Human papillomavirus vaccination 2020 guideline update: American Cancer Society guideline adaptation.

The American Cancer Society (ACS) presents an adaptation of the current Advisory Committee on Immunization Practices recommendations for human papillomavirus (HPV) vaccination. The ACS recommends routine HPV vaccination between ages 9 and 12 years to achieve higher on-time vaccination rates, which will lead to increased numbers of cancers prevented. Health care providers are encouraged to start offering the HPV vaccine series at age 9 or 10 years. Catch-up HPV vaccination is recommended for all persons through age 26 years who are not adequately vaccinated. Providers should inform individuals aged 22 to 26 years who have not been previously vaccinated or who have not completed the series that vaccination at older ages is less effective in lowering cancer risk. Catch-up HPV vaccination is not recommended for adults aged older than 26 years. The ACS does not endorse the 2019 Advisory Committee on Immunization Practices recommendation for shared clinical decision making for some adults aged 27 through 45 years who are not adequately vaccinated because of the low effectiveness and low cancer prevention potential of vaccination in this age group, the burden of decision making on patients and clinicians, and the lack of sufficient guidance on the selection of individuals who might benefit.

TRIP12 and UBR5 suppress spreading of chromatin ubiquitylation at damaged chromosomes.

Histone ubiquitylation is a prominent response to DNA double-strand breaks (DSBs), but how these modifications are confined to DNA lesions is not understood. Here, we show that TRIP12 and UBR5, two HECT domain ubiquitin E3 ligases, control accumulation of RNF168, a rate-limiting component of a pathway that ubiquitylates histones after DNA breakage. We find that RNF168 can be saturated by increasing amounts of DSBs. Depletion of TRIP12 and UBR5 allows accumulation of RNF168 to supraphysiological levels, followed by massive spreading of ubiquitin conjugates and hyperaccumulation of ubiquitin-regulated genome caretakers such as 53BP1 and BRCA1. Thus, regulatory and proteolytic ubiquitylations are wired in a self-limiting circuit that promotes histone ubiquitylation near the DNA lesions but at the same time counteracts its excessive spreading to undamaged chromosomes. We provide evidence that this mechanism is vital for the homeostasis of ubiquitin-controlled events after DNA breakage and can be subverted during tumorigenesis.

TM4SF19 controls GABP-dependent YAP transcription in head and neck cancer under oxidative stress conditions.

Tobacco and alcohol are risk factors for human papillomavirus-negative head and neck squamous cell carcinoma (HPV- HNSCC), which arises from the mucosal epithelium of the upper aerodigestive tract. Notably, despite the mutagenic potential of smoking, HPV- HNSCC exhibits a low mutational load directly attributed to smoking, which implies an undefined role of smoking in HPV- HNSCC. Elevated YAP (Yes-associated protein) mRNA is prevalent in HPV- HNSCC, irrespective of the YAP gene amplification status, and the mechanism behind this upregulation remains elusive. Here, we report that oxidative stress, induced by major risk factors for HPV- HNSCC such as tobacco and alcohol, promotes YAP transcription via TM4SF19 (transmembrane 4 L six family member 19). TM4SF19 modulates YAP transcription by interacting with the GABP (Guanine and adenine-binding protein) transcription factor complex. Mechanistically, oxidative stress induces TM4SF19 dimerization and topology inversion in the endoplasmic reticulum membrane, which in turn protects the GABPß1 subunit from proteasomal degradation. Conversely, depletion of TM4SF19 impairs the survival, proliferation, and migration of HPV- HNSCC cells, highlighting the potential therapeutic relevance of targeting TM4SF19. Our findings reveal the roles of the key risk factors of HPV- HNSCC in tumor development via oxidative stress, offering implications for upcoming therapeutic approaches in HPV- HNSCC.

The Deubiquitinase USP46 Is Essential for Proliferation and Tumor Growth of HPV-Transformed Cancers.

High-risk human papilloma viruses (HPVs) cause cervical, anal, and oropharyngeal cancers, unlike the low-risk HPVs, which cause benign lesions. E6 oncoproteins from the high-risk strains are essential for cell proliferation and transformation in HPV-induced cancers. We report that a cellular deubiquitinase, USP46, is selectively recruited by the E6 of high-risk, but not low-risk, HPV to deubiqutinate and stabilize Cdt2/DTL. Stabilization of Cdt2, a component of the CRL4Cdt2 E3 ubiquitin ligase, limits the level of Set8, an epigenetic writer, and promotes cell proliferation. USP46 is essential for the proliferation of HPV-transformed cells, but not of cells without HPV. Cdt2 is elevated in human cervical cancers and knockdown of USP46 inhibits HPV-transformed tumor growth in xenografts. Recruitment of a cellular deubiquitinase to stabilize key cellular proteins is an important activity of oncogenic E6, and the importance of E6-USP46-Cdt2-Set8 pathway in HPV-induced cancers makes USP46 a target for the therapy of such cancers.

Deficiency in Ever2 does not increase susceptibility of mice to pathogenesis by the mouse papillomavirus, MmuPV1.

Epidermodysplasia verruciformis (EV) is a rare genetic skin disorder that is characterized by the development of papillomavirus-induced skin lesions that can progress to squamous cell carcinoma (SCC). Certain high-risk, cutaneous ß-genus human papillomaviruses (ß-HPVs), in particular HPV5 and HPV8, are associated with inducing EV in individuals who have a homozygous mutation in one of three genes tied to this disease: EVER1, EVER2, or CIB1. EVER1 and EVER2 are also known as TMC6 and TMC8, respectively. Little is known about the biochemical activities of EVER gene products or their roles in facilitating EV in conjunction with ß-HPV infection. To investigate the potential effect of EVER genes on papillomavirus infection, we pursued in vivo infection studies by infecting Ever2-null mice with mouse papillomavirus (MmuPV1). MmuPV1 shares characteristics with ß-HPVs including similar genome organization, shared molecular activities of their early, E6 and E7, oncoproteins, the lack of a viral E5 gene, and the capacity to cause skin lesions that can progress to SCC. MmuPV1 infections were conducted both in the presence and absence of UVB irradiation, which is known to increase the risk of MmuPV1-induced pathogenesis. Infection with MmuPV1 induced skin lesions in both wild-type and Ever2-null mice with and without UVB. Many lesions in both genotypes progressed to malignancy, and the disease severity did not differ between Ever2-null and wild-type mice. However, somewhat surprisingly, lesion growth and viral transcription was decreased, and lesion regression was increased in Ever2-null mice compared with wild-type mice. These studies demonstrate that Ever2-null mice infected with MmuPV1 do not exhibit the same phenotype as human EV patients infected with ß-HPVs.IMPORTANCEHumans with homozygous mutations in the EVER2 gene develop epidermodysplasia verruciformis (EV), a disease characterized by predisposition to persistent ß-genus human papillomavirus (ß-HPV) skin infections, which can progress to skin cancer. To investigate how EVER2 confers protection from papillomaviruses, we infected the skin of homozygous Ever2-null mice with mouse papillomavirus MmuPV1. Like in humans with EV, infected Ever2-null mice developed skin lesions that could progress to cancer. Unlike in humans with EV, lesions in these Ever2-null mice grew more slowly and regressed more frequently than in wild-type mice. MmuPV1 transcription was higher in wild-type mice than in Ever2-null mice, indicating that mouse EVER2 does not confer protection from papillomaviruses. These findings suggest that there are functional differences between MmuPV1 and ß-HPVs and/or between mouse and human EVER2.

The membrane-associated ubiquitin ligase MARCHF8 stabilizes the human papillomavirus oncoprotein E7 by degrading CUL1 and UBE2L3 in head and neck cancer.

The human papillomavirus (HPV) oncoprotein E7 is a relatively short-lived protein required for HPV-driven cancer development and maintenance. E7 is degraded through ubiquitination mediated by cullin 1 (CUL1) and the ubiquitin-conjugating enzyme E2 L3 (UBE2L3). However, E7 proteins are maintained at high levels in most HPV-positive cancer cells. A previous proteomics study has shown that UBE2L3 and CUL1 protein levels are increased by the knockdown of the E3 ubiquitin ligase membrane-associated ring-CH-type finger 8 (MARCHF8). We have recently demonstrated that HPV16 upregulates MARCHF8 expression in HPV-positive keratinocytes and head and neck cancer (HPV+ HNC) cells. Here, we report that MARCHF8 stabilizes the HPV16 E7 protein by degrading the components of the S-phase kinase-associated protein 1-CUL1-F-box ubiquitin ligase complex in HPV+ HNC cells. We found that MARCHF8 knockdown in HPV+ HNC cells drastically decreases the HPV16 E7 protein level while increasing the CUL1 and UBE2L3 protein levels. We further revealed that the MARCHF8 protein binds to and ubiquitinates CUL1 and UBE2L3 proteins and that MARCHF8 knockdown enhances the ubiquitination of the HPV16 E7 protein. Conversely, the overexpression of CUL1 and UBE2L3 in HPV+ HNC cells decreases HPV16 E7 protein levels and suppresses tumor growth in vivo. Our findings suggest that HPV-induced MARCHF8 prevents the degradation of the HPV16 E7 protein in HPV+ HNC cells by ubiquitinating and degrading CUL1 and UBE2L3 proteins.IMPORTANCESince human papillomavirus (HPV) oncoprotein E7 is essential for virus replication; HPV has to maintain high levels of E7 expression in HPV-infected cells. However, HPV E7 can be efficiently ubiquitinated by a ubiquitin ligase and degraded by proteasomes in the host cell. Mechanistically, the E3 ubiquitin ligase complex cullin 1 (CUL1) and ubiquitin-conjugating enzyme E2 L3 (UBE2L3) components play an essential role in E7 ubiquitination and degradation. Here, we show that the membrane ubiquitin ligase membrane-associated ring-CH-type finger 8 (MARCHF8) induced by HPV16 E6 stabilizes the E7 protein by degrading CUL1 and UBE2L3 and blocking E7 degradation through proteasomes. MARCHF8 knockout restores CUL1 and UBE2L3 expression, decreasing E7 protein levels and inhibiting the proliferation of HPV-positive cancer cells. Additionally, overexpression of CUL1 or UBE2L3 decreases E7 protein levels and suppresses in vivo tumor growth. Our results suggest that HPV16 maintains high E7 protein levels in the host cell by inducing MARCHF8, which may be critical for cell proliferation and tumorigenesis.

Unfurling the functional association between long intergenic noncoding RNAs (lincRNAs) and HPV16-related cervical cancer pathogenesis through weighted gene co-expression network analysis of differentially expressed lincRNAs and coding genes.

Long intergenic noncoding RNAs (lincRNAs) do not overlap annotated coding genes and are located in intergenic regions, as opposed to antisense and sense-intronic lncRNAs, located in genic regions. LincRNAs influence gene expression profiles and are thereby key to disease pathogenesis. In this study, we assessed the association between lincRNAs and HPV16-positive cervical cancer (CaCx) pathogenesis using weighted gene co-expression network analysis (WGCNA) with coding genes, comparing differentially expressed lincRNA and coding genes (DElincGs and DEcGs, respectively) in HPV16-positive patients with CaCx (n = 44) with those in HPV-negative healthy individuals (n = 34). Our analysis revealed five DElincG modules, co-expressing and correlating with DEcGs. We validated a substantial number of such module-specific correlations in the HPV16-positive cancer TCGA-CESC dataset. Four such modules, displayed significant correlations with patient traits, such as HPV16 physical status, lymph node involvement and overall survival (OS), highlighting a collaborative effect of all genes within specific modules on traits. Using the DAVID bioinformatics knowledgebase, we identified the underlying biological processes associated with these modules as cancer development and progression-associated pathways. Next, we identified the top 10 DElincGs with the highest connectivity within each functional module. Focusing on the prognostic module hub genes, downregulated CTD-2619J13.13 expression was associated with poor patient OS. This lincRNA gene interacted with 25 coding genes of its module and was associated with such biological processes as keratinization loss and keratinocyte differentiation, reflecting severe disease phenotypes. This study has translational relevance in fighting various cancers with high mortality rates in underdeveloped countries.

Copy Number Profiling Implicates Thin High-Grade Squamous Intraepithelial Lesions as a True Precursor of Cervical Human Papillomavirus-Induced Squamous Cell Cancer.

Full-thickness high-grade squamous intraepithelial lesions (HSIL) are precursors of invasive cervical squamous cell carcinoma (SCC). The World Health Organization and Lower Anogenital Squamous Terminology Standardization Project for human papilloma virus (HPV)-associated lesions divide full-thickness HSIL of the cervix into thin HSIL with thickness of 1 to 9 cell layers and the typical full-thickness HSIL of >10 cell layers. Although HPV oncogene transcripts and p16ink4a overexpression, as markers of transforming HPV infection, are detectable in thin HSIL, the biological significance of thin HSIL in cervical carcinogenesis remains poorly understood. To further characterize thin HSIL, we performed a comparative study of chromosomal copy number variations (CNV), an analysis of dysregulated genes present in the segments with CNV, and a generalized genetic complexity calculation for 31 thin HSIL, 31 thick HSIL, 24 microinvasive SCC (pT1a SCC), and 22 highly invasive SCC samples. Thin HSIL share various CNV and specific dysregulated gene pathways with thick HSIL and invasive SCC. Thin HSIL exhibited an average CNV of 11.6% compared with 14.1% for thick HSIL, 15.5% for pT1a SCC, and 26.6% for highly invasive SCC. The CNV included gains at 1q and 3q (40% and 43%, respectively), partial loss of 3p, and loss of chromosomes 11 (18%), 16 (50%), 20 (35%), and 22 (40%). Pathways affected solely in thin HSIL were those enhancing immune evasion and primarily involved the (interleukin) IL6, IL21, and IL23 genes. ILs are transiently upregulated in response to infection and play a crucial role in mounting antitumor T-cell activity. Deregulation reflects an attempt by the HPV to evade the initial immune response of the host. The primary pathways shared by thick HSIL and invasive SCC were interactions between lymphoid and nonlymphoid cells, NOTCH2 signaling, tight junction interactions (primarily of the claudin family), and FGR2 alternative splicing. Our results show that thin HSIL carry similar genetic changes as thick HSIL and SCC, indicating that thin HSIL are true precursor lesions that can progress to thick HSIL and SCC.

Human malignancies associated with persistent HPV infection.

Human papillomavirus (HPV)-associated malignancies account for ~5% of human cancers worldwide. Thirteen, or more, HPV types are oncogenic, but infection with these viruses is common and usually cleared within 2 years. Only infections that become persistent are associated with the development of cancer, often occurring several decades later. These cancers mostly arise in 6 different anatomical regions: 5 are anogenital (anus, cervix, penis, vagina, and vulva) and the sixth is the oropharynx. Oncogenic HPVs promote cellular proliferation and genomic instability, but the anatomical niche of the target tissue also plays an important role in the development of cancer. Cells that reside in transitional regions between different types of epithelia, such as in the anus, cervix, and oropharynx, are particularly vulnerable to oncogenesis.

Advances in testing for human papillomavirus -mediated head and neck cancer.

PURPOSE OF REVIEW: New evidence has recently emerged regarding the utility and benefits of dual p16 INKa (p16) and Human papillomavirus (HPV) status testing when determining the diagnosis and prognosis of patients with oropharyngeal cancer. RECENT FINDINGS: HPV RNA polymerase chain reaction (PCR) is the most accurate diagnostic test. The other assays (HPV DNA PCR, HPV DNA/RNA in-situ hybridization (ISH) and p16) applied to formalin fixed tumour tissue have varying but high sensitivities and specificities. Dual p16 and HPV testing identifies discordant (p16+/HPV- or p16-/HPV+) results in 9.2% of cases, who have significantly poorer prognoses than p16+/HPV+, particularly in smokers. The proportion of discordant cases varies by region, and appears to be highest in regions with lowest attributable (p16+/HPV+) fractions. Dual testing improves prognostication for oropharyngeal cancer cases by identifying discordant cases and improving the prognostic power of the Tumour Node Metastasis (TNM) classification, especially in regions with high discordant rates. SUMMARY: Dual testing is essential when considering patients for clinical trials of treatment de-escalation, and may be important when counselling patients on prognosis, especially in regions with high discordant rates and in smokers.

Scattering-Based Light-Sheet Microscopy Imaging of Human Papillomavirus-Associated Squamous Lesions of the Anal Canal: A Proof-of-Principle Study.

Demand for anal cancer screening is expected to rise following the recent publication of the Anal Cancer-HSIL Outcomes Research trial, which showed that treatment of high-grade squamous intraepithelial lesions significantly reduces the rate of progression to anal cancer. While screening for human papillomavirus-associated squamous lesions in the cervix is well established and effective, this is less true for other sites in the lower anogenital tract. Current anal cancer screening and prevention rely on high-resolution anoscopy with biopsies. This procedure has a steep learning curve for providers and may cause patient discomfort. Scattering-based light-sheet microscopy (sLSM) is a novel imaging modality with the potential to mitigate these challenges through real-time, microscopic visualization of disease-susceptible tissue. Here, we report a proof-of-principle study that establishes feasibility of dysplasia detection using an sLSM device. We imaged 110 anal biopsy specimens collected prospectively at our institution's dysplasia clinic (including 30 nondysplastic, 40 low-grade squamous intraepithelial lesion, and 40 high-grade squamous intraepithelial lesion specimens) and found that these optical images are highly interpretable and accurately recapitulate histopathologic features traditionally used for the diagnosis of human papillomavirus-associated squamous dysplasia. A reader study to assess diagnostic accuracy suggests that sLSM images are noninferior to hematoxylin and eosin images for the detection of anal dysplasia (sLSM accuracy = 0.87; hematoxylin and eosin accuracy = 0.80; P = .066). Given these results, we believe that sLSM technology holds great potential to enhance the efficacy of anal cancer screening by allowing accurate sampling of diagnostic tissue at the time of anoscopy. While the current imaging study was performed on ex vivo biopsy specimens, we are currently developing a handheld device for in vivo imaging that will provide immediate microscopic guidance to high-resolution anoscopy providers.

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

A 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.

High-grade vulvar intraepithelial neoplasia: comprehensive characterization and long-term vulvar carcinoma risk.

AIMS: Adequate diagnosis of human papillomavirus (HPV)-associated high-grade squamous intraepithelial lesion (HSIL) and HPV-independent vulvar intraepithelial neoplasia (VIN) is essential but can be challenging. We comprehensively characterized a large population-based series of vulvar lesions, originally reported as high-grade VIN, and assessed the cancer risk. METHODS AND RESULTS: Baseline high-grade VIN of 751 patients were categorized by histopathological reassessment, integrating the results of immunohistochemistry (p16INK4a , p53, Ki-67) and HPV DNA testing. Integrated analyses resulted in 88.4% HPV-associated lesions (77.0% HSIL, 10.9% low-grade SIL [LSIL], and 0.4% vulvar squamous cell carcinoma [VSCC]), 10.9% HPV-independent lesions (6.1% HPV-independent VIN, 4.7% nondysplastic lesions, and 0.1% VSCC) and 1.1% inconclusive lesions. HSIL demonstrated p16INK4a block-positivity in 99.0%, increased Ki-67 in ≥2/3rd of the epithelium in 93.6%, and HPV positivity in 99.6%. In HSIL, a p53 wildtype mid-epithelial staining pattern was common (51.6%) while this was not observed in HPV-independent lesions. HPV-independent VIN harboured mutant p53 patterns in 65.2% and showed a wide morphological spectrum, ranging from differentiated to nondifferentiated ('HPV-associated-like', in 41.3%). Kaplan-Meier analyses showed a 10-year cancer risk of 8.0% in HPV-associated HSIL, 67.4% in HPV-independent VIN/p53mutant, and 27.8% in HPV-independent VIN/p53wildtype. Strikingly, the 10-year cancer risk was 73.3% in HPV-independent VIN with nondifferentiated ('HPV-associated-like') morphology. CONCLUSION: Immunohistochemistry by p16INK4a and p53 is highly recommended for optimal categorization into HPV-associated and HPV-independent VIN, which is of utmost importance given the different cancer risk. The high cancer risk of HPV-independent VIN underscores the need for surgical treatment and close follow-up, especially in case of a p53 mutant pattern and/or nondifferentiated morphology.

Clinical analysis of 314 patients with high-grade squamous intraepithelial lesion who underwent total hysterectomy directly: a multi-center, retrospective cohort study.

OBJECTIVE: To identify the risk factors of cervical high-grade squamous intraepithelial lesion(HSIL) complicated with occult cervical cancer and standardize the management of initial treatment for HSIL. METHOD: The clinical data of patients who underwent total hysterectomy directly due to HSIL in the obstetrics and gynecology department of two tertiary hospitals and three secondary hospitals from 2018 to 2023 were collected. Their general characteristics, pathological parameters and survival status were analyzed. Logistic regression model was used to analyze the correlation between clinical parameters and postoperative pathological upgrading. RESULT: 1. Among the 314 patients with HSIL who underwent total hysterectomy directly, 73.2% were from primary hospitals. 2. 25 patients (7.9%) were pathologically upgraded to cervical cancer, all of which were early invasive cancer. 3. Up to now, there was no recurrence or death in the 25 patients with early-stage invasive cancer, and the median follow-up period was 21 months(range 2-59 months). 4. Glandular involvement(OR 3.968; 95%CI 1.244-12.662) and lesion range ≥ 3 quadrants (OR 6.527; 95% CI 1.78-23.931), HPV 16/18 infection (OR 5.382; 95%CI 1.947-14.872), TCT ≥ ASC-H (OR 4.719; 95%CI 1.892-11.766) were independent risk factors that affected the upgrading of postoperative pathology. 5. The area under the curve (AUC) calculated by the Logistic regression model was 0.840, indicating that the predictive value was good. CONCLUSION: There is a risk of occult cervical cancer in patients with HSIL. Glandular involvement, Lesion range ≥ 3 quadrants, HPV 16/18 infection and TCT ≥ ASC-H are independent risk factors for HSIL combined with occult cervical cancer. The prognosis of biopsy-proved HSIL patients who underwent extrafascial hysterectomy and unexpected early invasive cancer was later identified on specimen may be good.

Relationship between p16/ki67 immunoscores and PAX1/ZNF582 methylation status in precancerous and cancerous cervical lesions in high-risk HPV-positive women.

BACKGROUND: The risk of cervical cancer progression in high-risk human papillomavirus (HR-HPV)-positive women is associated with cervical lesion severity and molecular heterogeneity. Classification systems based on p16 and Ki67 expression cumulative scores (0-3 each)-p16/Ki67 collectively known as an immunoscore [IS]-are an accurate and reproducible method for grading cervical intraepithelial neoplasia (CIN) lesions. Meanwhile, DNA methylation is an early event in the development of cervical cancer. Hence, this study evaluated the relationship among CIN, p16/Ki-67 IS, and PAX1/ZNF582 methylation. METHODS: In this study, 414 HPV-positive paraffin-embedded specimens were collected, and PAX1/ZNF582 methylation and the p16/ki67 IS were determined. A total of 43 invalid samples were excluded and 371 were included in the statistical analyses. There were 103 cervicitis, 95 CIN1, 71 CIN2, 89 CIN3, and 13 squamous cell carcinoma (SCC) cases. The association between PAX1/ZNF582 methylation and p16/Ki6 immunohistochemical staining scores was analyzed. RESULTS: The ΔCp of PAX1m (PAX1 methylation) and ZNF582m (ZNF582 methylation) decreased with cervical lesion severity (Cuzick trend test, all P < 0.001). The severity of the cervical lesions and p16, Ki67, and p16/Ki67 IS showed an increasing trend (Multinomial Cochran-Armitage trend test, all P < 0.001). The prevalence of PAX1m/ZNF582m increased with an increase in the IS of p16, Ki67, and p16/Ki67 (Cochran-Armitage trend test, all P < 0.001). In cervical SCC, the IS was 5-6, and the PAX1m/ZNF582m was positive. Meanwhile, heterogeneity was observed in CIN lesions: 10 cases had an IS of 3-4 and were PAX1m/ZNF582m-positive in ≤ CIN1; 1 case had an IS of 0-2 and was PAX1m/ZNF582m-positive in CIN2/3. CONCLUSIONS: Significant heterogeneity was observed in CIN lesions for p16 and Ki67 immunohistochemical staining scores and PAX1/ZNF582 methylation. This may help clinicians personalize the management of CIN based on the predicted short-term risk of cancer progression, minimizing the rate of missed CIN1 diagnoses and incorrect treatment of CIN2/3.

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.

Circular RNAs and cervical cancer: friends or foes? A landscape on circRNA-mediated regulation of key signaling pathways involved in the onset and progression of HPV-related cervical neoplasms.

Cervical cancer (CC) is a common gynecologic malignancy, accounting for a significant proportion of women death worldwide. Human papillomavirus (HPV) infection is one of the major etiological causes leading to CC onset; however, genetic, and epigenetic factors are also responsible for disease expansion. Circular RNAs (circRNAs), which are known as a particular subset of non-coding RNA (ncRNA) superfamily, with covalently closed loop structures, have been reported to be involved in the progression of diverse diseases, especially neoplasms. In this framework, abnormally expressed circRNAs are in strong correlation with CC pathogenesis through regulating substantial signaling pathways. Also, these RNA molecules can be considered as promising biomarkers and therapeutic targets for CC diagnosis/prognosis and treatment, respectively. Herein, we first review key molecular mechanisms, including Wnt/ß-catenin, MAPK, and PI3K/Akt/mTOR signaling pathways, as well as angiogenesis and metastasis, by which circRNAs interfere with CC development. Then, diagnostic, prognostic, and therapeutic potentials of these ncRNA molecules will be highlighted in depth.

Genomic characterization of vulvar squamous cell carcinoma reveals differential gene expression based on clinical outcome.

OBJECTIVE: The greatest challenge in the management of vulvar squamous cell carcinoma (VSCC) is treatment of recurrent disease where options for surgery and radiation have been exhausted, or treatment of disease where distant metastasis is present. Identification of mutations differentially expressed between tumor from patients who died of aggressive disease and tumor from patients with an indolent course could reveal novel prognostic indicators and guide development of therapeutic drugs. METHODS: From 202 consecutive patients with VSCC, patients who recurred and died of disease (group A) were identified and matched by age, tumor size, depth of invasion and nodal status with those whose disease did not recur (group B). Tumors from 21 patients were subjected to whole exome sequencing of DNA and RNA, immunohistochemistry (IHC) antibodies of PD-L1 and P16, and in-situ hybridization (ISH) for high-risk HPV. RESULTS: Analysis of DNA and RNA revealed six genes that were strongly differentially expressed between group A and B: TGM3, ACVR2A, ROS1, NFEL2, CCND1 and BCL6. Clinically relevant DNA mutations were significantly greater in group A versus B: 7 vs 2.3 mutations per patient. The most common genomic alterations were mutations in TP53 and the promoter region of TERT. Other common genomic events include alterations of FAT1, CDKN2A, PIK3CA, CCND1, and LRP1B. All samples were MSI stable and tumor mutational burden (TMB) was similar in groups A and B. Most VSCC specimens (81%) were positive for PD-L1. CONCLUSIONS: ACVR2A and TGM3 are significantly under-expressed in tumors with poor outcome, suggesting they may play a role in tumor suppression. Clinical outcome of VSCC appears independent of MSI, TMB, or PD-L1 status.

High concordance of molecular subtyping between pre-surgical biopsy and surgical resection specimen (matched-pair analysis) in patients with vulvar squamous cell carcinoma using p16- and p53-immunostaining.

OBJECTIVE: Vulvar squamous cell carcinoma (VSCC) can be stratified into three molecular subtypes based on the immunoexpression of p16 and p53: HPV-independent p53-abnormal (p53abn) (most common, biologically aggressive), HPV-associated, with p16-overexpression (second most common, prognostically more favourable) and more recently recognised HPV-independent p53-wildtype (p53wt) (rarest subtype, prognostically intermediate). Our aim was to determine whether molecular subtypes can be reliably identified in pre-operative biopsies and whether these correspond to the subsequent vulvectomy specimen. METHODS: Matched-paired pre-surgical biopsies and subsequent resection specimen of 57 patients with VSCC were analysed for the immunohistochemical expression of p16 and p53 by performing a three-tiered molecular subtyping to test the accuracy rate. RESULTS: Most cases 36/57 (63.2%) belonged to the HPV-independent (p53-abn) molecular subtype, followed by HPV-associated 17/57 (29.8%) and HPV-independent (p53wt) 4/57 (7.0%). The overall accuracy rate on biopsy was 91.2% (52/57): 97.3% for p53-abnormal, 94.1% for p16-overexpression and 50% for p16-neg/p53-wt VSCC. Incorrect interpretation of immunohistochemical p53 staining pattern was the reason for discordant results in molecular subtyping in all five cases. In one case there was an underestimation of p53 pattern (wildtype instead of abnormal/aberrant) and in one case an overestimation of the p53 staining pattern (abnormal/aberrant instead of wildtype). In 3/5 there was a "double positive" staining result (p16 overexpression and abnormal/aberrant p53 staining pattern). In that cases additional molecular workup is required for correct molecular subtyping, resulting in an overall need for molecular examination of 3/57 (3.5%). CONCLUSIONS: Compared to the final resections specimen, the three-tiered molecular classification of VSCC can be determined on pre-surgical biopsies with a high accuracy rate. This enables more precise surgical planning, prediction of the response to (chemo) radiation, selection of targeted therapies and planning of the optimal follow-up strategy for patients in the age of personalised medicine.

Calendar-period trends in cervical precancer and cancer diagnoses since the introduction of human papillomavirus and cytology co-testing into routine cervical cancer screening at Kaiser Permanente Northern California.

OBJECTIVES: The longer-term impact of introducing human papillomavirus (HPV) testing into routine cervical cancer screening on precancer and cancer rates by histologic type has not been well described. Calendar trends in diagnoses were examined using data from Kaiser Permanente Northern California, which introduced triennial HPV and cytology co-testing in 2003 for women aged ≥30 years. METHODS: We examined trends in cervical precancer (cervical intraepithelial neoplasia grade 3 [CIN3] and adenocarcinoma in situ [AIS]) and cancer (squamous cell carcinoma [SCC] and adenocarcinoma [ADC]) diagnoses per 1000 screened during 2003-2018. We examined ratios of squamous vs. glandular diagnoses (SCC:ADC and CIN3:AIS). RESULTS: CIN3 and AIS diagnoses increased approximately 2% and 3% annually, respectively (ptrend < 0.001 for both). While SCC diagnoses decreased by 5% per annually (ptrend < 0.001), ADC diagnoses did not change. These patterns were generally observed within each age group (30-39, 40-49, and 50-64 years). ADC diagnoses per 1000 screened did not change even among those who underwent co-testing starting in 2003-2006. SCC:ADC decreased from approximately 2.5:1 in 2003-2006 to 1.3:1 in 2015-2018 while the CIN3:AIS remained relatively constant, ∼10:1. CONCLUSIONS: Since its introduction at KPNC, co-testing increased the detection of CIN3 over time, which likely caused a subsequent reduction of SCC. However, there has been no observed decrease in ADC. One possible explanation for lack of effectiveness against ADC is the underdiagnosis of AIS. Novel strategies to identify and treat women at high risk of ADC need to be developed and clinically validated.