Comparison of high-risk HPV detection by the AmpFire® HPV Screening 16/18/HR technique (Atila Biosystems) and the hybrid capture 2 test (Qiagen).

BACKGROUND: Detection of high-risk human papillomaviruses (hrHPV) is widely used at the first line of cervical cancer screening, requiring rigorous validation of the clinical performance of commercial kits designed for this indication. METHODS: Performance of the AmpFire HPV Screening 16/18/HR test (AF, Atila Biosystems) and the Hybrid Capture 2 test (HC2, Qiagen) for detecting hrHPV was cross-compared in 200 cervical samples in our institution. RESULTS: The global percentage of agreement between the 2 techniques was 95.0% (95%CI 92-98%) with a Cohen's kappa coefficient of 0.85 (95%CI 0.75-0.94). Ten samples showed discordant results between the 2 techniques in both directions (5 HC2+/AF- and 5 HC2-/AF+). Among possible explanations for these discrepancies was the detection of HPV66 and HPV53 genotypes in two samples, since these genotypes are targeted by the Ampfire test but not by the HC2 test, as well as intrinsic differences in analytical performance to target specific genotypes. CONCLUSIONS: A high level of agreement was observed between the two techniques, which encourages further testing in order to definitively validate the use of the Ampfire kit for primary cervical cancer screening.

Betapapillomavirus natural history and co-detection with alphapapillomavirus in cervical samples of adult women.

Human papillomaviruses (HPV) of the genus Betapapillomavirus can infect both cutaneous and mucosal sites, but research on their natural history at mucosal sites remains scarce. We examined the risk factors and co-detection patterns of HPVs of the Betapapillomavirus and Alphapapillomavirus genera in cervical samples of the Ludwig-McGill cohort study. We assessed a subset of 505 women from the Ludwig-McGill cohort study from São Paulo, Brazil. Cervical samples over the first year of follow-up were tested for DNA of over 40 alphapapillomavirus types and 43 betapapillomavirus types using a type-specific multiplex genotyping polymerase chain reaction assay. We assessed the risk factors for prevalent and incident betapapillomavirus type detection, and whether types were detected more frequently together than expected assuming independence using permutation tests, logistic regression, and Cox regression. We observed significant within-genus clustering but not cross-genus clustering. Multiple betapapillomavirus types were co-detected in the same sample 2.24 (95% confidence interval [CI]: 1.65-3.29) times more frequently than expected. Conversely, co-detections of alphapapillomavirus and betapapillomavirus types in the same sample occurred only 0.64 (95% CI: 0.51-0.83) times as often as expected under independence. In prospective analyses, positivity to one HPV genus was associated with a nonsignificant lower incidence of detection of types in the other genus. Lifetime number of sex partners and new sex partner acquisition were associated with lower risks of prevalent and incident betapapillomavirus detection. Betapapillomaviruses are commonly found in the cervicovaginal tract. Results suggest potentially different mechanisms of transmission for betapapillomavirus genital infections other than vaginal sex.

Characterization of HPV subtypes in invasive cervical cancer in Botswana patients using a pan-pathogen microarray technology.

Human papillomavirus (HPV) plays a significant role in the development of cervical cancers in the setting of co-infection with HIV. Botswana has a high prevalence of HIV and cervical cancer. In this study, we investigated the distribution of HPV subtypes in cervical cancer biopsy samples from patients in Botswana using a highly sensitive pan-pathogen microarray technology, PathoChip, to detect both high- (HR-HPV) and low-risk HPV (LR-HPV) subtypes in women living with HIV (WLWH) and women living without HIV. We analyzed samples from 168 patients, of which 73% (n = 123) were WLWH with a median CD4 count of 479.5 cells/µL. Five HR-HPV subtypes were detected in the cohort: HPV 16, 18, 26, 34, and 53. The most prevalent subtypes were HPV 26 (96%) and HPV 34 (92%); 86% of WLWH (n = 106) had co-infection with four or more HR-HPV subtypes compared to 67% (n = 30) of women without HIV (p < 0.01). We detected 66 LR-HPV subtypes among all cervical cancer patients, with HPV 6b and 48 being most prevalent. Notably, signatures for LR-HPV subtypes 10, 41, 90, and 129 were only detected in WLWH. Signal intensity for HPV 18 was significantly weaker in WLWH with CD4 levels ≤200 cells/µL as compared to patients with >200 cells/µL and HIV-negative patients. Although the majority of cervical cancer specimens in this cohort were determined to have multiple HPV infections, the most prevalent HR-HPV subtypes (HPV 26 and HPV34) found in these cervical cancer samples are not covered in the current HPV vaccines. Though no conclusions can be made on the direct carcinogenicity of these subtypes the results do underlie the need for continued screening for prevention of cervical cancer.

Human papillomavirus genotyping using next generation sequencing (NGS) in cervical lesions: Genotypes by histologic grade and their relative proportion in multiple infections.

Sensitive and specific genotyping of human papillomaviruses (HPVs) is critical for the surveillance and monitoring of the vaccine effectiveness. Here, HPV genotypes were identified in 137 cervical samples with different histology (79 ≤CIN1 and 58 CIN3+) using Nested-PCR followed by Next-Generation sequencing (NGS) and relative proportions for each genotype in multiple infections were computed. All samples had been previously genotyped by PCR-Reverse Blotting Hybridization (PCR-RBH) thus allowing for a concordance analysis between both techniques. Multiple infections were present in 85% of ≤CIN1 cases compared to only 41% in CIN3+ cases (p<0.001). Among ≤CIN1 cases a towering genotypic diversity was observed, considering both low (LR-) and high risk (HR-) HPV genotypes; while among CIN3+, diversity was lower, HR-HPVs prevailing in most cases, especially HPV16. Furthermore, the predominance of HR-HPV genotypes in the proportions identified in each sample was higher in CIN3+ cases [(HPV16 (62.5%), followed by HPV31 and HPV58 (8.3% each)], than in ≤CIN1 cases [(HPV16 (17.7%), followed by HPV52 (14.7%) and HPV31 (10.3%)]. Agreement between PCR-RBH and NGS was higher than 90% for all genotypes (with an overall Kappa of 0.7), even though NGS identified eighty-nine positive results for HPV genotypes that had not been detected by PCR-RBH, evidencing its greater sensitivity. These results suggest that a reduction in genotypic diversity and/or an increase in the relative proportion of HR-HPVs in multiple infections can be considered as a biomarker for the potential risk of malignant progression.

Fluid Biomarkers in HPV and Non-HPV Related Oropharyngeal Carcinomas: From Diagnosis and Monitoring to Prognostication-A Systematic Review.

Biomarkers are crucial in oncology, from detection and monitoring to guiding management and predicting treatment outcomes. Histological assessment of tissue biopsies is currently the gold standard for oropharyngeal cancers, but is technically demanding, invasive, and expensive. This systematic review aims to review current markers that are detectable in biofluids, which offer promising non-invasive alternatives in oropharyngeal carcinomas (OPCs). A total of 174 clinical trials from the PubMed search engine in the last 5 years were identified and screened by 4 independent reviewers. From these, 38 eligible clinical trials were found and subsequently reviewed. The biomarkers involved, categorized by human papillomavirus (HPV)-status, were further divided according to molecular and cellular levels. Recent trials investigating biomarkers for both HPV-positive and HPV-negative OPCs have approaches from various levels and different biofluids including plasma, oropharyngeal swabs, and oral rinse. Promising candidates have been found to aid in detection, staging, and predicting prognosis, in addition to well-established factors including HPV-status, drinking and smoking status. These studies also emphasize the possibility of enhancing prediction results and increasing statistical significance by multivariate analyses. Liquid biopsies offer promising assistance in enhancing personalized medicine for cancer treatment, from lowering barriers towards early screening, to facilitating de-escalation of treatment. However, further research is needed, and the combination of liquid biopsies with pre-existing methods, including in vivo imaging and invasive techniques such as neck dissections, could also be explored in future trials.

Mechanistic Contributions of lncRNAs to Cellular Signaling Pathways Crucial to the Lifecycle of Human Papillomaviruses.

Papillomaviruses are ubiquitous epitheliotropic viruses with double-stranded circular DNA genomes of approximately 8000 base pairs. The viral life cycle is somewhat unusual in that these viruses can establish persistent infections in the mitotically active basal epithelial cells that they initially infect. High-level viral genome replication ("genome amplification"), the expression of capsid proteins, and the formation of infectious progeny are restricted to terminally differentiated cells where genomes are synthesized at replication factories at sites of double-strand DNA breaks. To establish persistent infections, papillomaviruses need to retain the basal cell identity of the initially infected cells and restrain and delay their epithelial differentiation program. To enable high-level viral genome replication, papillomaviruses also need to hold the inherently growth-arrested terminally differentiated cells in a replication-competent state. To provide ample sites for viral genome synthesis, they target the DNA damage and repair machinery. Studies focusing on delineating cellular factors that are targeted by papillomaviruses may aid the development of antivirals. Whilst most of the current research efforts focus on protein targets, the majority of the human transcriptome consists of noncoding RNAs. This review focuses on one specific class of noncoding RNAs, long noncoding RNAs (lncRNAs), and summarizes work on lncRNAs that may regulate the cellular processes that are subverted by papillomavirus to enable persistent infections and progeny synthesis.

Molecular Mechanisms of MmuPV1 E6 and E7 and Implications for Human Disease.

Human papillomaviruses (HPVs) cause a substantial amount of human disease from benign disease such as warts to malignant cancers including cervical carcinoma, head and neck cancer, and non-melanoma skin cancer. Our ability to model HPV-induced malignant disease has been impeded by species specific barriers and pre-clinical animal models have been challenging to develop. The recent discovery of a murine papillomavirus, MmuPV1, that infects laboratory mice and causes the same range of malignancies caused by HPVs provides the papillomavirus field the opportunity to test mechanistic hypotheses in a genetically manipulatable laboratory animal species in the context of natural infections. The E6 and E7 proteins encoded by high-risk HPVs, which are the HPV genotypes associated with human cancers, are multifunctional proteins that contribute to HPV-induced cancers in multiple ways. In this review, we describe the known activities of the MmuPV1-encoded E6 and E7 proteins and how those activities relate to the activities of HPV E6 and E7 oncoproteins encoded by mucosal and cutaneous high-risk HPV genotypes.

HPV infection and 5mC/5hmC epigenetic markers in penile squamous cell carcinoma: new insights into prognostics.

BACKGROUND: Penile cancer is one of the most aggressive male tumors. Although it is preventable, the main etiologic causes are lifestyle behaviors and viral infection, such as human papillomavirus (HPV). Long-term epigenetic changes due to environmental factors change cell fate and promote carcinogenesis, being an important marker of prognosis. We evaluated epidemiological aspects of penile squamous cell carcinoma (SCC) and the prevalence of HPV infection using high-risk HPV (hrHPV) and p16INK4A expression of 224 participants. Global DNA methylation was evaluated through 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC). RESULTS: The incidence of HPV was 53.2% for hrHPV and 22.32% for p16INK4a. hrHPV was not related to systemic or lymph node metastasis and locoregional recurrence, nor influenced the survival rate. P16INK4a seems to be a protective factor for death, which does not affect metastasis or tumor recurrence. Lymph node and systemic metastases and locoregional recurrence increase the risk of death. An increased 5mC mark was observed in penile SCC regardless of HPV infection. However, there is a reduction of the 5hmC mark for p16INK4a + (P = 0.024). Increased 5mC/5hmC ratio (> 1) was observed in 94.2% of penile SCC, irrespective of HPV infection. Despite the increase in 5mC, it seems not to affect the survival rate (HR = 1.06; 95% CI 0.33-3.38). CONCLUSIONS: P16INK4a seems to be a good prognosis marker for penile SCC and the increase in 5mC, an epigenetic mark of genomic stability, may support tumor progression leading to poor prognosis.

Human papillomavirus-related neoplasia of the ocular adnexa.

Human papillomaviruses (HPV) are involved in approximately 5% of solid cancers worldwide. The mucosotropic genotypes infect the stratified epithelium of various locations, where persistent infection may lead to invasive carcinomas. While the causative role of HPV in certain anogenital and head and neck carcinomas is well established, the role of HPV in carcinomas arising in the mucosal membranes of the ocular adnexal tissue (the lacrimal drainage system and the conjunctiva) has been a topic of great uncertainty. Therefore, we conducted a series of studies to assess the correlation between HPV and carcinomas arising in the mucosa of the ocular adnexal tissue and characterize the clinical, histopathological, and genomic features of the tumors in the context of HPV status in a Danish nationwide cohort. We collected clinical and histopathological data and tumor specimens from patients with carcinomas of the conjunctiva and the lacrimal drainage system, and their potential precursors, identified in Danish nationwide registries. The HPV status of the tumors was determined by the combined use of HPV DNA polymerase chain reaction (PCR), HPV E6/E7 mRNA in-situ hybridization, and p16 immunohistochemistry. The genomic profile was investigated by high-throughput DNA sequencing targeting 523 cancer-relevant genes. The literature to date on carcinomas of the lacrimal drainage system and the conjunctiva was summarized. In the Danish cohort, 67% of all carcinomas of the lacrimal drainage system and 21% of all conjunctival carcinomas were HPV-positive. HPV16 was the most frequently implicated genotype. A full-thickness expression of the viral oncogenes E6 and E7 was evident in almost all HPV DNA-positive cases. The HPV-positive carcinomas of the conjunctiva and the lacrimal drainage system shared histopathological and genomic features distinct from their HPV-negative counterparts. The HPV-positive carcinomas were characterized by a non-keratinizing morphology, p16 overexpression, high transcriptional activity of HPV E6/E7, and frequent pathogenic variants in the PI3K-AKT signaling cascade. In contrast, the HPV-negative carcinomas were characterized by a keratinizing morphology, lack of p16 and E6/E7 expression, and frequent somatic pathogenic variants in TP53, CDKN2A, and RB1. Among the patients with conjunctival tumors, HPV positivity was associated with a younger age at diagnosis and a higher risk of recurrence. In conclusion, the results support an etiological role of HPV in a subset of conjunctival and LDS carcinomas and their precursor lesions. Our investigations have shown that the HPV-positive carcinomas of the ocular adnexa share genomic and phenotypic characteristics with HPV-positive carcinomas of other anatomical locations. Therefore, these patients may be eligible for inclusion in future basket trials and future treatment regimens tailored to the more frequently occurring HPV-positive carcinomas of other locations. Future research will further elucidate the diagnostic, prognostic, and predictive role of HPV in these carcinomas.

Human papillomavirus (HPV) forårsager ca. 5% af alle non-haematologiske cancertilfaelde på verdensplan. De slimhindeafficerende genotyper inficerer flerlagede pladeepitheler i forskellige anatomiske lokalisationer, og en persisterende infektion kan medføre cancerudvikling. Den kausale rolle for HPV i udviklingen af visse anogenitale og for hoved-hals cancer er veletableret, men rollen i udviklingen af carcinomer i det okulaere adnexa (conjunctiva og tårevejene) er stadig behaeftet med usikkerhed. Vi udførte derfor en serie af studier for at undersøge sammenhaengen mellem HPV og udviklingen af carcinom i conjunctiva og tårevejene og karakterisere den kliniske, histologiske og genetiske profil af tumorerne baseret på HPV-status i en landsdaekkende, dansk kohorte. Ved brug af landsdaekkende patientregistre, indsamlede vi kliniske og histopatologiske data samt tumormateriale fra patienter diagnosticeret med carcinom i conjunctiva eller tårevejene og deres potentielle forstadier. Undersøgelser for HPV i tumormaterialet blev foretaget ved p16 immunhistokemi, HPV DNA polymerase chain reaction (PCR) og ved HPV E6/E7 mRNA in-situ hybridisering. Den genetiske profil blev undersøgt ved high-throughput DNA-sekventering målrettet 523 cancer-relevante gener. Litteraturen omhandlende associationen mellem HPV og conjunctival intraepithelial neoplasi og carcinom blev gennemgået. I det danske materiale var 67% af tårevejscarcinomerne og 21% af alle conjunctivale carcinomer HPV-positive. I begge lokalisationer var HPV16 den hyppigste genotype. Alle HPV-positive tumorer, fraset én, udtrykte ekspression af de virale onkogener E6 og E7. Histopatologiske og genetiske undersøgelser viste at de HPV-positive carcinomer udgået fra conjunctiva og tårevejene delte genotypiske og faenotypiske traek der adskilte dem fra de HPV-negative carcinomer. De HPV-positive carcinomer var karakterisereret af en ikke-keratiniserende morfologi, p16-ekspression, udtalt ekspression af HPV E6/E7 og hyppige patogene varianter i PI3K-AKT signalleringskaskaden. Derimod var de HPV-negative carcinomer karakteriseret af en keratiniserende morfologi og hyppige patogene varianter i TP53, CDKN2A, og RB1. For at konkludere, støtter vores resultater op om at HPV spiller en kausal rolle i subgrupper af carcinomer og deres forstadier der udgår fra conjunctiva og tårevejene. Vores undersøgelser har vist, at de HPV-positive carcinomer deler genetiske og faenotypiske karakteristika med HPV-positive carcinomer i andre anatomiske lokalisationer. Det er derfor muligt, at disse patienter kan indgå i fremtidige basket-trials og kan drage nytte af de behandlingsmetoder der udvikles til hyppigere forekomne HPV-positive carcinomer. Fremtidig forskning vil videre afgøre den diagnostiske, prognostiske, og praediktive vaerdi af HPV i carcinomer i det okulaere adnexa.

Genetic deletion of HPV E7 oncogene effectively regresses HPV driven oral squamous carcinoma tumour growth.

The major HPV oncogenes, E6 and E7, are known for its notoriety in driving the carcinogenic process in human papilloma virus (HPV) driven cancers. It is well-established that the removal of E7 dampens HPV cancer cell growth and proliferation. This has made E7 an attractive target for HPV cancers. Seminal work from our laboratory employed a CRISPR editing approach to delete E7 which resulted in the effective elimination of HPV+ cervical cancer tumours in vivo. We have also successfully delayed HPV+ tumour growth in vivo with aurora kinase (AURK) inhibitors, an effect which is strongly sensitized by the presence of E7. Unlike our previous observations in cervical cancer cells, in vitro targeting of E6/E7 have only resulted in partial killing of HPV+ oral squamous carcinoma (OSC) cells. However, the effect of sustained removal of E7 on HPV+ OSC tumour growth have not been explored. In this study, we investigated a staggered combination of aurora kinase inhibition, using alisertib, followed by CRISPR editing of E7, to determine if this would lead to better HPV+ OSC killing. Remarkably, genetic deletion of E7 alone was sufficient to effectively regress established HPV+ OSC tumours in vivo suggesting that E7 is essential in the maintenance of HPV+ OSC cancers.

Investigation of molecular mechanisms underlying JAK/STAT signaling pathway in HPV-induced cervical carcinogenesis using 'omics' approach.

The precise mechanism of action of Janus Kinases (JAK)/Signal Transducer and activator of Transcription (STAT) signaling in human papillomavirus (HPV)-associated cervical cancer (CaCx) is poorly defined. The present study dissected the underlying components of JAK/STAT signaling in HPV-positive cervical neoplasms. Whole transcriptome profile of CaCx cohort from TCGA database revealed elevated STAT3 and its impact on CaCx patients' survival. Using the RT2 Profiler PCR Array, we analyzed 84 genes of interest associated with JAK/STAT signaling in mRNA derived from HPV-negative and HPV-positive cervical lesions which revealed 21 differentially expressed genes (DEGs). Analyses of DEGs using the Database for Annotation, Visualization and Integrated Discovery tool indicated maximum genes enriched in immune response and negative regulation of apoptotic process. Protein-protein network analysis indicated IL4, STAT5A, STAT4, and JAK3 to be the key genes in the interaction network. Further, 7 key DEGs (IL4R, IRF1, EGFR, OAS1, PIAS1, STAT4, and STAT5A) were validated in TCGA cohort using R2 platform. These genes were differentially expressed among HPV-positive cervical tissues and their correlation with STAT3 was established. EGFR and IL4R showed a comparatively strong correlation with STAT3 that supports their involvement in pathogenesis of CaCx. Finally, the Kaplan-Meier analysis established the prognostic association of the key DEGs, in CaCx cohort. The STAT3 and associated key genes discovered from our study establish a strong pathogenic role of JAK/STAT3 pathway in HPV-mediated cervical carcinogenesis.

Full genotyping and FAM19A4/miR124-2 methylation analysis in high-risk human papillomavirus-positive samples from women over 30 years participating in cervical cancer screening in Örebro, Sweden.

Currently, cervical cancer prevention is undergoing comprehensive development regarding human papillomavirus (HPV) vaccination and cervical cancer screening. In Sweden and many other countries, high coverage vaccinated cohorts are entering screening within the next few years. This entails demands for baseline HPV genotype data across the screening age range for surveillance and a basis for screening program adjustment. In 2016, Örebro County, Sweden, changed to primary HPV screening using HPV mRNA testing followed by cytology triage. An alternative triage method to cytology could allow for a fully molecular screening algorithm and be implemented in a screening program where self-sampling is included. Hypermethylation analysis of the human genes FAM19A4/miR124-2 has been suggested as a promising triage method. HPV mRNA-positive screening samples (n = 529) were included and subjected to genotyping targeting a broad range of both low-risk and high-risk genotypes in addition to hypermethylation analysis of the two human genes FAM19A4/miR124-2. Data were connected to cytological and histological status and age. The most commonly detected genotypes were HPV31, 16, and 52. In addition, HPV18 was one of the most common genotypes in high-grade squamous intraepithelial lesions (HSILs) samples. In relation to available vaccines, 26% of the women with histological HSIL or cancer (≥HSIL) tested positive for only hrHPV included in the quadrivalent vaccine and 77% of the genotypes in the nonavalent vaccine. According to these figures, a relatively large proportion of the HSILs will probably remain, even after age cohorts vaccinated with the quadrivalent vaccine enter the screening program. Hypermethylation positivity was associated with increasing age, but no HPV-related independently predictive factors were found. Accordingly, age needs to be considered in development of future screening algorithms including triage with hypermethylation methodology.

Dickkopf-1 expression is repressed by oncogenic human papillomaviruses (HPVs) and regulates the Cisplatin sensitivity of HPV-positive cancer cells in a JNK-dependent manner.

Oncogenic human papillomavirus (HPV) types control the phenotype of cervical cancer cells through the sustained expression of the viral E6/E7 oncogenes. Here, we show that they strongly restrain expression of the putative tumor suppressor protein Dkk1 (Dickkopf-1) in HPV-positive cervical cancer cells through the restriction of p53 expression by the continuously expressed endogenous E6 oncoprotein. Moreover, our study reveals that compromised Dkk1 expression is linked to increased resistance of HPV-positive cervical cancer cells toward the proapoptotic activity of Cisplatin. Although Dkk1 can act as a Wnt antagonist, the antiapoptotic effect resulting from Dkk1 repression is not linked to an activation of this pathway. Rather, transcriptome and functional analyses uncover that Dkk1 repression leads to a strongly diminished stimulation of c-Jun N-terminal kinase (JNK) signaling which is required for efficient apoptosis induction by Cisplatin in cervical cancer cells. Further, we observed that Dkk1-depleted cervical cancer cells induce senescence under Cisplatin treatment instead of apoptosis, suggesting that Dkk1 levels can strongly influence the phenotypic response of these cells toward Cisplatin. Collectively, these results provide new insights into the virus/host cell crosstalk in cervical cancer cells by identifying Dkk1 as a cellular target which is maintained under strong negative control by the continuous expression of the HPV oncogenes. Moreover, they identify Dkk1 as a critical determinant for the sensitivity of cervical cancer cells toward Cisplatin, showing that Dkk1 repression leads to increased Cisplatin resistance by impairing proapoptotic JNK signaling.

Single-Cell Transcriptome Analysis Reveals Different Immune Signatures in HPV- and HPV + Driven Human Head and Neck Squamous Cell Carcinoma.

Background: Head and neck squamous cell carcinoma (HNSCC) is a significant health problem and related to poor long-term outcomes, indicating more research to be done to deeply understand the underlying pathways. Objective: This current study aimed in the assessment of the viral- (especially human papilloma virus [HPV]) and carcinogen-driven head and neck squamous cell carcinoma (HNSCC) microenvironment based on single-cell sequencing analysis. Methods: Data were downloaded from GEO database (GSE139324), including 131224 cells from 18 HP- HNSCC patients and 8 HPV+ HNSCC patients. Following data normalization, all highly variable genes in single cells were identified, and batch correction was applied. Differentially expressed genes were identified using Wilcoxon rank sum test. A gene enrichment analysis was performed in each cell cluster using KEGG analysis. Single-cell pseudotime trajectories were constructed with MONOCLE (version 2.6.4). Cell-cell interactions were analyzed with CellChat R package. Additionally, cell-cell communication patterns in key signal pathways were compared in different tissue groups. A hidden Markov model (HMM) was used to predict gene expression states (on or off) throughout pseudotime. Five-year overall survival outcomes were compared in both HPV+ and HPV- subsets. Results: 20,978 high-quality individual cells passed quality control. RNA-seq data were used from 522 HNSCC primary tumor samples. 1,137 differentially expressed genes between HPV+ and HPV- HNSCC patients were investigated. 96 differentially expressed genes were associated with overall survival and highly enriched in B cell associated biological process. Cell composition differed between types of samples. MHC-I, MHC-II, and MIF signaling pathways were found to be most relevant. Within these pathways, some cells were either signal receiver or signal sender, depending on sample type, respectively. Six genes were obtained, AREG and TGFBI (upregulation), CD27, CXCR3, MS4A1, and CD19 (downregulation), whose expression and HPV types were highly associated with worse overall survival. AREG and TGFBI were pDC marker genes, CXCR3 and CD27 were significantly expressed in T cell-related cells, while MS4A1 and CD19 were mainly expressed in B naïve cells. Conclusions: This study revealed dynamic changes in cell percentage and heterogeneity of cell subtypes of HNSCC. AREG, TGFBI, CD27, CXCR3, MS4A1, and CD19 were associated with worse overall survival in HPV-related HNSCC. Especially B-cell related pathways were revealed as particularly relevant in HPV-related HNSCC. These findings are a basis for the development of biomarkers and therapeutic targets in respective patients.

Detection of High-Risk Human Papillomavirus DNA in Invasive Ductal Carcinoma Specimens.

BACKGROUND: According to several studies, there is an association between human papillomavirus (HPV) and breast cancer. Therefore, detection and genotyping of HPV seem important. The present study aimed to investigate the presence of HPV DNA in breast tissues  by analyzing the L1 gene. MATERIALS AND METHODS: This case-control study was conducted on 63 formalin-fixed paraffin-embedded (FFPE) tissues of invasive ductal carcinoma (IDC) as the case group and 32 FFPE tissues of fibroadenoma as the control group. HPV DNA was detected using the polymerase chain reaction assay. Positive samples were then subjected to genotyping. All statistical analyses were performed in SPSS version 22.0. RESULTS: The patients' age ranged from 15 to 92 years, with a mean age of 43.54±16.36 years. HPV DNA was detected in 17/95 (17.89%) samples, including 9/32 (28.12%) fibroadenoma samples and 8/63 (12.69%) IDC samples. No significant difference was observed regarding the presence of HPV DNA between the IDC and fibroadenoma tissues (P=0.08). However, a significant difference was found in the detection of high-risk HPV (HR-HPV) between the case and control groups (P=0.03). In the case group, 87.5% of the detected viruses (7/8 samples) were HR-HPV, while in the control group, 22.22% of positive samples (2/9 samples) were HR-HPV (P=0.03). Based on the results, HR-HPV and low-risk HPV genotypes were detected in 53% (9/17) and 47% (8/17) of positive samples, respectively. CONCLUSION: In this study, 12.69% of IDC samples were positive for HPV genomes, and HR-HPV was detected in 87.5% of these samples. The present results suggest the important role of HR-HPV in the development of breast cancer.

Gene Expression and DNA Methylation in Human Papillomavirus Positive and Negative Head and Neck Squamous Cell Carcinomas.

High-risk human papillomaviruses (HPV) are important agents, responsible for a large percentage of the 745,000 cases of head and neck squamous cell carcinomas (HNSCC), which were identified worldwide in 2020. In addition to being virally induced, tobacco and heavy alcohol consumption are believed to cause DNA damage contributing to the high number of HNSCC cases. Gene expression and DNA methylation differ between HNSCC based on HPV status. We used publicly available gene expression and DNA methylation profiles from the Cancer Genome Atlas and compared HPV positive and HPV negative HNSCC groups. We used differential gene expression analysis, differential methylation analysis, and a combination of these two analyses to identify the differences. Differential expression analysis identified 1854 differentially expressed genes, including PCNA, TNFRSF14, TRAF1, TRAF2, BCL2, and BIRC3. SYCP2 was identified as one of the top deregulated genes in the differential methylation analysis and in the combined differential expression and methylation analyses. Additionally, pathway and ontology analyses identified the extracellular matrix and receptor interaction pathway as the most altered between HPV negative and HPV positive HNSCC groups. Combining gene expression and DNA methylation can help in elucidating the genes involved in HPV positive HNSCC tumorigenesis, such as SYCP2 and TAF7L.

Metatranscriptome analysis in human papillomavirus negative cervical cancers.

Human papillomavirus (HPV) negative cancers are associated with symptomatic detection, late-stage diagnosis, and worse prognosis. It is thus essential to investigate all possible infectious agents and biomarkers that could early identify these HPV negative cancers. We aimed to analyze and compare the metatranscriptome present in HPV positive and HPV negative cervical cancers. We analyzed the whole RNA sequencing files from 223 HPV negative cervical cancers (negativity established after confirming cervical cancer diagnosis, sample adequacy and subjecting specimens to PCR and unbiased RNA sequencing), 223 HPV positive tumors and 11 blank paraffin block pools (used as controls) using Kraken2 software. Overall, 84 bacterial genera were detected, with 6/84 genera showing a positive median number of reads/sample and being present in both cervical tumor groups (HPV positive and negative). Viral reads belonged to 63 different viral genera, with 6/63 genera showing a positive median annotated read/sample value. No significant difference among genera was detected except for the presence of alpha-papillomaviruses. Metatranscriptome of bacteria and viruses present in HPV positive and HPV negative cervical cancers show no significant difference, except for HPV. Further studies are needed to early identify this biologically distinct group of cervical cancers.

Genotype prevalence and age distribution of human papillomavirus from infection to cervical cancer in Japanese women: A systematic review and meta-analysis.

BACKGROUND: National HPV vaccination coverage in Japan is less than one percent of the eligible population and cervical cancer incidence and mortality are increasing. This systematic review and meta-analysis aimed to provide a comprehensive estimate of HPV genotype prevalence for Japan. METHODS: English and Japanese databases were searched to March 2021 for research reporting HPV genotypes in cytology and histology samples from Japanese women. Summary estimates were calculated by disease stage from cytology only assessment - Normal, ASCUS, LSIL, HSIL and from histological assessment - CIN1, CIN2, CIN3/AIS, ICC (ICC-SCC, and ICC-ADC), and other. A random-effects meta-analysis was used to calculate summary prevalence estimates of any-HPV, high-risk (HR) and low-risk (LR) vaccine types, and vaccine genotypes (bivalent, quadrivalent, or nonavalent). This study was registered with PROSPERO: CRD42018117596. RESULTS: A total of 57759 women with normal cytology, 1766 ASCUS, 3764 LSIL, 2017 HSIL, 3130 CIN1, 1219 CIN2, 869 CIN3/AIS, and 4306 ICC (which included 1032 ICC-SCC, and 638 ICC-ADC) were tested for HPV. The summary estimate of any-HPV genotype in women with normal cytology was 15·6% (95% CI: 12·3-19·4) and in invasive cervical cancer (ICC) was 85·6% (80·7-89·8). The prevalence of HR-HPV was 86·0% (95% CI: 73·9-94·9) for cytological cases of HSIL, 76·9% (52·1-94·7) for histological cases of CIN3/AIS, and 75·7% (68·0-82·6) for ICC. In women with ICC, the summary prevalence of bivalent vaccine genotypes was 58·5% (95% CI: 52·1-64·9), for quadrivalent genotypes was 58·6% (52·2-64·9) and for nonavalent genotypes was 71·5% (64·9-77·6), and of ICC cases that were HPV positive over 90% of infections are nonavalent vaccine preventable. There was considerable heterogeneity in all HPV summary estimates and for ICC, this heterogeneity was not explained by variability in study design, sample type, HPV assay type, or HPV DNA detection method, although studies published in the 1990s had lower prevalence estimates of any-HPV and HR HPV genotypes. INTERPRETATIONS: HPV prevalence is high among Japanese women. The nonavalent vaccine is likely to have the greatest impact on reducing cervical cancer incidence and mortality in Japan.

Discrimination of human papillomavirus genotypes using innovative technique nested-high resolution melting.

The prompt detection of human papillomavirus and discrimination of its genotypes by combining conventional methods in new molecular laboratories is essential to achieve the global call of eliminating cervical cancer. After predicting the melting temperature of an approximately 221 bp region of the L1 gene from different HPV genotypes by bioinformatics software, an innovative technique based on the nested- high resolution melting was designed with three approaches and using conventional PCR, qPCR, and diagnostic standards. HPV-positive samples identified by microarray along with diagnostic standards were evaluated by qPCR-HRM and discordant results were subjected to sequencing and analyzed in silico using reference types. In addition to screening for human papillomavirus, nested-qPCR-HRM is one of the modified HRM techniques which can discriminate some genotypes, including 6, 16, 18, 52, 59, 68 and 89. Despite the differences in diagnostic capabilities among HRM, microarray and sequencing, a number of similarities between HRM, and sequencing were diagnostically identified as the gold standard method. However, the bioinformatics analysis and melting temperature studies of the selected region in different HPV genotypes showed that it could be predicted. With numerous HPV genotypes and significant genetic diversity among them, determining the virus genotype is important. Therefore, our goal in this design was to use the specific molecular techniques with several specific primers to increase sensitivity and specificity for discriminating a wide range of HPV genotypes. This approach led to new findings to evaluate the ability of different approaches and procedures in accordance with bioinformatics.

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

The 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.