Maria Kyrgiou

Lipidomic profiling of endometrial cancer using desorption electrospray ionization mass spectrometry imaging

Novel technologies are required to improve endometrial cancer diagnosis and enhance the early detection of preinvasive precursors. Herein, desorption electrospray ionization mass spectrometry imaging (DESI-MSI) was used to differentiate malignant from benign endometrial tissues and assess lipidomic differences among patients with obesity and diabetes. Reverse phase protein array (RPPA) analysis was performed in the same patients to gain insights into the altered signaling pathways and investigate the protein expression levels in endometrial cancer cases and controls. Tissues from 64 women (50 cancer, 14 benign) were analyzed with DESI-MSI achieving 90% sensitivity and 93% specificity. Discriminatory spectral features were primarily phospholipids [phosphatidic acid (PA), phosphatidylethanolamine (PE), phosphatidylserine (PS), and phosphatidylinositol (PI)], all elevated in cancer. Proteomics revealed upregulated proteins linked to commonly dysregulated pathways in endometrial cancer, namely PI3K/AKT/mTOR, MAPK/RAS, and Wnt signaling pathways. Lipidomic differences were found between high- (obesity/diabetes) and low-risk phenotypes (no obesity/diabetes), with PE and PS elevated in high-risk benign and PE reduced in high-risk cancer cases. A single phospholipid (PE(O-38:4)) was found as discriminatory in both normal and cancer cohorts, which may serve as biomarker in women with benign histology at high-risk of developing endometrial cancer. This study reports the application of DESI-MSI for lipidomic characterisation of endometrial cancer.

DNA methylation signatures of cervical pre‐invasive and invasive disease: An epigenome‐wide association study

AbstractEpigenetic alterations are essential in the development of cancers, while epigenome‐wide exploration in cervical cancer has been limited. In this epigenome‐wide association study (EWAS) we explore differential DNA methylation signatures associated with CIN (cervical intraepithelial neoplasia) grade 3 and cervical cancer to better understand potential drivers and biomarkers of cervical carcinogenesis. 247 women were recruited between 2014 and 2020 (N = 119 benign, N = 74 CIN3/CGIN [cervical glandular intraepithelial neoplasia] and N = 54 cancer). Methylation signatures were obtained from exfoliated cervical cells and sequenced using the Illumina 850 k array. Logistic regression and conditional analyses were used to test for independent associations between Cytosine‐phosphate‐Guanine (CpG) sites and case–control status, with adjustment for batch, chip, age, and human papillomavirus (HPV) status. 409 CpG sites were strongly associated with CIN3/cancer (p‐value <5 × 10−8). Following conditional analysis, two CpG sites located in PAX1 (cg16767801) and NREP‐AS1 genes (cg23642047) were independently associated with case status, yielding an area under the curve (AUC) of 0.92 (AUC = 0.97 for invasive disease). In a validation dataset (CIN3 only) PAX1/NREP‐AS1 yielded a combined AUC of 0.77. Methylation markers offer promise for use in cervical screening, particularly as triage tests and self‐sampling. We have identified a novel combined methylation marker that offers a high accuracy for the detection of CIN3 or worse.

Treatment methods for cervical intraepithelial neoplasia in England: A cost‐effectiveness analysis

AbstractObjectiveTo compare the cost‐effectiveness of different treatments for cervical intraepithelial neoplasia (CIN).DesignA cost‐effectiveness analysis based on data available in the literature and expert opinion.SettingEngland.PopulationWomen treated for CIN.MethodsWe developed a decision‐analytic model to simulate the clinical course of 1000 women who received local treatment for CIN and were followed up for 10 years after treatment. In the model we considered surgical complications as well as oncological and reproductive outcomes over the 10‐year period. The costs calculated were those incurred by the National Health Service (NHS) of England.Main outcome measuresCost per one CIN2+ recurrence averted (oncological outcome); cost per one preterm birth averted (reproductive outcome); overall cost per one adverse oncological or reproductive outcome averted.ResultsFor young women of reproductive age, large loop excision of the transformation zone (LLETZ) was the most cost‐effective treatment overall at all willingness‐to‐pay thresholds. For postmenopausal women, LLETZ remained the most cost‐effective treatment up to a threshold of £31,500, but laser conisation became the most cost‐effective treatment above that threshold.ConclusionsLLETZ is the most cost‐effective treatment for both younger and older women. However, for older women, more radical excision with laser conisation could also be considered if the NHS is willing to spend more than £31,500 to avert one CIN2+ recurrence.

Laser-assisted rapid evaporative ionisation mass spectrometry (LA-REIMS) as a metabolomics platform in cervical cancer screening

The introduction of high-risk human papillomavirus (hrHPV) testing as part of primary cervical screening is anticipated to improve sensitivity, but also the number of women who will screen positive. Reflex cytology is the preferred triage test in most settings but has limitations including moderate diagnostic accuracy, lack of automation, inter-observer variability and the need for clinician-collected sample. Novel, objective and cost-effective approaches are needed. In this study, we assessed the potential use of an automated metabolomic robotic platform, employing the principle of laser-assisted Rapid Evaporative Ionisation Mass Spectrometry (LA-REIMS) in cervical cancer screening. In a population of 130 women, LA-REIMS achieved 94% sensitivity and 83% specificity (AUC: 91.6%) in distinguishing women testing positive (n = 65) or negative (n = 65) for hrHPV. We performed further analysis according to disease severity with LA-REIMS achieving sensitivity and specificity of 91% and 73% respectively (AUC: 86.7%) in discriminating normal from high-grade pre-invasive disease. This automated high-throughput technology holds promise as a low-cost and rapid test for cervical cancer screening and triage. The use of platforms like LA-REIMS has the potential to further improve the accuracy and efficiency of the current national screening programme. Work was funded by the MRC Imperial Confidence in Concept Scheme, Imperial College Healthcare Charity, British Society for Colposcopy and Cervical Pathology, National Research Development and Innovation Office of Hungary, Waters corporation and NIHR BRC.

The European response to the WHO call to eliminate cervical cancer as a public health problem

AbstractThe age‐standardised incidence of cervical cancer in Europe varies widely by country (between 3 and 25/100000 women‐years) in 2018. Human papillomavirus (HPV) vaccine coverage is low in countries with the highest incidence and screening performance is heterogeneous among European countries. A broad group of delegates of scientific professional societies and cancer organisations endorse the principles of the WHO call to eliminate cervical cancer as a public health problem, also in Europe. All European nations should, by 2030, reach at least 90% HPV vaccine coverage among girls by the age of 15 years and also boys, if cost‐effective; they should introduce organised population‐based HPV‐based screening and achieve 70% of screening coverage in the target age group, providing also HPV testing on self‐samples for nonscreened or underscreened women; and to manage 90% of screen‐positive women. To guide member states, a group of scientific professional societies and cancer organisations engage to assist in the rollout of a series of concerted evidence‐based actions. European health authorities are requested to mandate a group of experts to develop the third edition of European Guidelines for Quality Assurance of Cervical Cancer prevention based on integrated HPV vaccination and screening and to monitor the progress towards the elimination goal. The occurrence of the COVID‐19 pandemic, having interrupted prevention activities temporarily, should not deviate stakeholders from this ambition. In the immediate postepidemic phase, health professionals should focus on high‐risk women and adhere to cost‐effective policies including self‐sampling.

The intelligent knife (iKnife) and its intraoperative diagnostic advantage for the treatment of cervical disease

Clearance of surgical margins in cervical cancer prevents the need for adjuvant chemoradiation and allows fertility preservation. In this study, we determined the capacity of the rapid evaporative ionization mass spectrometry (REIMS), also known as intelligent knife (iKnife), to discriminate between healthy, preinvasive, and invasive cervical tissue. Cervical tissue samples were collected from women with healthy, human papilloma virus (HPV) ± cervical intraepithelial neoplasia (CIN), or cervical cancer. A handheld diathermy device generated surgical aerosol, which was transferred into a mass spectrometer for subsequent chemical analysis. Combination of principal component and linear discriminant analysis and least absolute shrinkage and selection operator was employed to study the spectral differences between groups. Significance of discriminatory m/z features was tested using univariate statistics and tandem MS performed to elucidate the structure of the significant peaks allowing separation of the two classes. We analyzed 87 samples (normal = 16, HPV ± CIN = 50, cancer = 21 patients). The iKnife discriminated with 100% accuracy normal (100%) vs. HPV ± CIN (100%) vs. cancer (100%) when compared to histology as the gold standard. When comparing normal vs. cancer samples, the accuracy was 100% with a sensitivity of 100% (95% CI 83.9 to 100) and specificity 100% (79.4 to 100). Univariate analysis revealed significant MS peaks in the cancer-to-normal separation belonging to various classes of complex lipids. The iKnife discriminates healthy from premalignant and invasive cervical lesions with high accuracy and can improve oncological outcomes and fertility preservation of women treated surgically for cervical cancer. Larger in vivo research cohorts are required to validate these findings.

The vaginal microbiota and innate immunity after local excisional treatment for cervical intraepithelial neoplasia

Abstract Background Vaginal microbiota (VMB) composition is altered in women with cervical intra-epithelial neoplasia (CIN) compared to healthy controls and is associated with disease progression. However, the impact of CIN excision on the VMB and innate immunity is not known. This observational study aims to explore the impact of CIN excision on the VMB, antimicrobial peptides (AMP) and proinflammatory cytokines. Methods We sampled 103 non-pregnant, premenopausal women at the time of excisional treatment for CIN and at their 6-month follow-up visit. A further 39 untreated controls with normal cytology were also sampled. We used metataxonomics to group vaginal swab samples into community state types (CSTs) and ELISA to quantify cytokine and AMP levels in matched vaginal secretions. Analyses were performed to compare the bacterial composition and immune analyte levels before and after CIN excision and in healthy controls. Results Women with CIN had significantly higher rates of Lactobacillus species depletion pre-treatment compared to healthy controls (CST IV 21/103, 20% vs 1/39, 3%, p = 0.0081). Excision did not change the VMB composition, with CST IV remaining significantly more prevalent after excision compared to untreated, healthy controls (CST IV 19/103, 20% vs 1/39, 3%, p = 0.0142). Prevotella bivia and Sneathia amnii were significantly higher in samples before treatment compared to untreated controls, and Prevotella bivia remained significantly higher amongst the treated, with less Lactobacillus crispatus compared to untreated controls. IL-1β and IL-8 remained significantly elevated pre- (p < 0.0001 and p = 0.0014, respectively) and post-treatment (p < 0.0001 and p = 0.0035, respectively) compared to untreated controls. Levels of human beta-defensin-1 and secretory leukocyte protease inhibitor were both significantly reduced following CIN excision (p < 0.0001); however, their levels remained lower than controls post-treatment. Conclusions Women with CIN have an increased prevalence of Lactobacillus sp. depletion, high-diversity VMB composition, and higher levels of proinflammatory cytokines and AMPs compared to normal controls. Surgical excision of the disease reduces levels of vaginal AMPs but does not alter VMB composition or cytokine levels. These findings suggest that women with CIN have an inherent predisposition to a high-diversity proinflammatory environment that is not corrected by disease excision. The failure to re-establish a Lactobacillus-enriched CST may explain why women remain at high risk of pre-invasive and invasive disease recurrence.

Role of human papillomavirus (HPV) vaccination on HPV infection and recurrence of HPV related disease after local surgical treatment: systematic review and meta-analysis

Objective To explore the efficacy of human papillomavirus (HPV) vaccination on the risk of HPV infection and recurrent diseases related to HPV infection in individuals undergoing local surgical treatment. Design Systematic review and meta-analysis Data sources PubMed (Medline), Scopus, Cochrane, Web of Science, and ClinicalTrials.gov were screened from inception to 31 March 2021. Review methods Studies reporting on the risk of HPV infection and recurrence of disease related to HPV infection after local surgical treatment of preinvasive genital disease in individuals who were vaccinated were included. The primary outcome measure was risk of recurrence of cervical intraepithelial neoplasia grade 2 or higher (CIN2+) after local surgical treatment, with follow-up as reported by individual studies. Secondary outcome measures were risk of HPV infection or other lesions related to HPV infection. Independent and in duplicate data extraction and quality assessment were performed with ROBINS-I and RoB-2 tools for observational studies and randomised controlled trials, respectively. Grading of Recommendations Assessment, Development, and Evaluation (GRADE) was implemented for the primary outcome. Observational studies and randomised controlled trials were analysed separately from post hoc analyses of randomised controlled trials. Pooled risk ratios and 95% confidence intervals were calculated with a random effects meta-analysis model. The restricted maximum likelihood was used as an estimator for heterogeneity, and the Hartung-Knapp-Sidik-Jonkman method was used to derive confidence intervals. Results 22 articles met the inclusion criteria of the review; 18 of these studies also reported data from a non-vaccinated group and were included in the meta-analyses (12 observational studies, two randomised controlled trials, and four post hoc analyses of randomised controlled trials). The risk of recurrence of CIN2+ was reduced in individuals who were vaccinated compared with those who were not vaccinated (11 studies, 19 909 participants; risk ratio 0.43, 95% confidence interval 0.30 to 0.60; I 2 =58%, τ 2 =0.14, median follow-up 36 months, interquartile range 24-43.5). The effect estimate was even stronger when the risk of recurrence of CIN2+ was assessed for disease related to HPV subtypes HPV16 or HPV18 (six studies, 1879 participants; risk ratio 0.26, 95% confidence interval 0.16 to 0.43; I 2 =0%, τ 2 =0). Confidence in the meta-analysis for CIN2+ overall and CIN2+ related to HPV16 or HPV18, assessed by GRADE, ranged from very low to moderate, probably because of publication bias and inconsistency in the studies included in the meta-analysis. The risk of recurrence of CIN3 was also reduced in patients who were vaccinated but uncertainty was large (three studies, 17 757 participants; 0.28, 0.01 to 6.37; I 2 =71%, τ 2 =1.23). Evidence of benefit was lacking for recurrence of vulvar, vaginal, and anal intraepithelial neoplasia, genital warts, and persistent and incident HPV infections, although the number of studies and participants in each outcome was low. Conclusion HPV vaccination might reduce the risk of recurrence of CIN, in particular when related to HPV16 or HPV18, in women treated with local excision. GRADE assessment for the quality of evidence indicated that the data were inconclusive. Large scale, high quality randomised controlled trials are required to establish the level of effectiveness and cost of HPV vaccination in women undergoing treatment for diseases related to HPV infection. Systematic review registration PROSPERO CRD42021237350.

Multizonal intraepithelial neoplasia of the lower genital tract and anus in women: terminology for defining the disease, an introduction by the British Society for Colposcopy and Cervical Pathology (BSCCP), International Anal Neoplasia Society (IANS), European Federation for Colposcopy (EFC) and British Society for the Study of Vulval Disease (BSSVD) scientific committees

Abstract Multizonal anogenital intraepithelial neoplasia (MZIN) is an uncommon chronic pre-malignant condition. In the United Kingdom (UK) and elsewhere MZIN is managed by a variety of clinical specialities with differing strategies, resulting in a lack of standardisation in diagnosis and treatment which ultimately disadvantages those affected. Screening for anogenital precancerous conditions is sporadic rather than nationalised in the UK and elsewhere in Europe, with the exception of the cervix. To address this lack of standardisation, the BSCCP brought together a panel of stakeholders from aligned expert society committees (IANS, EFC and BSSVD) to review existing evidence and provide a framework for national UK guidelines. Here, we define terminology and scope, as a platform for subsequent guideline development and guide further research. We define MZIN as Human Papillomavirus (HPV)-related squamous intraepithelial lesions occurring in two or more anogenital regions. People with MZIN are a high-risk group for anogenital cancers and subsequently may require tailored monitoring in specialist multi-disciplinary clinics. Centralisation of care and education for primary care providers may improve management. The development of guidelines which incorporate all clinical stakeholders are now needed to provide an international framework regarding the screening, diagnosis, treatment and future prevention of MZIN.