ABSTRACT
Methylation markers have shown potential for triaging high-risk HPV-positive (hrHPV+) women to identify those at increased risk of invasive cervical cancer (ICC). Our aim was to assess the performance of the S5 DNA methylation classifier for predicting incident high-grade cervical intraepithelial neoplasia (CIN) and ICC among hrHPV+ women in the ARTISTIC screening trial cohort. The S5 classifier, comprising target regions of tumour suppressor gene EPB41L3 and L1 and L2 regions of HPV16, HPV18, HPV31, and HPV33, was assayed by pyrosequencing in archived hrHPV+ liquid-based samples from 343 women with high-grade disease (139 CIN2, 186 CIN3, and 18 ICC) compared to 800 hrHPV+ controls. S5 DNA methylation correlated directly with increasing severity of disease and inversely with lead time to diagnosis. S5 could discriminate between hrHPV+ women who developed CIN3 or ICC and hrHPV+ controls (p <.0001) using samples taken on average 5 years before diagnosis. This relationship was independent of cytology at baseline. The S5 test showed much higher sensitivity than HPV16/18 genotyping for identifying prevalent CIN3 (93% vs. 61%, p = .01) but lower specificity (50% vs. 66%, p <.0001). The S5 classifier identified most women at high risk of developing precancer and missed very few prevalent advanced lesions thus appearing to be an objective test for triage of hrHPV+ women. The combination of methylation of host and HPV genes enables S5 to combine the predictive power of methylation with HPV genotyping to identify hrHPV-positive women who are at highest risk of developing CIN3 and ICC in the future.
Subject(s)
DNA Methylation , Papillomavirus Infections , Uterine Cervical Dysplasia , Uterine Cervical Neoplasms , Humans , Female , Uterine Cervical Dysplasia/virology , Uterine Cervical Dysplasia/genetics , Uterine Cervical Dysplasia/diagnosis , Uterine Cervical Dysplasia/pathology , Uterine Cervical Neoplasms/virology , Uterine Cervical Neoplasms/genetics , Uterine Cervical Neoplasms/pathology , Uterine Cervical Neoplasms/diagnosis , Papillomavirus Infections/virology , Papillomavirus Infections/genetics , Papillomavirus Infections/complications , Adult , Middle Aged , Cohort Studies , Early Detection of Cancer/methods , Human papillomavirus 16/genetics , Human papillomavirus 16/isolation & purificationABSTRACT
Cervical cancer screening programs, including triage tests, need redesigning as human papillomavirus (HPV)-vaccinated women are entering the programs. Methylation markers offer a potential solution to reduce false-positive rates by identifying clinically relevant cervical lesions with progressive potential. In a nested case-control study, 9242 women who received the three-dose HPV16/18-vaccine at ages 12-15 or 18 in a community-randomized trial were included. Subsequently, they were re-randomized for either frequent or infrequent cervical cancer screening trials. Over a 15-year post-vaccination follow-up until 2022, 17 high-grade squamous intraepithelial lesion (HSIL) and 15 low-grade (LSIL) cases were identified at the 25-year screening round, alongside 371 age and community-matched HPV16/18-vaccinated controls. Methylation analyses were performed on cervical samples collected at age 25, preceding histologically confirmed LSIL or HSIL diagnoses. DNA methylation of viral (HPV16/18/31/33) and host-cell genes (EPB41L3, FAM19A4, and miR124-2) was measured, along with HPV-genotyping. No HPV16/18 HSIL cases were observed. The predominant HPV-genotypes were HPV52 (29.4%), HPV59/HPV51/HPV58 (each 23.5%), and HPV33 (17.7%). Methylation levels were generally low, with no significant differences in mean methylation levels of viral or host-cell genes between the LSIL/HSIL and controls. However, a significant difference in methylation levels was found between HSIL cases and controls when considering a combination of viral genes and EPB41L3 (p value = .0001). HPV-vaccinated women with HSIL had HPV infections with uncommon HPV types that very rarely cause cancer and displayed low methylation levels. Further investigation is warranted to understand the likely regressive nature of HSIL among HPV-vaccinated women and its implications for management.
ABSTRACT
CpG site methylation patterns have potential to improve differentiation of high-grade screening-detected cervical abnormalities. We assessed CpG differential methylation (DM) and differential variability (DV) in high-grade (CIN2+) vs. low-grade (≤CIN1) lesions. In ≤CIN1 (n=117) and CIN2+ (n=31) samples, cervical sample DNA underwent testing with Illumina HumanMethylation arrays. We assessed DM and DV of CpG methylation M values among nine cervical cancer-associated genes. We fit CpG-specific linear models and estimated empirical Bayes standard errors and false discovery rates (FDR). An exploratory epigenome-wide association study (EWAS) aimed to detect novel DM and DV CpGs (FDR<0.05) and Gene Ontology (GO) term enrichment. Compared to ≤CIN1, CIN2+ exhibited greater methylation at CCNA1 Cluster 1 (M value difference 0.24; 95% CI 0.04, 0.43) and RARB Cluster 2 (0.16; 95% CI 0.05, 0.28), and lower methylation at CDH1 Cluster 1 (-0.15; 95% CI -0.26, -0.04). CIN2+ exhibited lower variability at CDH1 Cluster 2 (variation difference -0.24; 95% CI -0.41, -0.05) and FHIT Cluster 1 (-0.30; 95% CI -0.50, -0.09). EWAS detected 3,534 DM and 270 DV CpGs. Forty-four GO terms were enriched with DM CpGs related to transcriptional, structural, developmental, and neuronal processes. Methylation patterns may help triage screening-detected cervical abnormalities and inform US screening algorithms.
ABSTRACT
This review is based on the recent EUROGIN scientific session: "Assessing risk of cervical cancer in the post-vaccination era," which addressed the demands of cervical intraepithelial neoplasia (CIN)/squamous intraepithelial lesion (SIL) triage now that the prevalence of vaccine-targeted oncogenic high-risk (hr) human papillomaviruses (HPVs) is decreasing. Change in the prevalence distribution of oncogenic HPV types that follows national HPV vaccination programs is setting the stage for loss of positive predictive value of conventional but possibly also new triage modalities. Understanding the contribution of the latter, most notably hypermethylation of cellular and viral genes in a new setting where most oncogenic HPV types are no longer present, requires studies on their performance in vaccinated women with CIN/SIL that are associated with nonvaccine HPV types. Lessons learned from this research may highlight the potential of cervical cells for risk prediction of all women's cancers.
Subject(s)
Papillomavirus Infections , Papillomavirus Vaccines , Uterine Cervical Dysplasia , Uterine Cervical Neoplasms , Female , Humans , Uterine Cervical Neoplasms/epidemiology , Uterine Cervical Neoplasms/prevention & control , Uterine Cervical Neoplasms/pathology , Papillomavirus Infections/complications , Papillomavirus Infections/epidemiology , Papillomavirus Infections/prevention & control , Predictive Value of Tests , Papillomavirus Vaccines/therapeutic use , Vaccination , Papillomaviridae/geneticsABSTRACT
ABSTRACT: Using data from the Human Papillomavirus (HPV) Infection in Men cohort study, we demonstrate HPV-16 methylation associations with persistent oral HPV infection, the obligate precursor to oropharyngeal cancer. Human papillomavirus type 16 persistence was significantly associated with methylation of HPV-16 L2 CpG-4268 (Wilcoxon P = 0.04), and methylation of HPV-16 E2 CpG Pos 4 (Wilcoxon P = 0.04).
Subject(s)
Oropharyngeal Neoplasms , Papillomavirus Infections , Male , Humans , Methylation , Human papillomavirus 16/genetics , Cohort Studies , Oropharyngeal Neoplasms/epidemiology , Oropharyngeal Neoplasms/genetics , Papillomavirus Infections/epidemiology , Papillomavirus Infections/complicationsABSTRACT
BACKGROUND: Methylation levels may be associated with and serve as markers to predict risk of progression of precancerous cervical lesions. We conducted an epigenome-wide association study (EWAS) of CpG methylation and progression to high-grade cervical intraepithelial neoplasia (CIN2 +) following an abnormal screening test. METHODS: A prospective US cohort of 289 colposcopy patients with normal or CIN1 enrollment histology was assessed. Baseline cervical sample DNA was analyzed using Illumina HumanMethylation 450K (n = 76) or EPIC 850K (n = 213) arrays. Participants returned at provider-recommended intervals and were followed up to 5 years via medical records. We assessed continuous CpG M values for 9 cervical cancer-associated genes and time-to-progression to CIN2+. We estimated CpG-specific time-to-event ratios (TTER) and hazard ratios using adjusted, interval-censored Weibull accelerated failure time models. We also conducted an exploratory EWAS to identify novel CpGs with false discovery rate (FDR) < 0.05. RESULTS: At enrollment, median age was 29.2 years; 64.0% were high-risk HPV-positive, and 54.3% were non-white. During follow-up (median 24.4 months), 15 participants progressed to CIN2+. Greater methylation levels were associated with a shorter time-to-CIN2+ for CADM1 cg03505501 (TTER = 0.28; 95%CI 0.12, 0.63; FDR = 0.03) and RARB Cluster 1 (TTER = 0.46; 95% CI 0.29, 0.71; FDR = 0.01). There was evidence of similar trends for DAPK1 cg14286732, PAX1 cg07213060, and PAX1 Cluster 1. The EWAS detected 336 novel progression-associated CpGs, including those located in CpG islands associated with genes FGF22, TOX, COL18A1, GPM6A, XAB2, TIMP2, GSPT1, NR4A2, and APBB1IP. CONCLUSIONS: Using prospective time-to-event data, we detected associations between CADM1-, DAPK1-, PAX1-, and RARB-related CpGs and cervical disease progression, and we identified novel progression-associated CpGs. IMPACT: Methylation levels at novel CpG sites may help identify individuals with ≤CIN1 histology at higher risk of progression to CIN2+ and inform risk-based cervical cancer screening guidelines.
Subject(s)
Papillomavirus Infections , Uterine Cervical Dysplasia , Uterine Cervical Neoplasms , Female , Humans , United States , Adult , Uterine Cervical Neoplasms/pathology , Prospective Studies , Epigenome , Early Detection of Cancer , DNA Methylation , Uterine Cervical Dysplasia/diagnosis , Papillomavirus Infections/complications , Papillomaviridae/genetics , Cell Adhesion Molecule-1/geneticsABSTRACT
The shift towards primary human papillomavirus (HPV)-based screening has necessitated the search for a secondary triage test that provides sufficient sensitivity to detect high-grade cervical intraepithelial neoplasia (CIN) and cancer, but also brings an improved specificity to avoid unnecessary clinical work and colposcopy referrals. We evaluated the performance of the previously described DNA-methylation test (S5) in detecting CIN3 and cancers from diverse geographic settings in high-, medium- and low-income countries, using the cut-off of 0.80 and exploratory cut-offs of 2.62 and 3.70. Assays were performed using exfoliated cervical specimens (n = 808) and formalin-fixed biopsies (n = 166) from women diagnosed with cytology-negative results (n = 220), CIN3 (n = 204) and cancer stages I (n = 245), II (n = 249), III (n = 28) and IV (n = 22). Methylation increased proportionally with disease severity (Cuzick test for trend, P < .0001). S5 accurately separated women with negative-histology from CIN3 or cancer (P < .0001). At the 0.80 cut-off, 543/544 cancers were correctly identified as S5 positive (99.81%). At cut-off 3.70, S5 showed a sensitivity of 95.77% with improved specificity. The S5 odds ratios of women negative for cervical disease vs CIN3+ were significantly higher than for HPV16/18 genotyping at all cut-offs (all P < .0001). At S5 cut-off 0.80, 96.15% of consistently high-risk human papillomavirus (hrHPV)-negative cancers (tested with multiple hrHPV-genotyping assay) were positive by S5. These cancers may have been missed in current primary hrHPV-screening programmes. The S5 test can accurately detect CIN3 and malignancy irrespective of geographic context and setting. The test can be used as a screening and triage tool. Adjustment of the S5 cut-off can be performed considering the relative importance given to sensitivity vs specificity.
Subject(s)
Biomarkers, Tumor/genetics , DNA Methylation , Human papillomavirus 16/isolation & purification , Human papillomavirus 18/isolation & purification , Papillomavirus Infections/complications , Uterine Cervical Dysplasia/diagnosis , Uterine Cervical Neoplasms/diagnosis , Adult , Cross-Sectional Studies , Early Detection of Cancer , Female , Follow-Up Studies , Global Health , Humans , Middle Aged , Papillomavirus Infections/virology , Prognosis , Retrospective Studies , Uterine Cervical Neoplasms/epidemiology , Uterine Cervical Neoplasms/genetics , Young Adult , Uterine Cervical Dysplasia/epidemiology , Uterine Cervical Dysplasia/geneticsABSTRACT
Methylation of host and viral genes is promising for triage of women with high-risk human papillomavirus infections (hrHPV). Using a population-based sample of hrHPV positive women with cervical biopsies within 12 months after cervical screening, the clinical value of the S5 methylation classifier (S5), HPV genotyping and cytology were compared as potential triage tests, for outcomes of cervical intraepithelial neoplasia (CIN) grade 3 or greater (CIN3+), CIN2+ and CIN2, and the area under the curve (AUC) calculated. S5 scores increased with histopathology severity (Ptrend < .001). For CIN3+, the AUC was 0.780 suggesting S5 provides good discrimination between Subject(s)
Papillomavirus Infections
, Uterine Cervical Dysplasia
, Uterine Cervical Neoplasms
, DNA Methylation
, Early Detection of Cancer
, Female
, Human papillomavirus 16/genetics
, Human papillomavirus 18/genetics
, Humans
, Papillomaviridae/genetics
, Papillomavirus Infections/complications
, Papillomavirus Infections/diagnosis
, Papillomavirus Infections/genetics
, Triage
, Uterine Cervical Neoplasms/diagnosis
, Uterine Cervical Neoplasms/genetics
, Uterine Cervical Neoplasms/pathology
, Uterine Cervical Dysplasia/diagnosis
, Uterine Cervical Dysplasia/genetics
, Uterine Cervical Dysplasia/pathology
ABSTRACT
The S5-methylation test, an alternative to cytology and HPV16/18 genotyping to triage high-risk HPV-positive (hrHPV+) women, has not been widely validated in low-middle-income countries (LMICs). We compared S5 to HPV16/18 and cytology to detect cervical intraepithelial neoplasia Grade 2 or worse (CIN2+) and CIN3+ in hrHPV+ women selected from a randomized pragmatic trial of 2661 Colombian women with an earlier-borderline abnormal cytology. We included all hrHPV+ CIN2 and CIN3+ cases (n = 183) age matched to 183 Subject(s)
Early Detection of Cancer/methods
, Papillomavirus Infections/complications
, Uterine Cervical Dysplasia/diagnosis
, Uterine Cervical Neoplasms/diagnosis
, Adult
, Aged
, Atypical Squamous Cells of the Cervix/pathology
, Atypical Squamous Cells of the Cervix/virology
, Colombia
, DNA Methylation
, Female
, Genes, Viral/genetics
, Humans
, Middle Aged
, Papillomavirus Infections/diagnosis
, Sensitivity and Specificity
, Uterine Cervical Neoplasms/pathology
, Uterine Cervical Neoplasms/virology
, Uterine Cervical Dysplasia/pathology
, Uterine Cervical Dysplasia/virology
ABSTRACT
BACKGROUND: There is no prognostic test to ascertain whether cervical intraepithelial neoplasias (CINs) regress or progress. The majority of CINs regress in young women, and treatments increase the risk of adverse pregnancy outcomes. We investigated the ability of a DNA methylation panel (the S5 classifier) to discriminate between outcomes among young women with untreated CIN grade 2 (CIN2). METHODS: Baseline pyrosequencing methylation and human papillomavirus (HPV) genotyping assays were performed on cervical cells from 149 women with CIN2 in a 2-year cohort study of active surveillance. RESULTS: Twenty-five lesions progressed to CIN grade 3 or worse, 88 regressed to less than CIN grade 1, and 36 persisted as CIN1/2. When cytology, HPV16/18 and HPV16/18/31/33 genotyping, and the S5 classifier were compared to outcomes, the S5 classifier was the strongest biomarker associated with regression vs progression. The S5 classifier alone or in combination with HPV16/18/31/33 genotyping also showed significantly increased sensitivity vs cytology when comparing regression vs persistence/progression. With both the S5 classifier and cytology set at a specificity of 38.6% (95% confidence interval [CI], 28.4-49.6), the sensitivity of the S5 classifier was significantly higher (83.6%; 95% CI, 71.9-91.8) than of cytology (62.3%; 95% CI, 49.0-74.4; P = 0.005). The highest area under the curve was 0.735 (95% CI, 0.621-0.849) in comparing regression vs progression with a combination of the S5 classifier and cytology, whereas HPV genotyping did not provide additional information. CONCLUSIONS: The S5 classifier shows high potential as a prognostic biomarker to identify progressive CIN2.
Subject(s)
Papillomaviridae , Papillomavirus Infections , Uterine Cervical Dysplasia , Uterine Cervical Neoplasms , Cohort Studies , Female , Human papillomavirus 16/genetics , Human papillomavirus 18 , Humans , Methylation , Papillomaviridae/genetics , Papillomavirus Infections/diagnosis , Pregnancy , Uterine Cervical Neoplasms/diagnosis , Uterine Cervical Dysplasia/diagnosisABSTRACT
Oropharyngeal cancer (OPC) incidence is increasing significantly among men and often requires intensive therapy causing significant morbidity. Early detection of OPC is needed, when monotherapy can be safely delivered with less treatment-associated morbidity, while maintaining high cure rates. We conducted a study of 101 pretreatment male OPC cases matched 1:1 to 101 disease-free controls for age and smoking history. Oral gargles were collected from cases and controls with additional biopsies or aspirates from cases. The HPV SPF10 -LiPA25 PCR assay was utilized for HPV genotyping. Methylation of three CpG sites (438, 427 and 425) in the EPB41L3 gene and methylation status of the L1 (6,367, 6,389), L2 (4,257, 4,262, 4,266, 4,269, 4,275, 4,282) and E2 (3,412, 3,415, 3,417, 3,433, 3,436) CpG sites of HPV 16 positive specimens was assessed by pyrosequencing. Significant correlations were observed between tumor and oral specimens for all methylation biomarkers (p < 0.01). EPB41L3 and HPV 16 L1, L2 and E2 methylation were significantly (p < 0.0001) higher among cases than controls, regardless of early vs. late disease. When HPV 16 genes and EPB41L3 methylation status were combined in a logistic regression analysis, a sensitivity of 70.3% and a specificity of 90.9% were observed for the detection of OPC from an oral gargle. Our data suggest that methylation biomarkers measured in oral gargles may have utility in identifying OPC early. Future studies are needed to replicate these findings and to inform additional biomarkers that can maximize specificity and sensitivity for early OPC detection.
Subject(s)
Biomarkers, Tumor/genetics , Early Detection of Cancer/methods , Microfilament Proteins/genetics , Oncogene Proteins, Viral/genetics , Oropharyngeal Neoplasms/diagnosis , Papillomavirus Infections/diagnosis , Adult , Aged , Case-Control Studies , CpG Islands/genetics , DNA Methylation , DNA, Viral/genetics , DNA, Viral/isolation & purification , Human papillomavirus 16/genetics , Human papillomavirus 16/isolation & purification , Humans , Male , Middle Aged , Mouth Mucosa/pathology , Mouth Mucosa/virology , Oropharyngeal Neoplasms/genetics , Oropharyngeal Neoplasms/pathology , Oropharyngeal Neoplasms/virology , Papillomavirus Infections/genetics , Papillomavirus Infections/pathology , Papillomavirus Infections/virology , Proof of Concept Study , Sensitivity and SpecificityABSTRACT
The evolution of precancerous cervical lesions is poorly understood. A widely held model of cervical intraepithelial neoplasia grade 3 (CIN3) development is sequential progression from normal through CIN1 and CIN2 to CIN3. Another hypothesis, the "molecular switch" model, postulates that CIN3 can evolve directly from human papillomavirus (HPV)-infected normal epithelium without progressing through CIN1 and CIN2. To shed light on this process, we compared DNA methylation of selected human biomarkers and HPV types in two groups of CIN1: CIN1 that were near or adjacent to CIN3 (adjacent-CIN1) and CIN1 that were the principal lesions with no CIN3 detected (principal-CIN1). 354 CIN (CIN1 and CIN3) and normal tissue areas were dissected and typed for HPV from 127 women who underwent loop electrosurgical excision procedures (LEEP). Methylation of genes EPB41L3 and the viral regions of HPV16-L1/L2, HPV18-L2, HPV31-L1, and HPV33-L2 were determined by a highly accurate quantitative pyrosequencing of bisulfite converted DNA. There was a significant trend of increased methylation with disease grade comparing normal to CIN1 and CIN3 (p < 0.0001). Adjacent-CIN1 predominantly shared the same HPV types as the CIN3, however, methylation differed substantially between adjacent-CIN1 and CIN3 (p = 0.008). In contrast diagnostically principal-CIN1 had an indistinguishable methylation distribution compared to adjacent-CIN1 (EPB41L3: p = 0.49; HPVme-All: p = 0.11). Our results suggest that progression from normal epithelium to CIN1 or CIN3 is usually promoted by the same HPV type but occurs via distinct DNA epigenotypes, thus favoring the "molecular switch" model.
Subject(s)
Biomarkers, Tumor/genetics , Cervix Uteri/metabolism , DNA Methylation , Epigenomics , Papillomavirus Infections/complications , Uterine Cervical Dysplasia/pathology , Uterine Cervical Neoplasms/pathology , Cervix Uteri/pathology , Cervix Uteri/virology , Disease Progression , Female , Follow-Up Studies , Gene Expression Regulation, Neoplastic , Humans , Papillomaviridae/isolation & purification , Papillomavirus Infections/genetics , Papillomavirus Infections/virology , Pilot Projects , Prognosis , Risk Factors , Uterine Cervical Neoplasms/genetics , Uterine Cervical Neoplasms/virology , Uterine Cervical Dysplasia/genetics , Uterine Cervical Dysplasia/virologyABSTRACT
Inflammation occurs as a result of exposure of tissues and organs to harmful stimuli such as microbial pathogens, irritants, or toxic cellular components. The primary physical manifestations of inflammation are redness, swelling, heat, pain, and loss of function to the affected area. These processes involve the major cells of the immune system, including monocytes, macrophages, neutrophils, basophils, dendritic cells, mast cells, T-cells, and B-cells. However, examination of a range of inflammatory lesions demonstrates the presence of specific leukocytes in any given lesion. That is, the inflammatory process is regulated in such a way as to ensure that the appropriate leukocytes are recruited. These events are in turn controlled by a host of extracellular molecular regulators, including members of the cytokine and chemokine families that mediate both immune cell recruitment and complex intracellular signalling control mechanisms that characterise inflammation. This review will focus on the role of the main cytokines, chemokines, and their receptors in the pathophysiology of auto-inflammatory disorders, pro-inflammatory disorders, and neurological disorders involving inflammation.
ABSTRACT
Introduction: DNA methylation is proposed as a novel biomarker able to monitor molecular events in human papillomavirus (HPV) infection pathophysiology, enabling the distinction between HPV-induced lesions with regression potential from those that may progress to HPV-related cancer. Methods: This meeting report summarises the presentations and expert discussions during the HPV Prevention and Control Board-focused topic technical meeting on DNA methylation validation in clinician-collected and self-collected samples, novel DNA methylation markers discovery, implementation in cervical cancer screening programs, and their potential in women living with human immunodeficiency virus (HIV). Results: Data presented in the meeting showed that HPV-positive, baseline methylation-negative women have a lower cumulative cervical cancer incidence than baseline cytology-negative women, making DNA methylation an attractive triage strategy. However, additional standardised data in different settings (low- versus high-income settings), samples (clinician-collected and self-collected), study designs (prospective, modelling, impact) and populations (immunocompetent women, women living with HIV) are needed. Conclusion: Establishing international validation guidelines were identified as the way forward towards accurate validation and subsequent implementation in current screening programs.
ABSTRACT
BACKGROUND: Topical cidofovir and imiquimod can effectively treat approximately 55% of patients with vulval intraepithelial neoplasia (VIN), thus avoiding the need for surgery. Human papillomavirus (HPV) Eâ¢2 gene methylation predicts response to treatment but a methylation measurement is only obtainable in approximately 50% of patients. OBJECTIVE: This work aimed to determine if the applicability and predictive power of the Eâ¢2 methylation assay could be improved by combining it with the components of a host and viral DNA methylation panel (S5) that has been found to predict disease progression in patients with cervical intraepithelial neoplasia. METHODS: HPV E2 methylation and S5 classifier score were measured in fresh tissue samples collected pre-treatment from 132 patients with biopsy-proven VIN grade 3 who participated in a multicentre clinical trial and were randomised to treatment with cidofovir or imiquimod. RESULTS: Combining HPV16 Eâ¢2 and HPV16 Lâ¢1 methylation provides a biomarker that is both predictive of response to topical treatment and that can produce a clinically applicable result for all patients. Patients with HPV 16 Lâ¢1^high and HPV 16 Eâ¢2^high (36/132 (27.3%)) were more likely to respond to treatment with cidofovir (12/15 (80.0%)) than imiquimod (9/21 (42.9%)) (p= 0.026). Patients with HPV 16 Lâ¢1^low or HPV 16 Eâ¢2^low (including those with no HPV/unassessable methylation) were more likely to respond to imiquimod: 23/50 (46.0%) vs 31/46 (67.4%) (p= 0.035). CONCLUSIONS: Combined HPV Eâ¢2 and Lâ¢1 methylation is a potential predictive marker in treatment for all patients with VIN. These findings justify validation in a prospective trial.
Subject(s)
Papillomavirus Infections , Uterine Cervical Neoplasms , Vulvar Neoplasms , Female , Humans , Imiquimod/therapeutic use , Cidofovir/therapeutic use , Prospective Studies , Aminoquinolines/therapeutic use , Aminoquinolines/adverse effects , Vulvar Neoplasms/drug therapy , Vulvar Neoplasms/genetics , Human papillomavirus 16/genetics , DNA Methylation , Biomarkers , Papillomavirus Infections/complications , Papillomavirus Infections/drug therapy , Papillomavirus Infections/genetics , Uterine Cervical Neoplasms/drug therapy , Uterine Cervical Neoplasms/geneticsABSTRACT
As oropharyngeal cancer (OPC) associated with human papillomavirus (HPV) increases in men, the need for a screening test to diagnose OPC early is crucial. While HPV-associated OPC has a favorable prognosis, recurrence is likely, and metastatic OPC is often incurable regardless of HPV status. Our previous study of pretreatment, male OPC cases (n = 101) and age- and smoking-matched controls (n = 101) found methylation of the host EPB41L3 tumor suppressor gene and HPV16 in the oral gargle was correlated with these biomarkers in the tumor. Methylation of these genes in the oral gargle was significantly (p < 0.0001) higher among cases compared to controls. To further study the utility of HPV16/EPB41L3 methylation, we expanded the sample size and specifically increased the number of early OPC cases (T1-T2, N0-N1; small tumors with a single ipsilateral node <3 cm) to evaluate these biomarkers in early and late OPC. This study included 228 OPC cases, 92 of which were early cases and frequency matched to 142 healthy controls. In logistic regression, the AUC for HPV16/EPB41L3 methylation for all OPC cases was 0.82. Among early and late OPC cases, the AUC was 0.78 and 0.85, respectively. For early cases, 76% sensitivity was achieved, replicating results from our prior study, with a specificity of 65%, indicating room for improvement. The ability of HPV16/EPB41L3 methylation to distinguish OPC from healthy controls highlights its utility as a potential biomarker for OPC. However, the inability to predict early OPC better than late stage OPC indicates the need for additional biomarkers to improve screening performance.
Subject(s)
Oropharyngeal Neoplasms , Papillomavirus Infections , Male , Humans , Human papillomavirus 16/genetics , Methylation , Papillomaviridae , Biomarkers , Microfilament Proteins/geneticsABSTRACT
Tumor necrosis factor receptor-associated periodic syndrome (TRAPS) is an autosomal dominant autoinflammatory condition caused by mutations in the TNFRSF1A gene which encodes the tumor necrosis factor (TNF) receptor, TNFR1. We investigated the effect of three high penetrance and three low penetrance TNFRSF1A mutations upon NF-κB transcription factor family subunit activity, and the resulting impact upon secretion of 25 different cytokines. Whilst certain mutations resulted in elevated NF-κB p65 subunit activity, others instead resulted in elevated c-Rel subunit activity. Interestingly, high p65 activity was associated with elevated IL-8 secretion, whereas high c-Rel activity increased IL-1ß and IL-12 secretion. In conclusion, while all six TNFRSF1A mutations showed enhanced NF-κB activity, different mutations stimulated distinct NF-κB family subunit activities, and this in turn resulted in the generation of unique cytokine secretory profiles.
Subject(s)
Cytokines/immunology , Proto-Oncogene Proteins c-rel/immunology , Receptors, Tumor Necrosis Factor, Type I/immunology , Transcription Factor RelA/immunology , Adult , Child , Female , Fever , Hereditary Autoinflammatory Diseases/blood , Hereditary Autoinflammatory Diseases/genetics , Hereditary Autoinflammatory Diseases/immunology , Humans , Immunoassay , Male , Middle Aged , Mutation , Proto-Oncogene Proteins c-rel/blood , Receptors, Tumor Necrosis Factor, Type I/genetics , Signal Transduction , Transcription Factor RelA/blood , Young AdultABSTRACT
Methylation biomarkers are promising tools for diagnosis and disease prevention. The S5 classifier is aimed at the prevention of cervical cancer by the early detection of cervical intraepithelial neoplasia (CIN). S5 is based on pyrosequencing a promoter region of EPB41L3 and five late regions of HPV types 16, 18, 31, and 33 following bisulfite conversion of DNA. Good biomarkers should perform well in a variety of sample types such as exfoliated cells, fresh frozen or formalin-fixed paraffin-embedded (FFPE) materials. Here, we tested the performance of S5 on 315 FFPE biopsies with paired exfoliated cervical samples using four different conversion kits (Epitect Bisulfite, Epitect Fast Bisulfite, EZ DNA Methylation, and EZ DNA Methylation-Lightning). The S5 values from FFPE biopsies for all kits were significantly correlated with those obtained from their paired exfoliated cells. For the EZ DNA Methylation kit, we observed an average increased methylation of 4.4% in FFPE. This was due to incomplete conversion of DNA (73% for FFPE vs. 95% for cells). The other kits had a DNA conversion rate in FFPE similar to the cells (95%-97%). S5 performed well at discriminating Subject(s)
DNA Methylation
, Uterine Cervical Dysplasia/pathology
, Uterine Cervical Neoplasms/pathology
, Adolescent
, Adult
, Aged
, Alphapapillomavirus/genetics
, Area Under Curve
, Biopsy/methods
, Colposcopy
, Female
, Formaldehyde
, Humans
, Microfilament Proteins/genetics
, Middle Aged
, Precancerous Conditions/genetics
, Precancerous Conditions/pathology
, Sensitivity and Specificity
, Sulfites
, Tissue Fixation
, Uterine Cervical Neoplasms/genetics
, Young Adult
, Uterine Cervical Dysplasia/genetics
ABSTRACT
INTRODUCTION: Urine self-sampling for human papillomavirus (HPV)-based cervical cancer screening is a non-invasive method that offers several logistical advantages and high acceptability, reducing barriers related to low screening coverage. This study developed and evaluated the performance of a low-cost urine self-sampling method for HPV-testing and explored the acceptability and feasibility of potential implementation of this alternative in routine screening. METHODS: A series of sequential laboratory assays examined the impact of several pre-analytical conditions for obtaining DNA from urine and subsequent HPV detection. Initially, we assessed the effect of ethylaminediaminetetraacetic acid (EDTA) as a DNA preservative examining several variables including EDTA concentration, specimen storage temperature, time between urine collection and DNA extraction, and first-morning micturition versus convenience sample collection. We further evaluated the agreement of HPV-testing between urine and clinician-collected cervical samples among 95 women. Finally, we explored the costs of self-sampling supplies as well as the acceptability and feasibility of urine self-sampling among women and healthcare workers. RESULTS: Our results revealed higher DNA concentrations were obtained when using a 40mM EDTA solution, storing specimens at 25°C and extracting DNA within 72 hrs. of urine collection, regardless of using first-morning micturition or a convenience sampling. We observed good agreement (Kappa = 0.72) between urine and clinician-collected cervical samples for HPV detection. Furthermore, urine self-sampling was an affordable method (USD 1.10), well accepted among cervical cancer screening users, healthcare workers, and decision-makers. CONCLUSION: These results suggest urine self-sampling is feasible and appropriate alternative for HPV-testing in HPV-based screening programs in lower-resource contexts.
Subject(s)
Alphapapillomavirus , DNA, Viral , Early Detection of Cancer , Papillomavirus Infections , Urine Specimen Collection , Uterine Cervical Neoplasms , Adult , Alphapapillomavirus/genetics , Alphapapillomavirus/metabolism , Cervix Uteri/metabolism , Cervix Uteri/virology , DNA, Viral/genetics , DNA, Viral/urine , Female , Humans , Middle Aged , Papillomavirus Infections/diagnosis , Papillomavirus Infections/genetics , Papillomavirus Infections/urine , Papillomavirus Infections/virology , Uterine Cervical Neoplasms/diagnosis , Uterine Cervical Neoplasms/urine , Uterine Cervical Neoplasms/virologyABSTRACT
INTRODUCTION: Squamous cell carcinoma of the anus is a common cancer among sexual minority men, especially HIV-positive sexual minority men; however, there is no evidenced-based national screening protocol for detection of anal precancers. Our objective is to determine compliance with annual anal canal self-sampling or clinician-sampling for human papillomavirus (HPV) DNA. METHODS AND ANALYSIS: This is a prospective, randomised, two-arm clinical study to evaluate compliance with annual home-based versus clinic-based HPV DNA screening of anal canal exfoliated cells. The setting is primary care community-based clinics. Recruitment is ongoing for 400 HIV-positive and HIV-negative sexual minority men and transgender persons, aged >25 years, English or Spanish speaking, no current use of anticoagulants other than nonsteroidal anti-inflammatory drugs and no prior diagnosis of anal cancer. Participants are randomised to either receive a swab in the mail for home-based collection of an anal canal specimen at 0 and 12 months (arm 1) or attend a clinic for clinician collection of an anal canal specimen at 0 and 12 months (arm 2). Persons will receive clinic-based Digital Anal Rectal Examinations and high-resolution anoscopy-directed biopsy to assess precancerous lesions, stratified by study arm. Anal exfoliated cells collected in the study are assessed for high-risk HPV persistence and host/viral methylation. The primary analysis will use the intention-to-treat principle to compare the proportion of those who comply with 0-month and 12-month sampling in the home-based and clinic-based arms. The a priori hypothesis is that a majority of persons will comply with annual screening with increased compliance among persons in the home-based arm versus clinic-based arm. ETHICS AND DISSEMINATION: The study has been approved by the Medical College of Wisconsin Human Protections Committee. Results will be disseminated to communities where recruitment occurred and through peer-reviewed literature and conferences. TRIAL REGISTRATION NUMBER: NCT03489707.