The diagnostic accuracy of E6 & 7 mRNA detection as a primary screening test for the detection of severe cervical lesions.

PURPOSE: This prospective accuracy study aimed to assess the diagnostic accuracy of nucleic acid sequence amplification (NASBA) and flow cytometry for E6/7 human papillomavirus (HPV) mRNA detection as a primary screening test compared to cytology in the triage of severe cervical intraepithelial neoplasia (CIN) lesions. METHODS: 1083 women referred to our outpatient gynecology clinics for a routine Pap test were recruited. Residual material of the Pap smears was tested by NASBA and by flow cytometry for E6/7 mRNA expression. Biopsy results were used as reference standards. The accuracy indices of both techniques and of NASBA type-16 HPV were assessed for the detection of CIN2+ lesions and were compared to cytology. RESULTS: An increased lesion severity was associated with increased positivity rates of both NASBA and flow cytometry tests (x(2), p<0.001). A positive correlation between NASBA and flow cytometry was identified when these methods were examined with the Phi coefficient (value 0.369, 95% confidence interval [95%CI] : 0.307-0.426). Furthermore, NASBA (89.7 vs 57.7%, p<0.0005) and flow cytometry (77.3 vs 57.7%, p<0.0005) exhibited higher specificity rates than cytology. However, their sensitivity rates did not exceed those of cytology (NASBA:69.8 vs 84.6%, p=0.051; flow cytometry: 69.12 vs 84.6%, p=0.043). CONCLUSIONS: Both NASBA and flow cytometry exhibited increased specificity for the triage of CIN2+ lesions. However, their relatively lower sensitivity and higher positivity rates when compared to cytology do not make them ideal for a primary screening test. Hence, the role of mRNA detection in the screening for severe cervical lesions remains to be clarified.

The diagnostic accuracy of two methods for E6&7 mRNA detection in women with minor cytological abnormalities.

OBJECTIVE: To examine the diagnostic accuracy of nucleic acid sequence based amplification (NASBA) and flow cytometry for E6&7 human papillomavirus (HPV) mRNA detection in the triage of minor cytological abnormalities. DESIGN: Prospective diagnostic accuracy study. SETTING: Gynecology outpatient clinics of a university hospital. POPULATION: 472 women with low-grade squamous intraepithelial lesion (LSIL) or atypical squamous cells of undetermined significance (ASCUS). METHODS: Residual material of the liquid-based smears was tested by NASBA and by flow cytometry for E6&E7 mRNA expression. Histological diagnosis was used as reference standard. MAIN OUTCOME MEASURES: Accuracy indices of the two techniques and of type 16-specific NASBA for the detection of cervical intraepithelial neoplasia (CIN) 2+ and CIN3+, accuracy indices at age >35 years, correlation between NASBA and flow, comparison between integrated and episomal high-risk HPV infection for risk of CIN2+. RESULTS: Both tests showed increased positivity rates with increasing severity of the lesion (p < 0.05, chi-squared test for trend). There was a positive correlation between NASBA and flow results (phi coefficient = 0.325). NASBA-positive cases were more likely to have CIN2+ than were NASBA-negative/DNA-positive for types 16, 18, 31, 33, 45 (25/73 vs. 4/52, p= 0.0004; Fisher's exact test). In the LSIL group the NASBA accuracy indices for CIN3+ were: sensitivity 75%, specificity 78.7% and positivity rate 20.8%, and for flow 77.8%, 64.5% and 35.9%, respectively. CONCLUSIONS: NASBA has favorable specificity and positivity rates for triaging LSIL prior to colposcopy. A relatively low sensitivity warrants cytological surveillance of the NASBA-negative LSILs. Flow cytometry does not perform as well overall.

Performance evaluation of manual and automated (MagNA pure) nucleic acid isolation in HPV detection and genotyping using Roche Linear Array HPV Test.

Nucleic acids of human papillomavirus (HPV) isolated by manual extraction method (AmpliLute) and automated MagNA pure system were compared and evaluated with cytohistological findings in 253 women. The concordance level between AmpliLute and MagNA was very good 93.3% (κ = 0.864, P < .0001). Overall HPVpositivity detected by AmpliLute was 57.3% (30.4% as single and 27% as multiple infections) in contrast to MagNA 54.5% (32% and 23%, resp.). Discrepant results observed in 25 cases: 11 MagNA(-)/AmpliLute(+), 10 of which had positive histology; 5 MagNA(+)/AmpliLute(-) with negative histology; 8 MagNA(+)/AmpliLute(+): in 7 of which AmpliLute detected extra HPV genotypes and 1 MagNA(invalid)/AmpliLute(+) with positive histology. Both methods performed well when compared against cytological (area under curve (AUC) of AmpliLute 0.712 versus 0.672 of MagNA) and histological diagnoses (AUC of AmpliLute 0.935 versus 0.877 of MagNA), with AmpliLute showing a slightly predominance over MagNA. However, higher sensitivities, specificities, and positive/negative predictive values were obtained by AmpliLute.

Evaluation Analysis of miRNAs Overexpression in Liquid-Based Cytology Endometrial Samples.

Background: miRNAs have an important role as their deregulation is linked to endometrial cancer. Methods: A custom miScript® miRNA PCR Array was used to investigate for the first time the expression of eight miRNAs in forty-nine histologically confirmed Liquid Based cytology endometrial samples. The expression profile of the same miRNAs was also examined in sixty formalin-fixed tissue samples. Results: Expression of seven miRNAs was significantly higher in malignant samples with three of them (mir-182, mir-141 and mir-205) performing optimally. Conclusion: These results suggest the potential use of this non-invasive method of sampling for miRNA expression studies. Furthermore miRNA overexpression could serve as an ancillary or reflex test for optimal identification of malignant samples especially in morphologically inadequate samples.

γH2Ax Expression as a Potential Biomarker Differentiating between Low and High Grade Cervical Squamous Intraepithelial Lesions (SIL) and High Risk HPV Related SIL.

BACKGROUND: γH2AX is a protein biomarker for double-stranded DNA breakage; its expression was studied in cervical squamous intraepithelial lesions and carcinomas. METHODS: Immunostaining for phospho-γH2AX was performed in sections from histologically confirmed cervical SIL and carcinomas, as well as from normal cervices used as controls. In total, 275 cases were included in the study: 112 low grade SIL (LGSIL), 99 high grade SIL (HGSIL), 24 squamous cell carcinoma (SCC), 12 adenocarcinoma and 28 cervical specimens with no essential lesions. Correlation of histological grading, high risk vs. low risk HPV virus presence, activated vs. non-activated status (by high risk HPV mRNA expression) and γH2AX expression in both basal and surface segments of the squamous epithelium was performed. RESULTS: Gradual increase of both basal and surface γH2AX expression was noted up from normal cervices to LGSIL harboring a low risk HPV type, to LGSIL harboring a high risk virus at a non-activated state (p<0.05). Thereafter, both basal and surface γH2AX expression dropped in LGSIL harboring a high risk virus at an activated state and in HGSIL. CONCLUSIONS: γH2AX could serve as a potential biomarker discriminating between LGSIL and HGSIL, as well as between LGSIL harboring high risk HPV at an activated state.

A Pyrosequencing Assay for the Quantitative Methylation Analysis of GALR1 in Endometrial Samples: Preliminary Results.

Endometrial cancer is the most common malignancy of the female genital tract while aberrant DNA methylation seems to play a critical role in endometrial carcinogenesis. Galanin's expression has been involved in many cancers. We developed a new pyrosequencing assay that quantifies DNA methylation of galanin's receptor-1 (GALR1). In this study, the preliminary results indicate that pyrosequencing methylation analysis of GALR1 promoter can be a useful ancillary marker to cytology as the histological status can successfully predict. This marker has the potential to lead towards better management of women with endometrial lesions and eventually reduce unnecessary interventions. In addition it can provide early warning for women with negative cytological result.

The Application of Classification and Regression Trees for the Triage of Women for Referral to Colposcopy and the Estimation of Risk for Cervical Intraepithelial Neoplasia: A Study Based on 1625 Cases with Incomplete Data from Molecular Tests.

OBJECTIVE: Nowadays numerous ancillary techniques detecting HPV DNA and mRNA compete with cytology; however no perfect test exists; in this study we evaluated classification and regression trees (CARTs) for the production of triage rules and estimate the risk for cervical intraepithelial neoplasia (CIN) in cases with ASCUS+ in cytology. STUDY DESIGN: We used 1625 cases. In contrast to other approaches we used missing data to increase the data volume, obtain more accurate results, and simulate real conditions in the everyday practice of gynecologic clinics and laboratories. The proposed CART was based on the cytological result, HPV DNA typing, HPV mRNA detection based on NASBA and flow cytometry, p16 immunocytochemical expression, and finally age and parous status. RESULTS: Algorithms useful for the triage of women were produced; gynecologists could apply these in conjunction with available examination results and conclude to an estimation of the risk for a woman to harbor CIN expressed as a probability. CONCLUSIONS: The most important test was the cytological examination; however the CART handled cases with inadequate cytological outcome and increased the diagnostic accuracy by exploiting the results of ancillary techniques even if there were inadequate missing data. The CART performance was better than any other single test involved in this study.

An intelligent clinical decision support system for patient-specific predictions to improve cervical intraepithelial neoplasia detection.

Nowadays, there are molecular biology techniques providing information related to cervical cancer and its cause: the human Papillomavirus (HPV), including DNA microarrays identifying HPV subtypes, mRNA techniques such as nucleic acid based amplification or flow cytometry identifying E6/E7 oncogenes, and immunocytochemistry techniques such as overexpression of p16. Each one of these techniques has its own performance, limitations and advantages, thus a combinatorial approach via computational intelligence methods could exploit the benefits of each method and produce more accurate results. In this article we propose a clinical decision support system (CDSS), composed by artificial neural networks, intelligently combining the results of classic and ancillary techniques for diagnostic accuracy improvement. We evaluated this method on 740 cases with complete series of cytological assessment, molecular tests, and colposcopy examination. The CDSS demonstrated high sensitivity (89.4%), high specificity (97.1%), high positive predictive value (89.4%), and high negative predictive value (97.1%), for detecting cervical intraepithelial neoplasia grade 2 or worse (CIN2+). In comparison to the tests involved in this study and their combinations, the CDSS produced the most balanced results in terms of sensitivity, specificity, PPV, and NPV. The proposed system may reduce the referral rate for colposcopy and guide personalised management and therapeutic interventions.

Comparison of two commercially available methods for HPV genotyping: CLART HPV2 and Linear Array HPV Genotyping tests.

OBJECTIVE: To compare the clinical efficiency of two commercially available HPV DNA detection and typing tests. The CLART HPV2 test, a novel HPV test based on DNA microarrays that can identify 35 HPV genotypes, was compared to the Linear Array HPV Genotyping (LA) test, a more widely used test able to identify 37 HPV genotypes. STUDY DESIGN: The CLART test was evaluated by comparing the genotyping results of 538 ThinPrep Pap tests with the LA test as well as with the cytological and histological findings. RESULTS: The exact same types and results were identified in 86.1% of the samples (kappa = 0.74). The tests showed excellent agreement in HPV positivity and identification of single and multiple infections (concordance rate 88.7%, kappa = 0.827). CONCLUSION: The CLART test demonstrated results comparable to those of the LA test in clinical sensitivity as measured by the positive predictive value of CIN2+ in ASCUS (67.3% vs. 57.1%), while overall it exhibited higher sensitivity, specificity, positive and negative predictive values and area under curves than the LA test in all cytological and histological subgroups analyzed.

Clinical performance of human papillomavirus E6, E7 mRNA flow cytometric assay compared to human papillomavirus DNA typing.

OBJECTIVE: To use flow cytometry to screen cervical samples for the overexpression of human papillomavirus (HPV) E6 and E7 mRNA and compare the performance of this assay with an HPV DNA array for the detection of high-grade cervical lesions. STUDY DESIGN: Cervical samples were analyzed for HPV DNA by clinical arrays, and the overexpression of E6 and E7 viral oncogenes was monitored using an HPV mRNA detection kit that quantifies the intracellular HPV E6 and E7 mRNA on a cell-by-cell basis. RESULTS: HPV positivity increased with severity of histologic lesions. On the basis of histology-confirmed CIN 2+ cases the specificity of HPV assay was 73.9% (95% CI 66.07, 80.88), whereas it was 39.3% (95% CI 31.85, 47.1) for the DNA assay. CONCLUSION: The HPV assay provides an early predictor of persistent HPV infection and may improve cervical cancer screening by increasing the specificity of detecting high-grade lesions.

Use of flow cytometry as a quality control device for liquid-based cervical cytology specimens.

BACKGROUND: Cervical cancer is the second most common cancer in women worldwide comprising a major concern of public health. Liquid-based cytology provides significantly more effective detection of low-grade intraepithelial neoplasia or more severe lesions, without loss of diagnostic specificity and reduces the number of cases classified as unsatisfactory. The objective of the study is the evaluation of flow cytometry as a rapid tool for quality control of the liquid specimen adequacy for the purpose of precise cytological diagnosis in detecting cervical abnormalities. METHODS: A total of 1,854 women undergoing routine cervical cytology joined this cohort study. A monolayer smear was prepared and 1 ml aliquot was removed and prepared for flow cytometric analysis. Cells were fixed and run on a Partec CyFlow SL, with front scatter (FSS) and side scatter (SSC) set on logarithmic scale. The forward scatter versus side scatter dot-plot was used for the distinction of ectocervical, endocervical, and polymorphonuclear cells. RESULTS: In the total of 1,854 cases, 15 specimens (0.8%) were characterized by the cytopathologist as unsatisfactory. Inadequacy was confirmed by flow cytometric analysis for four samples. Monolayer smears were reprepared for the remaining 11 samples. After the second microscopic observation, six out of the 11 cases (54.5%) were found to be satisfactory, whereas the remaining five were still designated as unsatisfactory by the cytopathologist. CONCLUSION: The parallel screening of adequacy by quantification of cell populations with flow cytometry can serve as an internal control for diagnosis and can overall decrease the number of unsatisfactory samples.

A levonorgestrel-releasing intrauterine system embedded in the omentum in a woman with abdominal pain: a case report.

INTRODUCTION: The Mirena intrauterine system has been licensed as a contraceptive in the United Kingdom since May 1995. The use of an intrauterine system as a primary method of contraception among women has been slowly increasing over the last few years and they now account for about 3% of contraceptive use in England. The Mirena intrauterine system now also has a license for the management of idiopathic menorrhagia. Women may be informed that the rate of uterine perforation associated with intrauterine contraceptive use is low (0-2.3 per 1000 insertions). The rate of perforation reported with the Mirena intrauterine system in a large observational cohort study was 0.9 per 1000 insertions. CASE PRESENTATION: In this case report, the diagnosis of an intraperitoneal Mirena intrauterine system was noted nearly four years after its insertion, despite the patient having had a vaginal hysterectomy and admissions to hospital in the interim with complaints of abdominal pain. CONCLUSION: This case report demonstrates clearly that whenever there is a question of a intrauterine system having fallen out following an ultrasound scan report showing an empty uterus, clinicians should also perform an abdominal X-ray.