Methylation in predicting progression of untreated high-grade cervical intraepithelial neoplasia

BACKGROUND: There is no baseline prognostic test to ascertain whether cervical intraepithelial neoplasia (CIN) will regress or progress. The majority of CIN regress in young women and since local treatments are known to increase the risk of adverse pregnancy outcomes interventions need to be sparing. We investigated the ability of a DNA methylation panel (the S5-classifier) to discriminate between progression and regression among women of childbearing age with untreated CIN grade 2 (CIN2). METHODS: Pyrosequencing methylation and HPV genotyping assays were performed on exfoliated cervical cells from 149 young 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 lesions persisted as CIN1/2. When cytology, HPV16/18- and HPV16/18/31/33-genotyping, and S5 at baseline were compared to outcomes, S5 was the strongest biomarker associated with regression versus progression. S5 alone or in combination with HPV16/18/31/33-genotyping also showed significantly increased sensitivity versus cytology, comparing regression vs. persistence/progression. With both S5 and cytology tests set at a specificity of 38.6% (95% CI 28.4-49.6) the sensitivity of S5 was significantly higher (83.6%, 95% CI 71.9-91.8) than for cytology (62.3%, 95% CI 49.0-74.4) (p=0.005). The highest area under the curve (AUC) was 0.735 (95% CI 0.621-0.849) in the regression vs. progression outcome with a combination of S5 and cytology, whereas HPV16/18 or HPV16/18/31/33-genotyping did not provide additional prognostic information. CONCLUSIONS: The S5-classifier shows high potential as a prognostic biomarker to identify women with progressive CIN2.Peer reviewe

Combined HPV 16 E2 and L1 methylation predict response to treatment with cidofovir and imiquimod in patients with vulval intraepithelial neoplasia

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) E⁢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 E⁢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 E⁢2 and HPV16 L⁢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 L⁢1

Methylation in Predicting Progression of Untreated High-grade Cervical Intraepithelial Neoplasia

BackgroundThere 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).MethodsBaseline 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.ResultsTwenty-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.ConclusionsThe S5 classifier shows high potential as a prognostic biomarker to identify progressive CIN2.</div

Assessing the risk of cervical neoplasia in the post-HPV vaccination era

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.publishedVersionPeer reviewe

Performance of an affordable urine self-sampling method for human papillomavirus detection in Mexican women

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

Tumour necrosis factor receptor trafficking dysfunction opens the TRAPS door to pro-inflammatory cytokine secretion

The related TNFR1 studies within the Turner laboratory were funded by Barts and The London Charitable Foundatio

Genetic and functional studies of TNFRSF1A mutations in inflammatory disorders

EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Proinflammatory action of the antiinflammatory drug infliximab in tumor necrosis factor receptor-associated periodic syndrome

Objective. Tumor necrosis factor receptor (TNFR) -associated periodic syndrome (TRAPS) is an autosomal-dominant autoinflammatory condition caused by mutations in the TNFRSFIA gene. Unlike other autoinflammatory diseases in which anti-TNF therapy is largely a successful treatment option, therapy with the anti-TNF drug infliximab is often ineffective in patients with TRAPS. Moreover, in certain cases, infliximab actually triggers severe episodes of inflammation. The aim of this study was to elucidate the mechanisms underlying such a reaction. Methods. Peripheral blood mononuclear cells (PBMCs) were obtained from patients with TRAPS. Both caspase 3 activity and NF-kappa B subunit activity were determined by enzyme-linked immunosorbent assay. Cytokine secretion was assessed using a specific customized human multiplex bead immunoassay kit. Results. Unlike findings in controls, cells from a family of 9 patients, all of whom carried the T50M mutation in TNFRSFIA, failed to respond to infliximab through proapoptotic induction of caspase 3 activity. Instead, we observed enhanced antiapoptotic c-Rel subunit activity, accompanied by a significant increase in secretion of the proinflammatory cytokines interleukin-1 beta (IL-1 beta), IL-1 receptor, IL-6, IL-8, and IL-12. Conclusion. Altered extracellular conformation of TNFRI, resulting from the T50M mutation in TNFRSFIA, results in failure of PBMCs to induce an apoptotic response to infliximab. We hypothesize that failure to shed infliximab-bound TNF/TNFRI from the cell surface of cells from patients with the T50M mutation triggers c-Rel activation, and that this leads to a marked increase in cytokine secretion and an increased proinflammatory response. In light of these findings, we strongly advise caution when prescribing infliximab as anti-TNF therapy to patients with TRAPS

Proinflammatory action of the antiinflammatory drug infliximab in tumor necrosis factor receptor-associated periodic syndrome

Objective. Tumor necrosis factor receptor (TNFR) -associated periodic syndrome (TRAPS) is an autosomal-dominant autoinflammatory condition caused by mutations in the TNFRSFIA gene. Unlike other autoinflammatory diseases in which anti-TNF therapy is largely a successful treatment option, therapy with the anti-TNF drug infliximab is often ineffective in patients with TRAPS. Moreover, in certain cases, infliximab actually triggers severe episodes of inflammation. The aim of this study was to elucidate the mechanisms underlying such a reaction. Methods. Peripheral blood mononuclear cells (PBMCs) were obtained from patients with TRAPS. Both caspase 3 activity and NF-kappa B subunit activity were determined by enzyme-linked immunosorbent assay. Cytokine secretion was assessed using a specific customized human multiplex bead immunoassay kit. Results. Unlike findings in controls, cells from a family of 9 patients, all of whom carried the T50M mutation in TNFRSFIA, failed to respond to infliximab through proapoptotic induction of caspase 3 activity. Instead, we observed enhanced antiapoptotic c-Rel subunit activity, accompanied by a significant increase in secretion of the proinflammatory cytokines interleukin-1 beta (IL-1 beta), IL-1 receptor, IL-6, IL-8, and IL-12. Conclusion. Altered extracellular conformation of TNFRI, resulting from the T50M mutation in TNFRSFIA, results in failure of PBMCs to induce an apoptotic response to infliximab. We hypothesize that failure to shed infliximab-bound TNF/TNFRI from the cell surface of cells from patients with the T50M mutation triggers c-Rel activation, and that this leads to a marked increase in cytokine secretion and an increased proinflammatory response. In light of these findings, we strongly advise caution when prescribing infliximab as anti-TNF therapy to patients with TRAPS