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.

The effect of high-risk HPV E6/E7 mRNA on the efficacy of topical photodynamic therapy with 5-aminolevulinic acid for cervical high-grade squamous intraepithelial lesions.

BACKGROUND: E6 and E7 high-risk human papillomavirus (HR-HPV) oncoproteins are closely associated with the initiation and progression of cervical cancer (CC) and pre-cancerous lesions. Cervical high-grade squamous intraepithelial lesions (HSIL), as pre-cancerous lesions, have a 5% chance of progressing to invasive cancer. Topical 5-aminolevulinic acid photodynamic therapy (ALA-PDT) is a novel non-invasive targeted therapy for intraepithelial lesions. Herein, we analyzed the effect of HR-HPV E6/E7 mRNA on ALA-PDT for cervical HSIL. METHODS: A retrospective analysis of 148 HR-HPV-positive patients diagnosed with cervical HSIL and receiving ALA-PDT was carried out. ALA-PDT was performed with 20% ALA thermosensitive gel, and irradiation at wavelength of 635 nm and density of 80-100 J/cm2 for 20-30 min. The therapeutic instruments of LED and semiconductor laser were applied for cervical lesions and lesions in endocervical canal, respectively. All patients were tested for HPV E6/E7 mRNA before and after PDT, and then followed up at 3, 6, and 12 months after treatment, and every six months thereafter. RESULTS: At the 6-month follow up, the complete remission (CR) rate of patients' lesions was 86.5% (128/148), whereas the total HPV clearance rate was 72.3% (107/148). It was evident that positive E6/E7 mRNA before treatment had a significant effect on HPV clearance rate (66.3% VS 81.4%, P = 0.045) and CR rate (80.9% VS 94.9%, P = 0.015). The E6/E7 mRNA associated with HPV16/18 and HPV16/18 combined with other HR-HPV (HPV16/18 and other HR-HPV) affected HPV clearance (P = 0.035) and lesions CR (P = 0.039), respectively. Moreover, persistently positive E6/E7 mRNA after treatment was closely associated with poor efficacy (HPV clearance rate: P = 0.000, CR rate: P = 0.000). Throughout the follow up period, two cases recurred but none of the patients progressed. CONCLUSIONS: This study has shown that ALA-PDT is an effective, safe, and alternative treatment for cervical HSIL, especially for the patients of childbearing age. However, its efficacy is relatively poor in patients with persistently positive E6/E7 mRNA before and after treatment, who are relatively insensitive to ALA-PDT.

An internal class III PDZ binding motif in HPV16 E6* protein is required for Dlg degradation activity.

BACKGROUND: A splice product of the E6 oncoprotein, E6*, is found in cells infected with HPV associated with a high-risk for cervical cancer. Both E6* and E6 promote Dlg degradation, considered a contributing factor for the tumorigenic potential of high-risk HPVs. The full-length E6 utilizes a conserved PDZ binding motif (PBM) at the extreme C-terminus to promote Dlg degradation. In contrast, this PBM is absent in E6*. METHODS: We performed western blot analysis, site-directed mutagenesis and co-immunoprecipitation to identify the key elements required for Dlg degradation activity of high-risk HPVE6*, using HPV16E6* as a model. RESULTS: Our data indicate that only one of the two internal putative class III PBMs, located between amino acids 24-27 (HDII) of HPV16E6*, was required to facilitate degradation of Dlg protein. Substitution of the two consensus residues in this region (D25 and I27) to glycine greatly diminished activity. Whereas substitution of the two conserved residues in the putative internal class I PBM (amino acids 16-19) or the second putative class III PBM (amino acids 28-31) was without effect. Interestingly, HPV66E6* which does not promote Dlg degradation can be converted into a form capable of facilitating Dlg degradation through the insertion of nine amino acids (20-28) containing the class III PBM from HPV16E6*. HPV16E6*-induced Dlg degradation appeared independent of E6AP. CONCLUSIONS: The internal class III PBM of HPV16E6*I required for Dlg degradation is identified. GENERAL SIGNIFICANCE: This study highlights that a novel class III PBM as the domain responsible for Dlg degradation activity in high-risk HPVE6*.

Timing, number, and type of sexual partners associated with risk of oropharyngeal cancer.

BACKGROUND: Case-control studies from the early 2000s demonstrated that human papillomavirus-related oropharyngeal cancer (HPV-OPC) is a distinct entity associated with number of oral sex partners. Using contemporary data, we investigated novel risk factors (sexual debut behaviors, exposure intensity, and relationship dynamics) and serological markers on odds of HPV-OPC. METHODS: HPV-OPC patients and frequency-matched controls were enrolled in a multicenter study from 2013 to 2018. Participants completed a behavioral survey. Characteristics were compared using a chi-square test for categorical variables and a t test for continuous variables. Adjusted odds ratios (aOR) were calculated using logistic regression. RESULTS: A total of 163 HPV-OPC patients and 345 controls were included. Lifetime number of oral sex partners was associated with significantly increased odds of HPV-OPC (>10 partners: odds ratio [OR], 4.3 [95% CI, 2.8-6.7]). After adjustment for number of oral sex partners and smoking, younger age at first oral sex (<18 vs >20 years: aOR, 1.8 [95% CI, 1.1-3.2]) and oral sex intensity (>5 sex-years: aOR, 2.8 [95% CI, 1.1-7.5]) remained associated with significantly increased odds of HPV-OPC. Type of sexual partner such as older partners when a case was younger (OR, 1.7 [95% CI, 1.1-2.6]) or having a partner who had extramarital sex (OR, 1.6 [95% CI, 1.1-2.4]) was associated with HPV-OPC. Seropositivity for antibodies to HPV16 E6 (OR, 286 [95% CI, 122-670]) and any HPV16 E protein (E1, E2, E6, E7; OR, 163 [95% CI, 70-378]) was associated with increased odds of HPV-OPC. CONCLUSION: Number of oral sex partners remains a strong risk factor for HPV-OPC; however, timing and intensity of oral sex are novel independent risk factors. These behaviors suggest additional nuances of how and why some individuals develop HPV-OPC.

Highly Reproducible and Sensitive Electrochemiluminescence Biosensors for HPV Detection Based on Bovine Serum Albumin Carrier Platforms and Hyperbranched Rolling Circle Amplification.

Most DNA-based electrochemiluminescence (ECL) biosensors are established through the self-assembly of thiolated single-stranded DNA (ssDNA) probes on the Au electrode surface. Because of this random assembly process, a significant discrepancy exists in the distribution of a modified DNA film on different electrodes, which greatly affects the reproducibility of a biosensor. In this study, a porous bovine serum albumin (BSA) layer was first modified on the electrode surface, which can improve the position distribution and spatial orientation of the self-assembly ssDNA probe. It was then coupled with hyperbranched rolling circle amplification to develop the high-reproducibility-and-sensitivity ECL biosensor for human papillomavirus 16 E6 and E7 oncogene detection. In the presence of the target DNA, the surface of the electrode accumulates abundant amplified products through reaction, which contain double-stranded DNA (dsDNA) fragments of different lengths, followed by plentiful dichlorotris (1,10-phenanthroline) ruthenium(II) hydrate (Ru(phen)32+, acting as an ECL indicator) insertion into grooves of dsDNA fragments, and a strong signal can be detected. There is a linear relationship between the signal and the target concentration range from 10 fM to 15 pM, and the detection limit is 7.6 fM (S/N = 3). After the BSA modification step, the relative standard deviation was reduced from 9.20 to 3.96%, thereby achieving good reproducibility. The proposed ECL strategy provides a new method for constructing high-reproducibility-and-sensitivity ECL biosensors.

RPRD1B is a potentially molecular target for diagnosis and prevention of human papillomavirus E6/E7 infection-induced cervical cancer: A case-control study.

BACKGROUND: The objective of the study is to investigate the biomarkers for diagnosis and prevention of human papillomavirus (HPV) infection-induced cervical cancer. METHODS: Cervical cancer tissues were collected from patients with cervical cancer, while noncancer tissues were collected from patients diagnosed with cervical lesions or uterine fibroids at the Chinese PLA General Hospital 301 and 309, China from December 2017 to June 2018. The cancer tissues were collected from the site of lesion, while the noncancer tissues were collected from similar anatomical locations. Quantitative real-time PCR, Western blot (WB), and immunohistochemistry (IHC) were used to detect the mRNA and protein levels of HPV E6/E7, RPRD1B (regulation of nuclear pre-mRNA domain containing 1B), cyclin D1, and transcription factor 4 (TCF4) between cervical cancer tissues and noncancer tissues. The correlation of HPV E6/E7, RPRD1B, cyclin D1, and TCF4 expressions was analyzed. RESULTS: Twenty patients with cervical cancer and 27 controls without cervical cancer were included in this study. The mRNA expression of HPV E6/E7and RPRD1B was significantly higher in patients with cervical cancer than controls, while cyclin D1 mRNA expression was significantly lower in patients with cervical carcinoma in situ stage, compared with controls. RPRD1B protein expression was significantly higher in patients compared to controls when analyzed by IHC. TCF4 was significantly lower in clinical stage I and Ib of cervical cancer when analyzed by WB. The mRNA and protein expressions of RPRD1B and cyclin D1 were significantly different between patients younger than 50 years old, compared to patients 50 years and older. CONCLUSIONS: HPV E6/E7 expression was associated with RPRD1B level in cervical cancer. The expression of RPRD1B and cyclin D1 in patients with cervical cancer might be affected by age.

[The value of P16/ki67 double labeling, HPV E6/E7 mRNA detection and their combined application in cytological shunt diagnosis of low-grade squamous intraepithelial lesions].

Objective To explore the value of double labeling of P16/ki67, E6/E7 mRNA of human papillomavirus (HPV) and combined detection in shunt diagnosis of low-grade squamous intraepithelial lesions (LSIL) by thin-layer cervical cytology (TCT). Methods The study enrolled 239 patients who underwent colposcopy and biopsy within 4 weeks after primary TCT diagnosis. The remaining cytological samples were double-labeled with P16/ki67 immunocytochemical staining and the HPV E6/E7 mRNA was detected by Panther automatic HPV E6/E7 mRNA detection system. Using SPSS22.0 software, the positive rates of P16/ki67 double-labeling, HPV E6/E7 mRNA and combined detection were analyzed in different cervical lesions, and the positive rates in the same cervical lesions were compared horizontally to evaluate the efficiency of double labeling of P16/ki67, HPV E6/E7 and combined detection in the diagnosis of high-grade squamous intraepithelial lesions (HSIL) and above lesions. Results The diagnostic results of HE staining for the 239 cases of LSIL were 71 cases of chronic cervicitis (29.71%), 143 cases of LSIL (59.83%), 22 cases of HSIL (9.20%) and 3 cases of cervical cancer (1.26%). There were 46 cases of P16+ki67+ lesions (19.25%), 41 cases of ki67+P16- lesions (17.15%), 33 cases of ki67-P16+ lesions (13.81%) and 119 cases of P16-ki67- lesions (49.79%). The positive rates of P16/ki67 double-labeling, HPV E6/E7 mRNA and combined detection increased with the severity of cervical lesions. The positive rate of combined detection was the highest in the HSIL lesions, which was higher than that of P16/ki67 double-labeling and HPV E6/E7 mRNA detection. The sensitivity of combined detection was higher than that of P16/ki67 double-labeling and HPV E6/E7 mRNA detection. The Youden index of joint detection was 0.7850. Conclusion The combined detection of P16/ki67 double labeling, HPV E6/E7 mRNA and HPV E6/E7 mRNA had a certain clinical value in the management of cell LSIL shunt diagnosis. The combined detection significantly improved the sensitivity and Youden index of HSIL and above lesions, while maintaining a high specificity and coincidence rate.

Characterization of cervical biopsies of women with HIV and HPV co-infection using p16ink4a, ki-67 and HPV E4 immunohistochemistry and DNA methylation.

This study aims to characterize cervical intraepithelial neoplasia (CIN) in women living with HIV using biomarkers. Immunohistochemical (IHC) staining for human papillomavirus (HPV) E4 protein indicates CIN with productive HPV infection, whereas Ki-67 and p16ink4a indicate CIN with transforming characteristics, which may be further characterized using DNA hypermethylation, indicative for advanced transforming CIN. Cervical biopsies (n = 175) from 102 HPV positive women living with HIV were independently reviewed by three expert pathologists. The consensus CIN grade was used as reference standard. IHC staining patterns were scored for Ki-67 (0-3), p16ink4a (0-3), and E4 (0-2) and correlated to methylation levels of four cellular genes in corresponding cervical scrapes. Reference standards and immunoscores were obtained from 165 biopsies:15 no dysplasia, 91 CIN1, 31 CIN2, and 28 CIN3. Ki-67 and p16ink4a scores increased with increasing CIN grade, while E4 positivity was highest in CIN1 and CIN2 lesions. E4 positive CIN1 lesions had higher Ki-67 and p16ink4a scores and higher methylation levels compared with E4 negative CIN1 lesions. E4 positive biopsies with low cumulative Ki-67/p16 ink4a immunoscores (0-3) had significantly higher methylation levels compared with E4 negative biopsies. No significant differences in Ki-67 and p16ink4a scores and methylation levels were observed between E4 negative and positive CIN2 or CIN3 lesions. The presence of high methylation levels in scrapes of CIN lesions with IHC characteristics of both productive (E4 positive) and transforming infections (increased Ki-67/p16ink4a expression) in women living with HIV might indicate a rapid aggressive course of HPV infections towards cancer in these women.

Prognostic and diagnostic validity of p16/Ki-67, HPV E6/E7 mRNA, and HPV DNA in women with ASCUS: a follow-up study.

BACKGROUND: We evaluated the prognostic and diagnostic ability of p16/Ki-67 immunocytochemistry, HPV E6/E7 mRNA testing and HPV DNA assay in triaging ASCUS to find a way to manage cervical lesions more effectively. METHODS: We conducted a prospective study through follow-up. The detection methods of the three factors: p16/Ki-67 immunocytochemistry conducted by using the CINtec® Plus Kit, E6/E7 mRNA testing by QuantiVirus®HPV E6/E7 mRNA assay and DNA by Hybrid Capture 2 assay. RESULTS: One hundred three women with ASCUS satisfied requirements and completed the entire follow-up process. All CIN2+ occurred in women who were mRNA positive at baseline, none in mRNA negative. 100% (6/6) patients with CIN2+ were HPV DNA assay positive, 100% (6/6) were HPV E6/E7 mRNA testing positive and 50.0% (3/6) were p16/Ki-67 immunocytochemistry positive. The risk ratio of E6/E7 mRNA test was 57.306 (95% CI 0.077-42,400.545). For endpoint of CIN2+, the sensitivity between HPV DNA assay and HPV E6/E7 mRNA testing is no statistical difference, but statistical difference exists between HPV E6/E7 mRNA testing vs. p16/Ki-67 immunocytochemistry (χ2 = 5.718, P = 0.023) and HPV DNA assay vs. p16/Ki-67 immunocytochemistry (χ2 = 5.718, P = 0.023). The specificity of E6/E7 mRNA testing, p16/Ki-67 and DNA assay in triaging ASCUS was 44.33, 75.26 and 11.34% respectively and is all statistical difference (χ2 = 26.277, P < 0.001(HPV DNA assay vs. HPV E6/E7 mRNA testing), χ2 = 19.297, P < 0.001(HPV E6/E7 mRNA testing vs. p16/Ki-67 immunocytochemistry), χ2 = 80.707, P < 0.001(HPV DNA assay vs. p16/Ki-67 immunocytochemistry). The expression level of 2097.09 copies/ml was the optimal cut-off value for HPV E6/E7 mRNA testing to diagnose CIN2+, the sensitivity and specificity was 61.1 and 68.2%. CONCLUSIONS: High expression of HPV E6/E7 mRNA could be a good candidate as a diagnostic biomarker to triage ASCUS superseding HPV DNA. p16/Ki-67 immunocytochemistry is suggested to be a good tool to triage ASCUS, but it reduced the sensitivity of diagnosis when improves the diagnostic specificity.

Competitive adsorption on gold nanoparticles for human papillomavirus 16 L1 protein detection by LDI-MS.

The detection of the HPV L1 protein provides information about the infection status of the virus, reflects the replication status of the HPV virus in cervical cells, and helps understand the regression and progress of cervical lesions. Herein, we report a novel laser desorption ionization mass spectrometry (LDI MS) method for the sensitive detection of the HPV 16 L1 protein, based on non-covalent competitive adsorption between the HPV 16 L1 aptamer and melamine on gold nanoparticles (AuNPs). The intensity of the MS signal corresponding to the mass tag shows a linear relationship with the HPV 16 L1 concentration in the range 2-80 ng mL-1, with a limit of detection (LOD) of 58.8 pg mL-1. Using this method, the HPV 16 L1 protein is quantitatively analyzed in both clinical and vaccine samples. The described method is simple and has high sensitivity and good reliability.

Performance of OncoE6TM Cervical Test in detecting cervical precancer lesions in HIV-positive women attending an HIV clinic in Bujumbura, Burundi: a cross-sectional study.

OBJECTIVE: New rapid and low-cost molecular tests for cervical cancer screening, such as the OncoE6 Cervical Test, are emerging and could be alternatives for low-income and middle-income countries. To this end, we evaluated the clinical performance of the OncoE6 Cervical Test in detecting cervical intraepithelial neoplasia (CIN) among HIV-infected women in Bujumbura, Burundi. METHODS: From June to December 2017, a cross-sectional study was conducted in 680 HIV-positive women at the University Hospital. Women aged 25-65 years who declared having had vaginal intercourse were consecutively recruited, and cervical specimens for OncoE6, liquid-based cytology and human papillomavirus (HPV) genotyping were obtained and visual inspection with acetic acid performed. Thereafter, participants underwent a colposcopic examination. The sensitivity, specificity, and positive and negative predictive values of the different tests were calculated with reference to 'colposcopic-histological' diagnoses, and areas under the receiver operating curves of OncoE6 and cytology tests were compared. RESULTS: The prevalence of CIN was 4.9%, and OncoE6 positivity was 3.1%. OncoE6 sensitivity varied from poor to low with increasing disease severity (42.1%, 95% CI 19.9% to 64.3% at CIN2+ threshold; and 58.3%, 95% CI 30.4% to 86.2% at CIN3+ threshold). OncoE6 had the highest specificity compared with all other tests used together. The performance of the OncoE6 test was significantly lower compared with cytology at atypical squamous cell of undetermined significance (ASCUS+) cut-off (AUC=0.68 vs 0.85, p=0.03) and low-grade squamous intraepithelial lesion (LSIL+) cut-off (AUC=0.68 vs 0.83, p=0.04) for CIN2+ diagnoses. However, the performance of the OncoE6 test was similar to that of cytology at high-grade squamous intraepithelial lesion (HSIL+) cut-off (AUC=0.68 vs 0.76; p=0.30) for CIN2+ diagnoses and was also similar to that of cytology at all cut-offs (ASCUS+, LSIL+ and HSIL+) for CIN3+ diagnoses (p1=0.76, p2=0.95 and p3=0.50, respectively). CONCLUSION: The current OncoE6 test proved to be a point-of-care test. However, given its poor performance for CIN2+ diagnoses, we do not recommend it for primary screening. We recommend to enrich it with more oncogenic HPV types, which may improve the performance of the test akin to that of cytology.

Utility of human papillomavirus L1 capsid protein and HPV test as prognostic markers for cervical intraepithelial neoplasia 2+ in women with persistent ASCUS /LSIL cervical cytology.

Objective: Efficient and highly predictive biomarkers reflecting the prognosis of persistent atypical squamous cells of unknown significance(ASCUS) and low grade squamous intraepithelial lesion(LSIL)s are unavailable and need to be developed urgently. We aimed to develop a predictive model for diagnosis of cervical intraepithelial neoplasia(CIN)2+ by analyzing the immunocytochemical expression of the HPV L1 capsid protein in patients with persistent ASCUS and LSIL with a high risk of HPV infection. Methods: Cervical cytology samples comprising (70 ASCUS and 215 LSIL Pap smears) were analyzed. Immunocytochemical identification of the HPV L1 capsid protein in cervical cytology samples was performed. Expression levels of HPV L1 capsid protein in cervical cytology samples were measured, and the correlation between HPV L1 expression and cervical pathologic diagnosis was evaluated. The risk for CIN2+ was calculated using the results of immunocytochemistry and the HPV DNA test. Results: Negative results for HPV L1 immunochemistry test were more frequently observed in CIN2+, and expression of the HPV L1 capsid protein was higher in CIN1 or cervicitis (Fisher's exact test, p<0.05). Diagnosis rates for CIN2+ were highest for the combination of HPV L1 capsid protein immunocytochemistry, cytology and HPV test when compared with other combinations (Akaike information criterion (AIC): 191.7, Schwarz criterion(SC): 206.3, p<0.001). Conclusion: Absence of HPV L1 capsid expression and presence of HPV type 16 or 18 infection are reliable predictors of progression to CIN2+ in patients showing persistent ASCUS and LSIL.

Distribution of HPV-16 and HPV-18 from the Patients Attending At Mymensingh Medical College Hospital by Newly Developed Oncoprotein Detection Assay.

Cervical cancer is one of cause of death in women in many developing countries. Persistent infection with Human Papilloma Virus (HPV), primarily high risk types 16 and 18, is recognized as a causal and essential factor for the development of cervical cancer. The objective of this cross sectional observational study is to detect the distribution of HPV-16 and HPV-18 among Onco E6 positive cases. Following universal safety precautions a total of 180 endocervical swabs were collected from Colposcopy clinic of Obstetrics and Gynaecology Department of Mymensingh Medical College Hospital (MMCH), Mymensingh, Bangladesh from January 2016 to December 2016. Laboratory work was done in the department of Microbiology, Mymensingh Medical College. E6 strip test is an immunochromatographic test based on the detection of HPV-E6 oncoprotein in cervical swab samples. Onco E6 cervical test was done on 180cases. Among them 60% were VIA positive and 120% were VIA negative. From this VIA positive cases 12(16.25%) were On E6 cervical test positive and from VIA negative cases 3(2.5%) were positive by this On E6 cervical test. From this 12 Onco E6 cervical test positive cases 10(%) were HPV-16 and 2(%) were HPV-18 and from VIA negative cases 3 were only HPV-16 by this test. Histopathological test done on 35 suspected cases and out of 08 cervical carcinoma cases 07 were positive by this Onco E6 cervical test which was also HPV-16 type. It may be concluded that HPV-16 is most prevalent type to cause cervical cancer and by this newly developed protein detection assay will be helpful to reduce over treatment and save many lives.

Human papillomaviruses' proteins with clinical utility.

Cervical cancer, the fourth leading cause of cancer-associated deaths among women worldwide, is associated with human papilloma virus (HPV) infection. Despite the prophylactic HPV vaccination and the implementation of cervical and HPV-based screening programs, a significant increase in cervical cancer incidence is estimated by the year 2020. Thus, further development of diagnostic tools that allow detection and risk assesment in genital HPV infection is necessary. A special interest is focused on the HPV viral proteins whose expression might be of use either as primary screening tool or in conjunction with other markers (cellular proteins, HPV DNA, PAP test).

[The role of HPV E6/E7 mRNA combined with P16/ki67 immunocytochemistry in the diagnosis of atypical squamous cells of undetermined significance(ASCUS)].

Objective To investigate the diagnostic value of human papillomavirus (HPV) E6/E7 mRNA combined with P16/antigen KI-67(ki67) immunocytochemical double staining in the atypical squamous cells of undetermined significance (ASCUS). Methods A total of 272 patients were selected and the results of HPV E6/E7 and P16/ki67 immunocytochemical double staining in the remaining cytological specimens were retrospectively analyzed. HPV E6/E7 gene was detected by HPV E6/E7 gene detection kit and Panther molecular diagnostic instrument. P16/ki67 was detected by immunocytochemical staining and Ventana Benchmark Ultra immunohistochemical staining instrument. Then we analyzed the difference of positive rate between the two detection methods in the same grade of cervical epithelial lesions, explored the difference of the two detection methods and their combined detection in the diagnosis of high grade squamous intraepithelial lesions (HSIL), and finally evaluated the role of different detection methods in shunt diagnosis of ASCUS. Results Histopathological findings of cervical cytology ASCUS includes chronic cervicitis, low-grade squamous intraepithelial lesions (LSIL), HSIL and cervical cancer. The positive rate of simple molecular diagnosis or immunocytochemical staining increased with the severity of cervical lesions. In cervicitis and LSIL lesion group, the difference between the positive rates of the two methods was obvious, but in HSIL and cervical cancer lesion group, there was no significant difference between the positive rates of the two methods. The sensitivity, specificity, Yoden index, coincidence rate, positive predictive value and negative predictive value were 95.65%, 85.40%, 0.81, 87.13%, 57.14% and 98.97%, respectively. Conclusion The detection of HPV E6/E7 and P16/ki67 immunocytochemical staining has certain significance in ASCUS shunt diagnosis. The combined detection of HPV E6/E7 and P16/ki67 can significantly improve the sensitivity of shunt diagnosis and maintain a good specificity.

HPV E4 expression and DNA hypermethylation of CADM1, MAL, and miR124-2 genes in cervical cancer and precursor lesions.

In this study, we evaluate the expression of human papillomavirus E4 protein (marker for the onset of a productive infection) and hypermethylation of host-cell CADM1, MAL, and miR124-2 genes (marker for an advanced, transforming infection) in cervical intraepithelial neoplasia (CIN) and cancer. A total of 115 cervical lesions were categorized by 3 pathologists into no dysplasia, CIN1, CIN2, CIN3, or cancer by classical histomorphological grading criteria, and by an immunoscore (cumulative value: 0-6) grading system based on Ki-67 (score: 0-3) and p16ink4a (score: 0-3) expression. Lesions were immunostained for E4 protein and analyzed for hypermethylation of CADM1, MAL, or miR124-2 genes. Expression of E4 and hypermethylation levels were related to CIN grade based on both classical and immunoscore grading. Hypermethylation increased with severity of the lesion as defined by both classical histomorphological grading and immunoscore criteria, and was always present in carcinomas (22/22). Extensive E4 expression decreased with increasing CIN grade and immunoscore, being most frequent in classically graded CIN1 or in lesions with cumulative immunoscore 1-3 and absent in carcinomas. High-grade lesions (CIN2/3 or immunoscore: 4-6) showed less E4 expression, which was inversely related to an increasing hypermethylation. Extensive E4 expression, as observed in a small proportion of high-grade lesions (6/49 and 8/43, respectively), was mostly associated with a negative methylation marker status (5/6 and 7/8, respectively). Our results illustrate the gradual transition of productive CIN (reflected by extensive E4 expression), to advanced transforming CIN (reflected by extensive hypermethylation) and cancer. Expression patterns of E4 and hypermethylation status of host-cell genes, may be used to identify cervical lesions at risk for cervical cancer, providing a better guidance for clinicians on treatment decisions.

Self-sampling coupled to the detection of HPV 16 and 18 E6 protein: A promising option for detection of cervical malignancies in remote areas.

OBJECTIVE: To evaluate both the performance and acceptability of a method coupling self-sampling with detection of cervical malignancy via elevated HPV 16 and 18 E6 oncoproteins (OncoE6™ Cervical Test) in remote areas in Brazil. METHODS: Women living in rural villages in proximity to Coari city, Amazonas, Brazil were invited to participate in a cervical cancer screening study. 412 subjects were enrolled; there were no refusals. In addition to E6 protein detection, DNA was extracted from the brushes and evaluated for HPV genotypes by PCR (PGMY09/11), followed by typing by the Papillocheck™ if positive. Subjects who were found to be positive for OncoE6 or HPV-DNA were referred for colposcopy. RESULTS: For 110 subjects (27%) this was the first cervical cancer exam. Overall the HPV-DNA prevalence was 19.1% (n = 79); 1.4% (n = 6) were positive by the OncoE6 Test. Fifty-six women attended the invitation for colposcopy where nine had an abnormal cervix and were subsequently biopsied. Histopathological analysis revealed 2 CIN3, 2 carcinomas and 5 CIN1. OncoE6 called two out of the three HPV 16 or 18 associated CIN3+ lesions. CONCLUSIONS: The findings suggest that self-administered sample collection in combination with OncoE6 Test is feasible in this population. This could enable expanded screening coverage while ensuring a high specificity which is imperative given the remote geographic location, since women bearing abnormal test results would necessitate travel and logistical burden to access colposcopy and treatment.

Quantitative polymerase chain reaction-based detection of HPV 16 E6 and E7 DNA in oral squamous cell carcinoma.

BACKGROUND: Although human papillomavirus (HPV) is considered as a causative factor in oral squamous cell carcinoma (OSCCs), its pathogenetic role is not well established. Moreover, a limited number of studies have compared the techniques of detecting the HPV infection in OSCC. This study aimed at the detection of HPV 16 E6 and E7 DNA in OSCC by quantitative polymerase chain reaction (qPCR) technique. METHODOLOGY: This retrospective study included 297 tissue sections obtained from histopathologically confirmed OSCC patients. The classification of tumors as poorly differentiated, moderately differentiated and well differentiated was performed by H&E staining following the WHO criteria for OSCC. The presence of HPV infection was detected by p16INK4A expression, conventional PCR technique, HPV 16 E6, and E7 by qPCR and flow cytometry. All statistical analysis was performed using MedCalc software v.16.4.3. P < 0.05 is considered as statistically significant. RESULTS: Of 297 samples, 128 samples were found to be HPV-positive by p16. Of total 128 HPV-positive samples, PCR, E6, and E7 qPCR were positive in 19, 97, and 98 samples, respectively. qPCR techniques were found highly significant in the detection of moderately differentiated (P < 0.0001) and widely differentiated (P < 0.0001) cases. The positivity of E6 qPCR increased as the p16 expression increased. A significant variation in E6 DNA copies was observed in different grades of p16 expression (P < 0.0001). However, overall E7 (5.4 × 105 copies/µL) DNA copies were higher than E6 (7.7 × 103 copies/µL). CONCLUSION: qPCR detection of HPV infection is a fast, reliable, and accurate technique gives valuable information about the infection status in terms of viral load.

Performance of OncoE6 cervical test with collection methods enabling self-sampling.

BACKGROUND: The paradigm shift from cytological screening to Human Papillomavirus (HPV)-based screening for cervical cancer allows the introduction of new technologies in sample collection and diagnostics. The OncoE6™ Cervical Test (OncoE6 Test) is a rapid, easy-to-use lateral flow method detecting HPV16/18 E6 oncoproteins that has proven to detect high-grade cervical lesions with high specificity. If compatible with self-collection samples, this technology might allow for decentralized screening of hard-to-reach populations. METHODS: For technical validation, cervicovaginal lavages were collected from 20 patients with confirmed HPV16+ or HPV18+ invasive cervical cancer. Cervical smears were collected by polyester-tipped swabs and cytobrushes. All samples were applied to the OncoE6 Test and cytobrush samples additionally genotyped. RESULTS: Lavage, swab, and cytobrush revealed concordant outcome in 18/20 samples. HPV types corresponded with the HPV genotyping by GP5+/6+ PCR analyses. Due to a rare mutation found in the E6 antibody binding site one sample was not detected, another sample had very low cellularity. CONCLUSIONS: Overall, vaginal lavages are technically adequate for the OncoE6 Test. Combining self-sampling with oncoprotein rapid testing to detect women with highest risk for severe dysplasia or cancer may allow for secondary cancer prevention in settings where other screening modalities were unsuccessful to date.