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

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.

Revealing an association between HPV and systemic lupus erythematosus: A bidirectional two-sample Mendelian randomization study.

BACKGROUND: An increasing number of studies have focused on the association between Human papillomavirus (HPV) infection and systemic lupus erythematosus (SLE). However, current evidence is largely based on retrospective studies, which are susceptible to confounding factors and cannot establish causation. METHODS: A bidirectional two-sample Mendelian randomization (MR) design was used to evaluate the causal relationship between HPV and SLE. Mononucleoside polymers (SNPS) with strong evidence for genome-wide association studies (GWAS) were selected from the HPV exposure dataset and used as an instrumental variable (IV) for this study. For the MR Analysis results, the MR-Egger intercept P test, MR-Presso global test, CochranQ test and leave-one test were used for sensitivity analysis. RESULTS: Based on the evidence of MR Analysis, this study finally determined that there was no causal association between HPV16 and HPV18 and SLE. CONCLUSIONS: Possible regulation of HPV infection is not significantly associated with regulation of SLE. These findings provide new insights into the underlying mechanisms of HPV and SLE and need to be validated by further studies.

Manipulating host secreted protein gene expression: an indirect approach by HPV11/16 E6/E7 to suppress PBMC cytokine secretion.

Human papillomavirus (HPV) 11/16 E6/E7 proteins have been recognized to be pivotal in viral pathogenesis. This study sought to uncover the potential mechanisms of how HPV11/16 E6/E7-transfected keratinocytes inhibit cytokine secretion in peripheral blood mononuclear cells (PBMC). Upon co-culturing HPV11/16 E6/E7-transfected keratinocytes with PBMC in a non-contact manner, we observed a marked decrease in various cytokines secreted by PBMC. To determine if this suppression was mediated by specific common secreted factors, we conducted transcriptomic sequencing on these transfected cells. This analysis identified 53 common differentially secreted genes in all four HPV-transfected cells. Bioinformatics analysis demonstrated these genes were predominantly involved in immune regulation. Results from quantitative PCR (qPCR) and an extensive literature review suggested the downregulation of 12 genes (ACE2, BMP3, BPIFB1, CLU, CST6, CTF1, HMGB2, MMP12, PDGFA, RNASE7, SULF2, TGM2), and upregulation of 7 genes (CCL17, CCL22, FBLN1, PLAU, S100A7, S100A8, S100A9), may be crucial in modulating tumor immunity and combating pathogenic infections, with genes S100A8 and S100A9, and IL-17 signaling pathway being particularly noteworthy. Thus, HPV11/16 E6/E7 proteins may inhibit cytokine secretion of immune cells by altering the expression of host-secreted genes. Further exploration of these genes may yield new insights into the complex dynamics of HPV infection.

In Situ Colorimetric LAMP Based on One-Step Modified Filter Paper to Screen Human Papillomavirus (HPV)16/18 from Clinical Samples.

Cervical cancer is among the most common malignant tumors in women. The development of rapid screening techniques plays an important role in early screening for cancer treatment. We have developed an HPV screening method, which effectively combines the high-efficiency nucleic acid enrichment of chitosan-modified filter paper and the rapid visual detectability of colorimetric LAMP, along with the enhancement of the tolerance ability of the pH-sensitive LAMP reagent to acidic original samples, making the detection of HPV 16/18 easy to carry out and reliable, which is helpful for the epidemiological prevention and control strategies of HPV-induced cancer. This technique can simultaneously exhibit the "in situ amplification" capability of chitosan-modified filter paper and the nontemperature cycle dependence of visual LAMP detection. Therefore, DNA extraction and amplification can be performed efficiently and quickly within a single reaction where all DNA is concentrated in the QF paper disc. By embedding amino-modified filter paper into the plastic chip, a simple and reliable disposable chip was prepared for rapid HPV16 and HPV18 detection from clinical endometrial samples, and the results were 100% consistent with clinical diagnosis. More importantly, even after the sample was diluted 100-fold, HPV16/18-infected cells could be accurately identified, showing the advantages of the system in early cancer screening. Moreover, for endometrial samples containing plenty of cells, the filter paper could be used to enrich cells by filtration, preventing the acidic fluid from impacting pH-induced colorimetric LAMP detection and realizing direct amplification for HPV identification without nucleic acid extraction. This easy-to-operate system that can analyze a wide range of samples will be suitable for routine on-site HPV screening, dramatically extending the applications and utility for rapid, near-patient nucleic acid testing.

Detection of HPV DNA in Saliva of Patients with HPV-Associated Oropharyngeal Cancer Treated with Radiotherapy.

BACKGROUND: To investigate the technical feasibility of RT-PCR and direct sequencing to quantify HPV DNA in the saliva of patients with Human-Papilloma-Virus related oropharyngeal cancer (HPV-OPC), the level of which is known to predict prognosis after treatment. METHODS: Nine patients with locally advanced HPV-OPC treated with definitive radiotherapy with chemotherapy or cetuximab, or radiotherapy alone between April 2016 and September 2017, were enrolled, two of whom also received induction chemotherapy. Saliva was collected before (baseline), during (mid-RT) and after (post-RT) radiotherapy. HPV-16 DNAs (E6 and E7) in saliva were quantified by RT-PCR and sequencing, the latter using a custom cancer panel. Correlations between HPV DNA levels and clinical outcomes were assessed. RESULTS: Compared to the baseline, the relative cycle threshold (Ct) value of E6 and E7 reduced at the point of mid-RT in the majority of the patients (100% and 75% for E6 and E7, respectively). Similarly, the relative Ct value from the baseline to post-RT reduced in 86% and 100% of the patients for E6 and E7, respectively. During the follow-up period, three patients (33%) experienced disease progression. The relative baseline Ct values of these three patients were in the top 4 of all the patients. The sequences of HPV DNA were detected in five (83%) of six samples of the baseline saliva that underwent DNA sequencing, along with several gene mutations, such as TP53,CDKN2A and PIK3CA. CONCLUSIONS: This study demonstrates that, in addition to detection and quantification of HPV DNA by RT-PCR, detection by sequencing of HPV-DNA using a customized cancer panel is technically possible.

Identification of HPV16 Lineages in South African and Mozambican Women with Normal and Abnormal Cervical Cytology.

BACKGROUND: Human papillomavirus 16 (HPV16) is an oncogenic virus responsible for the majority of invasive cervical cancer cases worldwide. Due to genetic modifications, some variants are more oncogenic than others. We analysed the HPV16 phylogeny in HPV16-positive cervical Desoxyribonucleic Acid (DNA) samples collected from South African and Mozambican women to detect the circulating lineages. METHODS: Polymerase chain reaction (PCR) amplification of the long control region (LCR) and 300 nucleotides of the E6 region was performed using HPV16-specific primers on HPV16-positive cervical samples collected in women from South Africa and Mozambique. HPV16 sequences were obtained through Next Generation Sequencing (NGS) methods. Geneious prime and MEGA 11 software were used to align the sequences to 16 HPV16 reference sequences, gathering the A, B, C, and D lineages and generating the phylogenetic tree. Single nucleotide polymorphisms (SNPs) in the LCR and E6 regions were analysed and the phylogenetic tree was generated using Geneious Prime software. RESULTS: Fifty-eight sequences were analysed. Of these sequences, 79% (46/58) were from women who had abnormal cervical cytology. Fifteen SNPs in the LCR and eight in the E6 region were found to be the most common in all sequences. The phylogenetic analysis determined that 45% of the isolates belonged to the A1 sublineage (European variant), 34% belonged to the C1 sublineage (African 1 variant), 16% belonged to the B1 and B2 sublineage (African 2 variant), two isolates belonged to the D1-3 sublineages (Asian-American variant), and one to the North American variant. CONCLUSIONS: The African and European HPV16 variants were the most common circulating lineages in South African and Mozambican women. A high-grade squamous intraepithelial lesion (HSIL) was the most common cervical abnormality observed and linked to European and African lineages. These findings may contribute to understanding molecular HPV16 epidemiology in South Africa and Mozambique.

Biological and clinical relevance of correlated expression levels of coding and long noncoding RNAs in HPV16 positive cervical cancers.

Human papillomavirus (HPV) drives cervical cancer (CaCx) pathogenesis and viral oncoproteins jeopardize global gene expression in such cancers. In this study, our aim was to identify differentially expressed coding (DEcGs) and long noncoding RNA genes (DElncGs) specifically sense intronic and Natural Antisense Transcripts as they are located in the genic regions and may have a direct influence on the expression pattern of their neighbouring coding genes. We compared HPV16-positive CaCx patients (N = 44) with HPV-negative normal individuals (N = 34) by employing strand-specific RNA-seq and determined the relationships between DEcGs and DElncGs and their clinical implications. By performing Gene set enrichment and protein-protein interaction (PPI) analyses of DEcGs, we identified enrichment of processes crucial for abortive virus life cycle and cancer progression. The DEcGs formed 16 gene clusters which we identified through Molecular Complex Detection (MCODE) plugin of Cytoscape. All the gene clusters portrayed cancer-related functions. We recorded significantly correlated expression levels of 79 DElncGs with DEcGs at proximal genomic loci based on Pearson's Correlation coefficients. Of these gene pairs, 24 pairs portrayed significantly altered correlation coefficients among patients, compared to normal individuals. Of these, 6 DEcGs of 6 such gene pairs, belonged to 5 of the identified gene clusters, one of which was survival-associated. Out of the 24 correlated DEcG: DElncG pairs, we identified 3 pairs, where expression of both members was significantly associated with patient overall survival. The findings justify the cooperative roles of these gene pairs, in patient prognostication, thereby bearing immense potential for translation. Thus, elucidation of correlative strengths between paired DElncGs and DEcGs in patient and normal samples, could serve as a foundation for identification of therapeutic and prognostic targets of HPV16-positive CaCx.

Epidemiological distribution of high-risk human papillomavirus genotypes and associated factors among patients with esophageal carcinoma at Bugando medical center in Mwanza, Tanzania.

BACKGROUND: Esophageal carcinoma is a growing concern in regions that have a high incidence of human papillomavirus (HPV) infection such as East Africa. HPV, particularly the high-risk genotypes, is increasingly recognized as a risk factor for esophageal carcinoma. We set out to investigate the prevalence and associated factors of high-risk HPV in formalin-fixed paraffin-embedded (FFPE) tissue blocks with esophageal carcinoma at Bugando Medical Center, a tertiary referral hospital in Mwanza, Tanzania, East Africa. METHODS: A total of 118 esophageal carcinoma FFPE tissue blocks, collected from January 2021 to December 2022, were analyzed. Genomic DNA was extracted from these tissues, and multiplex polymerase chain reaction (PCR) was performed to detect HPV using degenerate primers for the L1 region and type-specific primers for detecting HPV16, HPV18, and other high-risk HPV genotypes. Data were collected using questionnaires and factors associated with high-risk HPV genotypes were analyzed using STATA version 15 software. RESULTS: Of the 118 patients' samples investigated, the mean age was 58.3 ± 13.4 years with a range of 29-88 years. The majority of the tissue blocks were from male patients 81/118 (68.7%), and most of them were from patients residing in Mwanza region 44/118 (37.3%). Esophageal Squamous Cell Carcinoma (ESCC) was the predominant histological type 107/118 (91.0%). Almost half of the tissue blocks 63/118 (53.3%) tested positive for high-risk HPV. Among these, HPV genotype 16 (HPV16) was the most common 41/63 (65.1%), followed by HPV genotype 18 (HPV18) 15/63 (23.8%), and the rest were other high-risk HPV genotypes detected by the degenerate primers 7/63 (11.1%). The factors associated with high-risk HPV genotypes were cigarette smoking (p-value < 0.001) and alcohol consumption (p-value < 0.001). CONCLUSION: A substantial number of esophageal carcinomas from Bugando Medical Center in Tanzania tested positive for HPV, with HPV genotype 16 being the most prevalent. This study also revealed a significant association between HPV status and cigarette smoking and alcohol consumption. These findings provide important insights into the role of high-risk HPV in esophageal carcinoma in this region.

High-risk human papillomavirus infection and cervical cytopathology: relationship with cervical nitric oxide levels.

BACKGROUND: Nitric oxide (NO) may contribute to the persistence of high-risk human papillomavirus (hrHPV) infection, which has been linked to the development of premalignant lesions and cervical cancer. Our study aimed to examine the relationship between cervical NO metabolite (NOx) levels, hrHPV infection, and cytopathological findings. Additionally, we assessed cervical NOx levels as a biomarker for predicting hrHPV infection and epithelial atypia. METHODS: The study involved 74 women who attended the Gynecology and Obstetrics outpatient clinics at Cairo University Hospitals between November 2021 and August 2022. Cervical samples were subjected to Pap testing, assessment of NOx levels by the Griess method, and detection of hrHPV DNA by real-time polymerase chain reaction. RESULTS: High-risk HPV was detected in 37.8% of women. EA was found in 17.1% of cases, with a higher percentage among hrHPV-positive than negative cases (35.7% vs. 4.3%, p = 0.001). The most prevalent hrHPV genotype was HPV 16 (89.3%). The cervical NOx level in hrHPV-positive cases was significantly higher (37.4 µmol/mL, IQR: 34.5-45.8) compared to negative cases (2.3 µmol/mL, IQR: 1.2-9.8) (p = < 0.001). Patients with high-grade atypia showed significantly higher NOx levels (38.0 µmol/mL, IQR: 24.6-94.7) in comparison to NILM and low-grade atypia cases (5.0 µmol/mL, IQR: 1.6-33.3 and 34.5 µmol/mL, IQR: 11.7-61.7, respectively) (p = 0.006). Although the NOx levels among hrHPV-positive cases with low-grade atypia (40.4 µmol/mL, IQR: 33.3‒61.8) were higher than those with NILM (36.2 µmol/mL, IQR: 35.7‒44.0) and high-grade atypia (38.0 µmol/mL, IQR: 24.6‒94.7), the difference was not significant (p = 0.771). ROC curve analysis indicated that the cervical NOx cut-off values of > 23.61 µmol/mL and > 11.35 µmol/mL exhibited good diagnostic accuracy for the prediction of hrHPV infection and EA, respectively. CONCLUSIONS: The high prevalence of hrHPV infection, particularly HPV 16, in our hospital warrants targeted treatment and comprehensive screening. Elevated cervical NOx levels are associated with hrHPV infection and high-grade atypia, suggesting their potential use as biomarkers for predicting the presence of hrHPV and abnormal cytological changes.

The Hippo pathway transcription factors YAP and TAZ play HPV-type dependent roles in cervical cancer.

Human papillomaviruses (HPVs) cause most cervical cancers and an increasing number of anogenital and oral carcinomas, with most cases caused by HPV16 or HPV18. HPV hijacks host signalling pathways to promote carcinogenesis. Understanding these interactions could permit identification of much-needed therapeutics for HPV-driven malignancies. The Hippo signalling pathway is important in HPV+ cancers, with the downstream effector YAP playing a pro-oncogenic role. In contrast, the significance of its paralogue TAZ remains largely uncharacterised in these cancers. We demonstrate that TAZ is dysregulated in a HPV-type dependent manner by a distinct mechanism to that of YAP and controls proliferation via alternative cellular targets. Analysis of cervical cancer cell lines and patient biopsies revealed that TAZ expression was only significantly increased in HPV18+ and HPV18-like cells and TAZ knockdown reduced proliferation, migration and invasion only in HPV18+ cells. RNA-sequencing of HPV18+ cervical cells revealed that YAP and TAZ have distinct targets, suggesting they promote carcinogenesis by different mechanisms. Thus, in HPV18+ cancers, YAP and TAZ play non-redundant roles. This analysis identified TOGARAM2 as a previously uncharacterised TAZ target and demonstrates its role as a key effector of TAZ-mediated proliferation, migration and invasion in HPV18+ cancers.

HPV DNA status and clinical history of patients are supplements for accurate reporting of the cytological Pap smear.

The present study was aimed at showing the importance of HPV DNA status and the clinical history of the patients required by the cytologist for accurate reporting. A total of 1250 symptomatic women who attended the gynaecology outpatient department of the Mahavir Cancer Sansthan and Nalanda Medical College, Patna, for pap smear examinations were screened and recruited for the study. Due to highly clinical symptoms out of the negative with inflammatory smears reported, one hundred and ten patients were randomly advised for biopsy and HPV 16/18 DNA analysis by a gynaecologist to correlate negative smears included in the study. Pap smear reports revealed that 1178 (94.24%) were negative for intraepithelial lesions (NILM) with inflammatory smears, 23 (1.84%) smears showed low-grade squamous intraepithelial lesions (LSIL), 12 (0.96%) smears showed high-grade squamous intraepithelial lesions, and 37 (2.96%) smears showed an atypical squamous cell of undetermined significance (ASC-US). A biopsy of 110 out of 1178 (NILM) patients revealed that 15 (13.63%) women had cervical cancer, 29 women had CIN I, 17 women had CIN II + CIN III, 35 women had benign cervical changes, and 14 women had haemorrhages. On the other hand, HPV 16/18 DNA was detected as positive in 87 out of 110. The high positivity of HPV in biopsied cases where frank cervical cancer and at-risk cancer were also observed in the negative smear-screened patients reveals that the HPV status and clinical history of the patients will be quite helpful to the cytologist for accurate reporting, and suggests that a negative HPV DNA result may be a stronger predictor of cervical cancer risk than a negative Pap test.

Analysis of human papillomavirus type 16 E4, E5 and L2 gene variations among women with cervical infection in Xinjiang, China.

BACKGROUND: There is a high incidence of cervical cancer in Xinjiang. Genetic variation in human papillomavirus may increase its ability to invade, spread, and escape host immune response. METHODS: HPV16 genome was sequenced for 90 positive samples of HPV16 infection. Sequences of the E4, E5 and L2 genes were analysed to reveal sequence variation of HPV16 in Xinjiang and the distribution of variation among the positive samples of HPV16 infection. RESULTS: Eighty-one of the 90 samples of HPV16 infection showed variation in HPV16 E4 gene with 18 nucleotide variation sites, of which 8 sites were synonymous variations and 11 missense variations. 90 samples of HPV16 infection showed variation in HPV16 E5 and L2 genes with 16 nucleotide variation sites (6 synonymous, 11 missense variations) in the E5 gene and 100 nucleotide variation sites in L2 gene (37 synonymous, 67 missense variations). The frequency of HPV16 L2 gene missense variations G3377A, G3599A, G3703A, and G3757A was higher in the case groups than in the control groups. CONCLUSIONS: Phylogenetic tree analysis showed that 87 samples were European strains, 3 cases were Asian strains, there were no other variations, and G4181A was related to Asian strains. HPV16 L2 gene missense variations G3377A, G3599A, G3703A, and G3757A were significantly more frequent in the case groups than in the control groups.

A Crosstalk Analysis of high-risk human papillomavirus, microbiota and vaginal metabolome in cervicovaginal microenvironment.

The microbial community has a profound effect on the host microenvironment by altering metabolites. Persistent high-risk human papillomavirus (HRHPV) infection has been implicated as contributors to the initiation and progression of cervical cancer, but the involved mechanisms are unknown. Assessing the metabolic profile of the cervicovaginal microenvironment has the potential to reveal the functional interactions among the host, metabolites and microbes in HRHPV persistence infection and progression to cancer. The vaginal swabs of women were collected and divided into three groups according to the HPV HybridenPture DNA test (HC2). The participants, include 9 who were categorized as HPV-negative, 8 as positive for HPV16, and 9 as positive for HPV18. 16S rRNA gene sequencing and metabolomics analyses were applied to determine the influence of the vaginal microbiota and host metabolism on the link between HPV and cervicovaginal microenvironment. These findings revealed that HRHPV groups have unique metabolic fingerprints that distinguish them from heathy controls. We showed that HRHPV affects changes in microbial metabolic function, which has important implications for the host. Our study further demonstrated metabolite-driven complex host-microbe interactions and assist in understanding the alterations in the HRHPV-induced cervicovaginal microenvironment.

The association between human papillomavirus and lung cancer: A Mendelian randomization study.

BACKGROUND: To investigate the causal relationship between human papillomavirus (HPV) and lung cancer, we conducted a study using the two-sample Mendelian randomization (TSMR). METHOD: Data from genome-wide association studies (GWAS) were analyzed with HPV E7 Type 16 and HPV E7 Type 18 as exposure factors. The outcome variables included lung cancer, small cell lung cancer, adenocarcinoma and squamous cell lung cancer. Causality was estimated using inverse variance weighted (IVW), MR-Egger and weighted median methods. Heterogeneity testing, sensitivity analysis, and multiple validity analysis were also performed.. RESULTS: The results showed that HPV E7 Type 16 infection was associated with a higher risk of squamous cell lung cancer (OR = 7.69; 95% CI:1.98-29.85; p = 0.0149). HPV E7 Type 18 infection significantly increased the risk of lung adenocarcinoma (OR = 0.71; 95% CI: 0.38-1.31; p = 0.0079) and lung cancer (OR = 7.69; 95% CI:1.98-29.85; p = 0.0292). No significant causal relationship was found between HPV E7 Type 16 and lung adenocarcinoma, lung cancer, or small cell lung carcinoma, and between HPV E7 Type 18 and squamous cell lung cancer or small cell lung carcinoma. CONCLUSIONS: This study has revealed a causal relationship between HPV and lung cancers. Our findings provide valuable insights for further mechanistic and clinical studies on HPV-mediated cancer.

Prevalence of HPV16 L1 protein in oral biopsies: A diagnostic study from Ecuador.

This study was designed to investigate the expression of HPV16 L1-protein in biopsies of oral mucosa samples. The expression of HPV16 L1 protein was investigated in biopsies taken from oral mucosa from patients who required pathological diagnosis of oral lesions. Seventy-two samples were incubated with anti-L1 protein monoclonal antibodies and protein detection was revealed with diaminobenzidine. Expression of L1 protein was performed by a pathologist blinded for tissue diagnosis under light microscopy. Most of the lesions of oral mucosa were present in lining mucosa (75 %) and the most frequent lesion were mucocele (n = 17, 23.6 %), epithelial hyperplasia (n = 6, 8.33 %), fibroma (n = 5, 6.9 %) and inflammatory hyperplasia (n = 5, 6.9 %). L1 protein expression was observed only in five (6.9 %) samples (two squamous cell carcinomas, two epithelial hyperplasia, and one gingival hyperplasia). We concluded that L1 expression in oral biopsies presented a low frequency in oral mucosal biopsies samples.

Quantification of human papillomavirus cell-free DNA from low-volume blood plasma samples by digital PCR.

The incidence rate of human papillomavirus-driven oropharyngeal cancer (HPV-OPC) is increasing in countries with high human development index. HPV cell-free DNA (cfDNA) isolated from 3 to 4 mL blood plasma has been successfully used for therapy surveillance. A highly discussed application of HPV-cfDNA is early detection of HPV-OPC. This requires sensitive and specific cfDNA detection as cfDNA levels can be very low. To study the predictive power of pre-diagnostic HPV-cfDNA, archived samples from epidemiological cohorts with limited plasma volume are an important source. To establish a cfDNA detection workflow for low plasma volumes, we compared cfDNA purification methods [MagNA Pure 96 (MP96) and QIAamp ccfDNA/RNA] and digital PCR systems (Biorad QX200 and QIAGEN QIAcuity One). Final assay validation included 65 low-volume plasma samples from oropharyngeal cancer (OPC) patients with defined HPV status stored for 2-9 years. MP96 yielded a 28% higher cfDNA isolation efficiency in comparison to QIAamp. Both digital PCR systems showed comparable analytical sensitivity (6-17 copies for HPV16 and HPV33), but QIAcuity detected both types in the same assay. In the validation set, the assay had 80% sensitivity (n = 28/35) for HPV16 and HPV33 and a specificity of 97% (n = 29/30). In samples with ≥750 µL plasma, the sensitivity was 85% (n = 17/20), while in samples with <750 µL plasma, it was 73% (n = 11/15). Despite the expected drop in sensitivity with decreased plasma volume, the assay is sensitive and highly specific even in low-volume samples and thus suited for studies exploring HPV-cfDNA as an early HPV-OPC detection marker in low-volume archival material.IMPORTANCEHPV-OPC has a favorable prognosis compared to HPV-negative OPC. However, the majority of tumors is diagnosed after regional spread, thus making intensive treatment necessary. This can cause lasting morbidity with a large impact on quality of life. One potential method to decrease treatment-related morbidity is early detection of the cancer. HPV cfDNA has been successfully used for therapy surveillance and has also been detected in pre-diagnostic samples of HPV-OPC patients. These pre-diagnostic samples are only commonly available from biobanks, which usually only have small volumes of blood plasma available. Hence, we have developed a workflow optimized for small-volume archival samples. With this method, a high sensitivity can be achieved despite sample limitations, making it suitable to conduct further large-scale biobank studies of HPV-cfDNA as a prognostic biomarker for HPV-OPC.

Differential requirement of the transcription factor HOXC13 for the stable maintenance of human papillomavirus genome among high-risk genotypes.

The viral genome of the high-risk human papillomavirus (HPV), the causative agent of cervical cancer, is stably maintained as extrachromosomal episomes that establish persistent infection. We previously identified homeobox-transcription factor HOXC13 as an important host protein mediating the short-term retention of the HPV16 and HPV18 genomes in normal human immortalized keratinocytes (NIKS). Here, we used CRISPR-Cas9 technology to construct HOXC13 knockout (KO) NIKS cells to determine whether HOXC13 is required for the long-term maintenance of high-risk HPV genomes. HPV16, HPV18, HPV52, and HPV58 whole genomes were transfected into HOXC13 KO cells, and the copy number of viral genomes per cell was monitored over cell passages. Copy numbers of HPV16, HPV52, and HPV58 genomes decreased continuously in HOXC13 KO cells, whereas HPV18 genomes remained stable throughout passages. Thus, HOXC13 is critical for the stable maintenance of the viral genomes of HPV16, HPV52, and HPV58, but not HPV18.

Assessment of malignant potential for HPV types 16, 52, and 58 in the uterine cervix within a Korean cohort.

High-risk human papillomavirus (HR-HPV) is the primary carcinogen in uterine cervical carcinoma. While genotype-specific carcinogenic risks have been extensively studied in Western populations, data from Korean are sparse. This study evaluates the malignant potential of the three most prevalent HR-HPVs in Korea: HPV16, HPV52, and HPV58. We analyzed 230 patients who underwent cervical conization and had been tested for HPV within a year prior to the procedure, excluding those with multiple infections. This analysis was confined to patients with single HPV infections and assessed outcomes of CIN3+, which includes carcinoma in situ (CIN3) and invasive carcinoma. The incidence of invasive cervical cancer was 6.7% for HPV16, 1.7% for HPV52, and 2.0% for HPV58; however, these differences were not statistically significant (p = 0.187). The rate of CIN3+ for HPV16, HPV52, and HPV58 were 70.6%, 51.7%, and 58.8%, respectively. Despite the small sample size, which may limit the robustness of statistical analysis, the data suggest a higher observed risk with HPV16. These findings highlight the need for vigilant clinical management tailored to specific HPV genotypes and support the implementation of a nine-valent vaccine in Korea. Physicians should be aware of these genotype-specific risks when treating patients.

Dual lateral flow assay based on PdRu nanocages for human Papillomavirus detection.

Cervical cancer is one of the most common gynecological malignancies, with the vast majority of which being caused by persistent infection with Human Papillomavirus (HPV) 16 and 18. The current available HPV detection methods are sensitive and genotyped but are restricted by expensive instruments and skilled personnel. The development of an easy-to-use, rapid, and cost-friendly analysis method for HPV is of great need. Herein, hollow palladium-ruthenium nanocages modified with two oligonucleotides (PdRu capture probes) were constructed for genotyping and simultaneous detection of target nucleic acids HPV16 and HPV18 by dual lateral flow assay (DLFA). PdRu capture probes were endowed with bi-functions for the first time, which could be used to output signals and hybridize target nucleic acids. Under optimized conditions, the PdRu based-DLFA with detection limits of 0.93 nM and 0.19 nM, respectively, exhibited convenient operation, and high sensitivity. Meanwhile, the DLFA achieved excellent rapid detection within 20 min, which was attributed to capture probes that can be directly bound to amplification-free target nucleic acids. Therefore, the development of PdRu-based DLFA can be utilized for rapid, sensitive, and simultaneous genotyping detection of HPV16 and HPV18, showing great application for nucleic acid detection.

Highly sensitive and specific detection of human papillomavirus type 16 using CRISPR/Cas12a assay coupled with an enhanced single nanoparticle dark-field microscopy imaging technique.

Human papillomavirus (HPV) is a prevalent sexually transmitted pathogen associated with cervical cancer. Detecting high-risk HPV (hr-HPV) infections is crucial for cervical cancer prevention, particularly in resource-limited settings. Here, we present a highly sensitive and specific sensor for HPV-16 detection based on CRISPR/Cas12a coupled with enhanced single nanoparticle dark-field microscopy (DFM) imaging techniques. Ag-Au satellites were assembled through the hybridization of AgNPs-based spherical nucleic acid (Ag-SNA) and AuNPs-based spherical nucleic acid (Au-SNA), and their disassembly upon target-mediated cleavage by the Cas12a protein was monitored using DFM for HPV-16 quantification. To enhance the cleavage efficiency and detection sensitivity, the composition of the ssDNA sequences on Ag-SNA and Au-SNA was optimized. Additionally, we explored using the SynSed technique (synergistic sedimentation of Brownian motion suppression and dehydration transfer) as an alternative particle transfer method in DFM imaging to traditional electrostatic deposition. This addresses the issue of inconsistent deposition efficiency of Ag-Au satellites and their disassembly due to their size and charge differences. The sensor achieved a remarkable limit of detection (LOD) of 10 fM, lowered by 9-fold compared to traditional electrostatic deposition methods. Clinical testing in DNA extractions from 10 human cervical swabs demonstrated significant response differences between the positive and negative samples. Our sensor offers a promising solution for sensitive and specific HPV-16 detection, with implications for cancer screening and management.