance. A total of 45 Santali tribal women and 10 Kora tribal women were enrolled in the present study and demographic variables were recorded during collection. Genomic DNA was extracted from cervical/vaginal swab samples. IL1RN variable number of tandem repeats (VNTR) polymorphisms and HPV types were determined by PCR‐based assay. Association between IL1RN VNTR polymorphisms with the HPV infections among the tribal communities was determined by logistic regression analysis. HPV18 prevalence was significantly higher among tribal women. We observed that the polymorphism A2*A2 (p = 0.022; odds ratio [OR] (95% confidence interval [CI]) = 0.16 (0.03–0.86)] were more resistant to oncogenic HPV infection. Use of oral contraceptives was associated with higher relative risk (p = 0.008; OR [95% CI] = 5.39 [1.47–19.8]) for oncogenic HPV18 positivity among the tribal women. The A2 allele homozygosity of IL1RN VNTR was identified to be associated with the protection from oncogenic HPV infection among various tribal communities of West Bengal and therefore may be a useful marker of host immune response among them.

HPV‐ CCDC106 integration promotes cervical cancer progression by facilitating the high expression of CCDC106 after HPV E6 splicing

Abstract Human papillomavirus (HPV) integration and high expression of HPV oncogenes ( E6 and E7 ) are important mechanisms for HPV carcinogenesis in cervical cancer. However, the relationship between HPV integration and HPV E6 spliced transcripts, as well as the underlying mechanisms of HPV integration in carcinogenesis after HPV E6 splicing remains unclear. We analyzed HPV‐coiled‐coil domain containing 106 ( CCDC106 ) integration samples to characterize the roles of HPV integration, E6 spliceosome I (E6*I), and high CCDC106 expression in cervical carcinogenesis. We found that E6 was alternatively spliced into the E6*I transcript in HPV‐ CCDC016 integration samples with low p53 expression, in contrast to the role of E6*I in preventing p53 degradation in cervical cancer cells. In addition, CCDC106 was highly expressed after HPV‐ CCDC106 integration, and interacted with p53, resulting in p53 degradation and cervical cancer cell progression in vitro and in vivo. Importantly, when E6*I was highly expressed in cervical cancer cells, overexpression of CCDC106 independently degraded p53 and promoted cervical cancer cell progression. In this study, we explored the underlying mechanisms of HPV‐ CCDC106 integration in HPV carcinogenesis after HPV E6 splicing, which should provide insight into host genome dysregulation in cervical carcinogenesis.

Usefulness of high‐risk HPV early oncoprotein (E6 and E7) serological markers in the detection of cervical cancer: A systematic review and meta‐analysis

Abstract We reviewed the literature on the importance of selected anti‐high‐risk human papillomavirus (HR‐HPV) antibodies (namely, 16/18 and early oncoproteins E6 and E7) as potential serological markers for early detection of individuals at high risk of cervical cancer. We searched for studies in PubMed and Embase databases published from 2010 to 2020 on antibodies against HR‐HPV E6 and E7 early proteins and cervical cancer. Pooled sensitivity and specificity for HPV16 and HPV18 antibodies were calculated using a bivariate hierarchical random‐effects model. A total of 69 articles were identified; we included three studies with 1550 participants. For the three HPV16/18 E6 and E7 antibody tests, enzyme‐linked immunosorbent assay‐based assays had a sensitivity of 18% for detecting CIN2+ (95% confidence interval [CI]: 15–21) and a specificity of 96% (95% CI: 92–98), for slot‐blot, sensitivity was 28.9% (95% CI: 23.3–35.1) and specificity was 72% (95% CI: 66.6–77.0) for detecting CIN2+, and for multiplex HPV serology assay based on a glutathione S ‐transferase, sensitivity was 16% (95% CI: 8.45–28.6) and specificity was 98% (95% CI: 97–99) for detecting invasive cervical cancer. HR‐HPV16/18 E6 and E7 serological markers showed high specificity, but sensitivity was suboptimal for the detection of cervical cancer in either population screening settings or as point‐of‐care screening tests.

Microbiota and HPV: The role of viral infection on vaginal microbiota

AbstractThe World Health Organization (WHO) estimates that the prevalence of human papillomaviruses (HPV) infection is between 9% and 13% of the world population and only in the United States, more than 6.2 million are positive every year. There are more than 100 types of HPV, among them, two serotypes (16 and 18) are related to 70% of cervical cancers and precancerous cervical lesions. The vaginal microbiota could play a considerable role in HPV infection and the genesis of cervical tumors caused by HPV. Moreover, bacteria are strongly associated with vaginal inflammation and oncogenic mutations in human cells. We aim to investigate whether HPV infection could influence the bacterial microbiota composition in the uterine cervix. A total of 31 women were enrolled in this study. The vaginal swabs were collected; the HPV‐DNA was extracted with QIAamp DNA Microbiome. The V3–V4–V6 region of the 16S rDNA gene was amplified by polymerase chain reaction (PCR) followed by sequencing with MiSeq Illumina. The main phylum identified in the vaginal microbiota were Firmicutes, Bacteroidetes, Proteobacteria, and Actinobacteria. The phylum of Actinobacteria, Proteobacteria, and Bacteroides was more represented in HPV‐positive patients. Lactobacilli represented the dominant genus, with a high percentage of Lactobacilli iners, Lactobacilli jensenii, and Lactobacilli crispatus as species. Gardnerella vaginalis, Enterococcus spp., Staphylococcus spp., Proteus spp., and Atopobium were the most represented in HPV‐positive patients. An altered vaginal microbiota might play a functional role in HPV cervical infection, progression, and clearance. The relationship between infection and microbiota could spur the development of new probiotics. However, further studies are needed to clarify the role of the vaginal microbiota in HPV infection.

AIB1 is a novel target of the high‐risk HPV E6 protein and a biomarker of cervical cancer progression

Abstract The high‐risk human papillomaviruses (HPV‐16, ‐18) are critical etiologic agents in human malignancy, most importantly in cervical cancer. These oncogenic viruses encode the E6 and E7 proteins that are uniformly retained and expressed in cervical cancers and required for maintenance of the tumorigenic phenotype. The E6 and E7 proteins were first identified as targeting the p53 and pRB tumor suppressor pathways, respectively, in host cells, thereby leading to disruption of cell cycle controls. In addition to p53 degradation, a number of other functions and critical targets for E6 have been described, including telomerase, Myc, PDZ‐containing proteins, Akt, Wnt, mTORC1, as well as others. In this study, we identified Amplified in Breast Cancer 1 (AIB1) as a new E6 target. We first found that E6 and hTERT altered similar profiling of gene expression in human foreskin keratinocytes (HFK), independent of telomerase activity. Importantly, AIB1 was a common transcriptional target of both E6 and hTERT. We then verified that high‐risk E6 but not low‐risk E6 expression led to increases in AIB1 transcript levels by real‐time RT‐PCR, suggesting that AIB1 upregulation may play an important role in cancer development. Western blots demonstrated that AIB1 expression increased in HPV‐16 E6 and E7 expressing (E6E7) immortalized foreskin and cervical keratinocytes, and in three of four common cervical cancer cell lines as well. Then, we evaluated the expression of AIB1 in human cervical lesions and invasive carcinoma using immunohistochemical staining. Strikingly, AIB1 showed positivity in the nucleus of cells in the immediate suprabasal epithelium, while nuclei of the basal epithelium were negative, as evident in the Cervical Intraepithelial Neoplasia 1 (CIN1) samples. As the pathological grading of cervical lesions increased from CIN1, CIN2, CIN3 carcinoma in situ and invasive carcinoma, AIB1 staining increased progressively, suggesting that AIB1 may serve as a novel histological biomarker for cervical cancer development. For cases of invasive cervical carcinoma, AIB1 staining was specific to cancerous lesions. Increased expression of AIB1 was also observed in transgenic mouse cervical neoplasia and cancer models induced by E6E7 and estrogen. Knockdown of AIB1 expression in E6E7 immortalized human cervical cells significantly abolished cell proliferation. Taken together, these data support AIB1 as a novel target of HPV E6 and a biomarker of cervical cancer progression.

Trends in cocirculation of oncogenic HPV genotypes in single and multiple infections among the unvaccinated community

AbstractCocirculation of multiple human papillomavirus (HPV) infections with low, probably high, and high‐risk genotypes are to be associated with various grades of infections and cancer progression. The oncogenic high‐risk HPVs are distributed and cocirculated throughout the world. This study was investigated to identify HPV genotypes related to genital disorders in unvaccinated women. The subjects were referred from clinics to a molecular lab for HPV testing in Iran as a low‐coverage vaccinated country. HPVs DNAs of cervical scrapping and genital tissue specimens of 1,133 un‐vaccinated women were genotyped using an in vitro diagnostic line probe (reverse hybridization) assay. In addition, phylogenetic trees were constructed on 100 MY09/MY11 polymerase chain reaction (PCR) amplicons of common genotypes of HPV L1 gene by Sanger sequencing. The mean age of the population study was 32.7 ± 8.0 and the mean age of HPV‐positive cases was 31.6 ± 7.8. HPV DNA was detected in 57.8% (655/1133) of women subjects and 42.2% (478/1133) of cases were undetected. Among 655 HPV‐positive cases, 639 subjects (56.4%) were related to defined genotypes and 16 subjects (1.4%) were untypeable. The highest prevalence rate of HPV genotypes was identified in the 25–34 years. The top 6 dominant HPVs in single and multiple genotypes were HPV6 (284/655 [43.4%]), HPV16 (111/655 [16.9%]), HPV31 (72/655 [11%]), HPV53 (67/655 [10.2%]), HPV11 (62/655 [9.5%]), and HPV52 (62/655 [9.5%]). Moreover, single, multiple and untypeable HPV genotypes were diagnosed as follows: 1 type (318/655 [48.5%]), 2 types (162/655 [24.8%]), 3 types (83/655 [12.7%]), 4 types (42/655 [6.5%]), more than 5 types (34/655 [5.3%]), and 1.4% un‐typeable subjects. The sequenced partial L1 gene of HPV genotypes (GenBank databases under the accession numbers: MH253467‐MH253566) confirmed and determined the cocirculated HPV genotypes' origins and addressed helpful insights into the future viral epidemiology investigations. Multiple HPV infections and cocirculation of various oncogenic HPV genotypes among the normal population (women and men) with asymptomatic forms are still challenging in unvaccinated communities. The preventive and organized surveillance programs for HPV screening are needed to be considered and compiled by health policy makers of low or unvaccinated countries.