Long-term prediction by DNA methylation of high-grade cervical intraepithelial neoplasia: Results of the ARTISTIC cohort.

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.

Mimush Sheep and the Spectre of Inbreeding: Historical Background for Festetics's Organic and Genetic Laws Four Decades Before Mendel's Experiments in Peas.

The upheavals of late eighteenth century Europe encouraged people to demand greater liberties, including the freedom to explore the natural world, individually or as part of investigative associations. The Moravian Agricultural and Natural Science Society, organized by Christian Carl André, was one such group of keen practitioners of theoretical and applied scientific disciplines. Headquartered in the "Moravian Manchester" Brünn (nowadays Brno), the centre of the textile industry, society members debated the improvement of sheep wool to fulfil the needs of the Habsburg armies fighting in the Napoleonic Wars. Wool, as the raw material of soldiers' clothing, could influence the war's outcome. During the early nineteenth century, wool united politics, economics, and science in Brno, where breeders and natural scientists investigated the possibilities of increasing wool production. They regularly discussed how "climate" or "seed" characteristics influenced wool quality and quantity. Breeders and academics put their knowledge into immediate practice to create sheep with better wool traits through consanguineous matching of animals and artificial selection. This apparent disregard for the incest taboo, however, was viewed as violating natural laws and cultural norms. The debate intensified between 1817 and 1820, when a Hungarian veteran soldier, sheep breeder, and self-taught natural scientist, Imre (Emmerich) Festetics, displayed his inbred Mimush sheep, which yielded wool extremely well suited for the fabrication of light but strong garments. Members of the Society questioned whether such "bastard sheep" would be prone to climatic degeneration, should be regarded as freaks of nature, or could be explained by natural laws. The exploration of inbreeding in sheep began to be distilled into hereditary principles that culminated in 1819 with Festetics's "laws of organic functions" and "genetic laws of nature," four decades before Gregor Johann Mendel's seminal work on heredity in peas.

DNA methylation testing with S5 for triage of high-risk HPV positive women.

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

Clinical performance of methylation as a biomarker for cervical carcinoma in situ and cancer diagnosis: A worldwide study.

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.

A Randomized Comparison of Different Vaginal Self-sampling Devices and Urine for Human Papillomavirus Testing-Predictors 5.1.

BACKGROUND: Human papillomavirus (HPV)-based screening is rapidly replacing cytology as the cervical screening modality of choice. In addition to being more sensitive than cytology, it can be done on self-collected vaginal or urine samples. This study will compare the high-risk HPV positivity rates and sensitivity of self-collected vaginal samples using four different collection devices and a urine sample. METHODS: A total of 620 women referred for colposcopy were invited to provide an initial stream urine sample collected with the Colli-Pee device and take two vaginal self-samples, using either a dry flocked swab (DF) and a wet dacron swab (WD), or a HerSwab (HS) and Qvintip (QT) device. HPV testing was performed by the BD Onclarity HPV Assay. RESULTS: A total of 600 vaginal sample pairs were suitable for analysis, and 505 were accompanied by a urine sample. Similar positivity rates and sensitivities for CIN2+ and CIN3+ were seen for DF, WD, and urine, but lower values were seen for QT and HS. No clear user preferences were seen between devices, but women found urine easiest to collect, and were more confident they had taken the sample correctly. The lowest confidence in collection was reported for HS. CONCLUSIONS: Urine, a DF swab, and WD swab all performed well and were well received by the women, whereas the Qvintip and HerSwab devices were less satisfactory. IMPACT: This is the first study to compare five self-sampling methods in the same women taken at the same time. It supports wider use of urine or vaginal self-sampling for cervical screening.

Adjunctive testing by cytology, p16/Ki-67 dual-stained cytology or HPV16/18 E6 oncoprotein for the management of HPV16/18 screen-positive women.

High-risk human papillomavirus type 16/18 (HPV16/18) genotyping is unable to accurately discriminate nonprogressive infections from those that will progress to cervical cancer. Our study aimed to assesses if additional testing either with liquid-based cytology (LBC) or the putative progression markers p16/Ki-67 and HPV16/18 E6 oncoprotein (E6) can improve the efficiency of HPV16/18 genotyping for triaging high-risk HPV (hrHPV)-positive women through better cancer risk stratification. Women attending colposcopy after positive HPV16/18 genotyping results within the Forwarding Research for Improved Detection and Access for Cervical Cancer Screening and Triage (FRIDA) hrHPV-based screening study in Tlaxcala, Mexico, underwent further testing with LBC, p16/Ki-67 dual-stained (DS) cytology and E6. We calculated measures of test performance for detecting histologically confirmed cervical intraepithelial neoplasia grade 2 or higher (CIN2+) and grade 3 or higher (CIN3+). A number of 475 (64.3%) of 739 HPV16/18-positive women had complete results for all tests. Triage positivity rates were 14.1%, 18.5% and 24.4%, for LBC, E6 and DS, respectively. Compared with LBC, DS had higher sensitivity (24.4% vs 60.0%) although lower specificity (87.0% vs 79.3%) for CIN3+ (P < .001), whereas E6 had a sensitivity of 37.8% and a specificity of 83.5%. No invasive cancer was missed by DS or E6, but 75% were in normal cytology. DS test was associated with nearly 75% reduction of colposcopy referrals compared with the direct referral of all HPV16/18-positive women, giving the least number of colposcopies (n = 4.3) per CIN3+ detected. We show that adjunctive testing of HPV16/18-positive women with DS may greatly reduce unnecessary colposcopy referrals within HPV-based screening employing HPV16/18 genotyping while retaining acceptable sensitivity for CIN2+ and CIN3+.

Effective methylation triage of HPV positive women with abnormal cytology in a middle-income country.

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

Methylation of HPV 16 and EPB41L3 in oral gargles: Associations with oropharyngeal cancer detection and tumor characteristics.

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.

Comparison of HPV-16 and HPV-18 Genotyping and Cytological Testing as Triage Testing Within Human Papillomavirus-Based Screening in Mexico.

Importance: Triage tests enhance the efficiency cervical cancer screening based on human papillomavirus (HPV), but the best approach for maximizing programmatic effectiveness is still uncertain, particularly in a real-world scenario. Objective: To compare the clinical performance of 6 triage strategies based on liquid-based cytology (LBC) and HPV-16 and HPV-18 genotyping individually or in combination as sequential triage tests to detect cervical intraepithelial neoplasia (CIN) grade 2 or higher among women with high-risk HPV. Design, Setting, and Participants: This diagnostic study of routine cervical cancer screening was conducted at 100 primary health centers in Tlaxcala, Mexico. Women aged 30 to 64 years were recruited from August 1, 2013, to February 24, 2016, as part of the Forwarding Research for Improved Detection and Access for Cervical Cancer Screening and Triage study. Six triage scenarios for referral to colposcopy were examined: (1) LBC testing that found atypical squamous cells of undetermined significance (ASC-US) or worse, (2) positive results in HPV-16 genotyping, (3) positive results in HPV-18 genotyping, (4) positive results in HPV-16/HPV-18 genotyping, (5) positive results in HPV-16 genotyping or, if genotyping results were negative, reflex LBC testing that found ASC-US or worse, and (6) positive results in HPV-16/HPV-18 genotyping or, if genotyping results were negative, reflex LBC testing that found ASC-US or worse. Data were analyzed from October 2017 to August 2018. Exposures: Liquid-based cytological testing with simultaneous HPV-16 and HPV-18 genotyping. Women whose HPV genotyping results were positive for HPV-16 or HPV-18 or whose LBC results found ASC-US or worse and a random set of negative and normal results were referred to colposcopy with histologic analysis used for disease confirmation. Main Outcomes and Measures: Clinical performance of each test strategy for detection of CIN grade 2 or higher. Secondary outcomes included resource utilization of each triage scenario, measured by the number of tests performed, the referral rate for colposcopy, and the numbers of colposcopies per CIN grade 2 or higher detected. Results: A total of 36 212 women (median [interquartile range] age, 40 [35-47] years) were screened, and 4051 women (11.2%) had high-risk HPV. Of these women, 1109 (24.6%) were found to have HPV-16, HPV-18, or ASC-US or worse. Further histologic testing detected CIN grade 2 or higher in 110 of 788 women (14.0%) who underwent follow-up colposcopy. Sensitivity and specificity for 3 main triage strategies were 42.9% and 74.0% for LBC; 58.3% and 54.4% for HPV-16/HPV-18 genotyping; and 86.6% and 34.0% for HPV-16/HPV-18 genotyping with reflex LBC. The referral rate to colposcopy was 29% for HPV-16/HPV-18 with reflex LBC, which was 2-fold higher than the referral rate of 12% for LBC. Conclusions and Relevance: Triage of women with high-risk HPV with HPV-16/HPV-18 genotyping with reflex LBC was significantly associated with improvement in detection of CIN grade 2 or higher compared with LBC alone. The benefit of disease prevented may outweigh the cost of increasing requirements for colposcopy services in settings with limited adherence to follow-up after a positive screening result.

Methylation estimates the risk of precancer in HPV-infected women with discrepant results between cytology and HPV16/18 genotyping.

BACKGROUND: Vigilant management of women with high-risk human papillomavirus (hrHPV) is necessary in cancer screening programs. To this end, we evaluated the performance of S5 (targeting DNA methylation in HPV16, HPV18, HPV31, HPV33, and human gene EPB41L3) to predict cervical intraepithelial neoplasia grade 2 or higher (CIN2+) in a sample of hrHPV-infected women referred to colposcopy in the FRIDA Study, a large screening trial in Mexico. A nested case-control sample with women referred to colposcopy either by atypical squamous cells of undetermined significance or higher (ASCUS+) in cytology and/or positive for HPV types 16 or 18 was tested by S5. Seventy-nine cases of CIN2+ were age-matched to 237 controls without a diagnosis of CIN2+ (

The emergence of genetics from Festetics' sheep through Mendel's peas to Bateson's chickens.

It is now common knowledge-but also a misbelief-that in 1905 William Bateson coined the term 'genetics' for the first time in his letter to Adam Sedgwick. This important term was already formulated 81 years ago in a paper written by a sheepbreeding noble called Imre (Emmerich) Festetics, who still remains somewhat mysterious even today. The articles written by Festetics summarized the results of a series of lasting and elegant breeding experiments he had conducted on his own property. Selecting the best rams, Festetics had painstakingly crossed and backcrossed his best sheep to reach better wool quality. These experiments later turned out to reveal a better understanding of inheritance outlining genetics as a new branch of natural sciences.

Human Papillomavirus Research: Where Should We Place Our Bets?

BACKGROUND: Massive strides have been made with respect to primary and secondary prevention of human papillomavirus (HPV)-associated disease as a result of prophylactic vaccination and cervical screening based on molecular HPV testing. However, cervical cancer continues to be an important clinical and societal burden. Additionally, other HPV-associated cancers, for which there are no screening programmes, are rising. Finally, the optimal combination of vaccination and screening strategies will require careful thinking. Considering this unprecedented and important time, we were keen to solicit the views of the expert community to determine what they perceived were the key priorities for HPV research. Our objective was to identify consensus and key priorities for HPV-based research through provision of a questionnaire disseminated to a multidisciplinary group of key opinion leaders (KOLs). SUMMARY: A structured survey composed of 46 HPV research "categories" was sent to 73 KOLs who were invited to "rank" the categories according to priority. The invitees represented clinical and public health disciplines as well as basic scientists. Scores were weighted according to the number of responses. Invitees also had the opportunity to comment on barriers to the research and suggest other research areas that required attention not reflected in the survey. We received 29 responses in total; overall, the 3 highest-ranked categories were "optimal cervical screening in low and middle-income countries (LMICs)," "primary disease prevention in LMICs" and "impact of vaccine on HPV infection and associated disease." "HPV and the microbiome" and "mechanisms of transformation" were the highest-ranked categories with respect to basic research. Consistent barriers to research were around governance on the use of samples and data and funding, particularly in an era of vaccination. Key Messages: Research to support the management of disease in LMICs is clearly perceived as a priority in the international community in addition to other diverse areas which necessitate an improved basic understanding of viral mechanisms and interactions. International, multidisciplinary efforts which articulate the broader HPV research agenda will be important when seeking funding in addition to international endeavours to support the efficient use of existing samples and cohorts to facilitate such research.

Evaluation of a validated methylation triage signature for human papillomavirus positive women in the HPV FOCAL cervical cancer screening trial.

Human papillomavirus (HPV)-based cervical cancer screening requires triage of HPV positive women to identify those at risk of cervical intraepithelial neoplasia grade 2 (CIN2) or worse. We conducted a blinded case-control study within the HPV FOCAL randomized cervical cancer screening trial of women aged 25-65 to examine whether baseline methylation testing using the S5 classifier provided triage performance similar to an algorithm relying on cytology and HPV genotyping. Groups were randomly selected from women with known HPV/cytology results and pathology outcomes. Group 1: 104 HPV positive (HPV+), abnormal cytology (54 CIN2/3; 50

Molecular progression to cervical precancer, epigenetic switch or sequential model?

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.

Methylation of HPV and a tumor suppressor gene reveals anal cancer and precursor lesions.

We studied DNA methylation patterns of human papillomavirus (HPV) and tumor suppressor gene EPB41L3 in 148 anal and perianal biopsies to determine whether high levels of methylation would be associated with anal intraepithelial neoplasia (AIN). The most prevalent HPV type was HPV16, detected in 54% of the 30 benign biopsies, 33% of the 43 low-grade AIN (lgAIN), 82% of the 59 high grade AIN (hgAIN) and 4 of the 5 anal cancers. A methylation score was developed (0.561*HPV16me+0.439*EPB41L3) which had increasing values with severity of disease: the mean was 8.1% in benign, 13.2% in lgAIN, 22.3% in hgAIN and 49.3% in cancers (p < 0.0001). The methylation score as a triage classifier at a cut-off of 8.8 gave a sensitivity of 90.6% (95% CI: 82.8, 96.9), specificity of 50.7% (95% CI: 39.7, 61.6) and area under the curve of 0.82 (95% CI: 0.75-0.89) for separating hgAIN and cancer from benign and lgAIN biopsies. We conclude that methylation of HPV16 and EPB41L3 show highly significant association with increasing severity of AIN and cancer and may be useful as biomarkers in anal disease.

Role of quantitative p16INK4A mRNA assay and digital reading of p16INK4A immunostained sections in diagnosis of cervical intraepithelial neoplasia.

Visual interpretation of cervical biopsies is subjective and variable, generally showing fair to moderate inter-reader agreement in distinguishing high from low grade cervical intraepithelial neoplasia (CIN). We investigated the performance of two objective p16 quantitative tests in comparison with visual assessment: (i) p16-mRNA assay and (ii) digital analysis of sections stained for p16 protein. The primary analysis considered 232 high-risk human papilloma virus positive (HPV+) samples from diagnostic cervical specimens. A p16 RT-qPCR (p16-mRNA assay) was run on mRNA extracted from formalin-fixed paraffin-embedded sections. Two p16 immunohistochemistry (IHC) readings, a visual read by a histopathologist (Visual IHC) and a digital read of a high-resolution scan (Digital IHC), were done on adjacent sections. The worst reviewed CIN grade (agreed by at least two histopathologists) from up to two biopsies and a loop excision was taken, with CIN2/3 as the primary endpoint. Visual IHC attained a specificity of 70% (95%CI 61-77) for 85% (95%CI 77-91%) sensitivity. The four-point Visual IHC staining area under the curve (AUC) was 0.77 (95%CI 0.71-0.82), compared with 0.71 (95%CI 0.64-0.77) for p16-mRNA and 0.67 (95%CI 0.60-0.74) for Digital IHC. Spearman rank-order correlations were: visual to p16-mRNA 0.41, visual to digital 0.49 and p16-mRNA to digital: 0.22. The addition of p16-mRNA assay to visual reading of p16 IHC improved the AUC from 0.77 to 0.84 (p = 0.0049). p16-mRNA testing may be complementary to visual IHC p16 staining for a more accurate diagnosis of CIN, or perhaps a substitute in locations with a lack of skilled pathologists.

Virtues and Weaknesses of DNA Methylation as a Test for Cervical Cancer Prevention.

Epigenetics is the study of heritable and non-heritable genetic coding that is additive to information contained within classical DNA base pair sequences. Differential methylation has a fundamental role in the development and outcome of malignancies, chronic and degenerative diseases and aging. DNA methylation can be measured accurately and easily via various molecular methods and has become a key technology for research and healthcare delivery, with immediate roles in the elucidation of disease natural history, diagnostics and drug discovery. This review focuses on cancers of the lower genital tract, for which the most epigenetic information exists. DNA methylation has been proposed as a triage for women infected with human papillomavirus (HPV) and may eventually directly complement or replace HPV screening as a one-step molecular diagnostic and prognostic test. Methylation of human genes is strongly associated with cervical intraepithelial neoplasia (CIN) and cancer. Of the more than 100 human methylation biomarker genes tested so far in cervical tissue, close to 20 have been reported in different studies, and approximately 10 have been repeatedly shown to have elevated methylation in cervical cancers and high-grade CIN (CIN2 and CIN3), most prominently CADM1, EPB41L3, FAM19A4, MAL, miR-124, PAX1 and SOX1. Obtaining consistent performance data from the literature is quite difficult because most methylation studies used a variety of different assay methodologies and had incomplete and/or biased clinical specimen sets, varying assay thresholds and disparate target gene regions. There have been relatively few validation studies of DNA methylation biomarkers in large population-based screening studies, but an encouraging development more recently is the execution of well-designed studies to test the true performance of the markers in real-world settings. Methylation of HPV genes, especially HPV16, HPV18, HPV31, HPV33 and HPV45, in disease progression has been a major focus of research. Elevated methylation of the HPV16 L1 and L2 open reading frames, in particular, is associated with CIN2, CIN3 and invasive cancer. Essentially all cancers have high levels of methylation for human genes and for driver HPV types, which suggests that quantitative methylation tests may have utility in predicting CIN2 and CIN3 that are likely to progress. It is still early in the process of development of methylation biomarkers, but already they are showing strong promise as a universal and systematic approach to molecular triage, applicable to all cancers, not just cancer of the cervix. DNA methylation testing is better than HPV genotyping triage and is competitive with or complementary to other approaches such as cytology and p16 staining. Genome-wide studies are underway to systematically expand methylation classifier panels and find the best combinations of biomarkers. Methylation testing is likely to show big improvements in performance in the next 5 years.

A novel DNA methylation score accurately predicts death from prostate cancer in men with low to intermediate clinical risk factors.

Clinically aggressive disease behavior is difficult to predict in men with low to intermediate clinical risk prostate cancer and methylation biomarkers may be a valuable adjunct for assessing the management of these patients. We set to evaluate the utility of DNA methylation to identify high risk disease in men currently considered as low or intermediate risk. DNA was extracted from formalin-fixed paraffin-embedded transurethral prostate resection tissues collected during the years 1990-96 in a watchful-waiting cohort of men in the UK. The primary end point was death of prostate cancer, assessed by reviewing cancer registry records from 2009. Methylation was quantified by pyrosequencing assays for six genes (HSPB1, CCND2, TIG1, DPYS, PITX2, and MAL) with established biomarker value in prostate cancer. A novel prognostic methylation score was developed by multivariate Cox modelling using the six methylation biomarkers in 385 men with low-and-intermediate clinical risk variables and its prognostic value compared to two previously defined clinically-derived risk scores. Methylation score was the most significant variable in univariate and bivariate analysis in men with low-to-intermediate CAPRA risk score. When combined with CAPRA score the hazard ratio was 2.02; 95% confidence interval, 1.40-2.92. For a methylation score sensitivity of 83% the specificity was 44%, while the maximum achieved sensitivity by CAPRA was 68% at a specificity of 44%. The derived methylation score is a strong predictor of aggressive prostate cancer that could have an important role in directing the management of patients with low-to-intermediate risk disease. The estimated areas under the curve (AUC) at 10 years of follow-up were 0.62 (95% CI: 0.51, 0.70) and 0.74 (95% CI: 0.65, 0.82) for CAPRA, and combined (CAPRA + methylation) risk score (CRS) respectively.

Validation of a DNA methylation HPV triage classifier in a screening sample.

High-risk human papillomavirus (hrHPV) DNA tests have excellent sensitivity for detection of cervical intraepithelial neoplasia 2 or higher (CIN2+). A drawback of hrHPV screening, however, is modest specificity. Therefore, hrHPV-positive women might need triage to reduce adverse events and costs associated with unnecessary colposcopy. We compared the performance of HPV16/18 genotyping with a predefined DNA methylation triage test (S5) based on target regions of the human gene EPB41L3, and viral late gene regions of HPV16, HPV18, HPV31 and HPV33. Assays were run using exfoliated cervical specimens from 710 women attending routine screening, of whom 38 were diagnosed with CIN2+ within a year after triage to colposcopy based on cytology and 341 were hrHPV positive. Sensitivity and specificity of the investigated triage methods were compared by McNemar's test. At the predefined cutoff, S5 showed better sensitivity than HPV16/18 genotyping (74% vs 54%, P = 0.04) in identifying CIN2+ in hrHPV-positive women, and similar specificity (65% vs 71%, P = 0.07). When the S5 cutoff was altered to allow equal sensitivity to that of genotyping, a significantly higher specificity of 91% was reached (P < 0.0001). Thus, a DNA methylation test for the triage of hrHPV-positive women on original screening specimens might be a valid approach with better performance than genotyping.

HPV33 DNA methylation measurement improves cervical pre-cancer risk estimation of an HPV16, HPV18, HPV31 and \textit{EPB41L3} methylation classifier.

BACKGROUND: Persistent infection %by with high risk human papillomavirus (hrHPV) types causes cervical cancer but most women who test positive are at very low risk of neoplasia. Strategies are needed which can retain high sensitivity of hrHPV testing but reduce the number of false-positives. We showed previously that a combination DNA methylation triage assay for HPV types 16, 18 and 31 and human gene EPB41L3 was useful to identify high grade cervical lesions. OBJECTIVE: Assess whether measurement of DNA methylation in HPV type 33 can improve the previous classifier. METHODS: A London colposcopy referral group of 1493 women of whom 556 (37%) had histologically-confirmed CIN (cervical intraepithelial neoplasia) 2 or 3 that included 114 HPV33 positive women with methylation measured for three L2 CpGs 5557, 5560 and 5566. Discrimination performance was assessed for the new classifier S5, built by adding HPV33 to the earlier classifier. RESULTS: HPV33 methylation measurement improved prediction among HPV33 positive women. Receiver operating characteristic analyses showed an area under the curve (AUC) for HPV33 methylation of 0.68 (95% CI 0.57-0.78). The earlier risk score was significantly improved by HPV33 methytlation (AUC = 0.82 vs 0.80; P < 0.001). For 90% sensitivity the specificity for CIN2/3 was 49% (95% CI 46-52%). CONCLUSIONS: Measurement of HPV33 DNA methylation contributes independent diagnostic information to EPB41L3 and HPV16, HPV18 and HPV31, and is superior to genotyping. Other HPV and human methylation target regions might be useful to further improve S5.