Maria Nasioutziki

High-risk human papillomavirus DNA test and p16INK4a in the triage of LSIL: A prospective diagnostic study

OBJECTIVE The detection of high-grade cervical intraepithelial neoplasia (CIN) amongst patients with low-grade cytology (LSIL) is challenging. This study evaluated the role of high-risk HPV (HR-HPV) DNA test and p16(INK4a) immunostaining in identifying women with LSIL cytology at risk of harboring CIN2 or worse (CIN2+) and the role of p16(INK4a) in the triage of a population of HR-HPV positive LSIL. METHODS We conducted a prospective study including women with LSIL cytology. Detection of HR-HPV was carried out by means of a polymerase chain reaction based assay. p16(INK4a) immunostaining was performed using the Dako CINtec cytology kit. All patients had colposcopically directed punch biopsies or large loop excision of the transformation zone of the cervix. The endpoint was detection of a biopsy-confirmed CIN2+. RESULTS A series of 126 women with LSIL cytology were included. HR-HPV test had sensitivity 75% and specificity 64% for an endpoint of CIN2+. p16(INK4a) had significantly higher specificity of 89% (p=0.0000) but low sensitivity of 42%. The role of p16(INK4a) immunostaining in the triage of LSIL positive for HR-HPV was also evaluated. p16(INK4a) triage had 70% positive predictive value (PPV); however, this was not significantly higher than the PPV (56%) of HR-HPV test alone (p=0.4). CONCLUSIONS The results indicate that HR-HPV or p16(INK4a) cannot be used as solitary markers for the assessment of LSIL. The addition of p16(INK4a) immunostaining led to an increase in HR-HPV specificity; however, the biomarker needs to be assessed further to establish its role as an adjunct test in the triage of LSIL.

The evaluation of p16INK4a immunoexpression/immunostaining and human papillomavirus DNA test in cervical liquid-based cytological samples.

Aim: To evaluate the role of p16INK4a immunoexpression and human papillomavirus (HPV) DNA test for the detection of dyskaryotic cells in high-risk women. Materials and Methods: This work was a retrospective diagnostic study conducted in the University Hospital of Thessaloniki from January to December 2008. The subjects were women with current or previous HPV infection and current or previous cervical intraepithelial lesion (with or without treatment) or clinical warts. All liquid-based cytological samples were tested for P16INKa and HPV DNA test. The accuracy parameters used for the outcome included sensitivity, specificity, and positive predictive value. Results: A total of 226 women were included; the mean age was 29 years. Expression of p16INK4a was detected in the cytological samples of 13% of the negative cases, 44% of the cases of atypical squamous cells of undetermined significance, 46% of the cases of low-grade squamous intraepithelial lesion, and 78% of the cases of high-grade squamous intraepithelial lesion. A total of 91 women tested positive for high-risk HPV infection, and 54 of those had p16INK4a-positive staining reaction cells. The concordance between the 2 tests, HPV DNA and p16, was 59% regarding infection-positive cases. Diffuse strong parabasal p16INK4a immunostaining (nuclear score >2) was observed in 17 cases of the abnormal cytological findings (atypical squamous cells of undetermined significance, 2 cases; low-grade squamous intraepithelial lesion, 8 cases; high-grade squamous intraepithelial lesion, 7 cases). Colposcopy-directed biopsies were used as the criterion standard for the detection of cervical intraepithelial neoplasia in 91 women. The sensitivity of p16INK4a was 95% and the specificity was 92%, whereas the sensitivity of high-risk HPV was 100% and the specificity was 78%. The positive predictive value of p16INK4a was 71%, whereas that of HPV DNA was 44%. Conclusion: The findings suggest that p16INK4a immunostaining can improve the accuracy of cytological examination and HPV DNA test and may be particularly useful in the triage of low-grade lesions.

Histology Verification Demonstrates That Biospectroscopy Analysis of Cervical Cytology Identifies Underlying Disease More Accurately than Conventional Screening: Removing the Confounder of Discordance

Background Subjective visual assessment of cervical cytology is flawed, and this can manifest itself by inter- and intra-observer variability resulting ultimately in the degree of discordance in the grading categorisation of samples in screening vs. representative histology. Biospectroscopy methods have been suggested as sensor-based tools that can deliver objective assessments of cytology. However, studies to date have been apparently flawed by a corresponding lack of diagnostic efficiency when samples have previously been classed using cytology screening. This raises the question as to whether categorisation of cervical cytology based on imperfect conventional screening reduces the diagnostic accuracy of biospectroscopy approaches; are these latter methods more accurate and diagnose underlying disease? The purpose of this study was to compare the objective accuracy of infrared (IR) spectroscopy of cervical cytology samples using conventional cytology vs. histology-based categorisation. Methods Within a typical clinical setting, a total of n = 322 liquid-based cytology samples were collected immediately before biopsy. Of these, it was possible to acquire subsequent histology for n = 154. Cytology samples were categorised according to conventional screening methods and subsequently interrogated employing attenuated total reflection Fourier-transform IR (ATR-FTIR) spectroscopy. IR spectra were pre-processed and analysed using linear discriminant analysis. Dunn’s test was applied to identify the differences in spectra. Within the diagnostic categories, histology allowed us to determine the comparative efficiency of conventional screening vs. biospectroscopy to correctly identify either true atypia or underlying disease. Results Conventional cytology-based screening results in poor sensitivity and specificity. IR spectra derived from cervical cytology do not appear to discriminate in a diagnostic fashion when categories were based on conventional screening. Scores plots of IR spectra exhibit marked crossover of spectral points between different cytological categories. Although, significant differences between spectral bands in different categories are noted, crossover samples point to the potential for poor specificity and hampers the development of biospectroscopy as a diagnostic tool. However, when histology-based categories are used to conduct analyses, the scores plot of IR spectra exhibit markedly better segregation. Conclusions Histology demonstrates that ATR-FTIR spectroscopy of liquid-based cytology identifies the presence of underlying atypia or disease missed in conventional cytology screening. This study points to an urgent need for a future biospectroscopy study where categories are based on such histology. It will allow for the validation of this approach as a screening tool.

Awareness of human papillomavirus infection, testing and vaccination in midwives and midwifery students in Greece

Summary Midwives play an important role in the implementation of cervical cancer screening. We assessed the knowledge of human papillomavirus (HPV) infection and of its relationship with cervical cancer in 107 midwives and 29 graduating midwifery students. The majority of midwives (78.5%) were aware that a viral infection causes cervical cancer, whereas only 48.3% of the students knew this (p = 0.003). Only one midwife (0.9%) was not aware of HPV infection compared with 10.3% of the students (p = 0.029). Midwives were also more knowledgeable of the relationship between HPV infection and cervical cancer and of the availability of a vaccine against HPV infection (p = 0.005 and p < 0.0001, respectively). In conclusion, Greek midwives have a satisfactory level of knowledge about cervical cancer and HPV infection, in contrast to midwifery students. It is important to better educate midwifery students in order to facilitate the incorporation of HPV testing and vaccination in clinical practice.

Clinical management of HPV‐related disease of the lower genital tract

Cytology remains the mainstay for cervical screening. The need to achieve effective management, limit complications, and preserve reproductive function led to the popularity of local treatment. Although the cure rates for ablative and excisional methods are similar, the excisional method provides a more reliable histopathological diagnosis. Recent evidence revealed increased perinatal morbidity after treatment that appears to be related to the proportion of cervix removed. The human papillomavirus (HPV) DNA test appears to enhance the detection of disease in primary screening, in the triage of minor cytological abnormalities, and in follow‐up. Further research on the clinical application of a scoring system is ongoing. The vaccines are now available and appear to be safe, well tolerated, and highly efficacious in HPV naive women. A synergy of vaccination and screening will be required. Treatment for early cervical cancer is increasingly shifting toward more fertility‐sparing surgical techniques. Careful selection of patients is essential.

Classification of cervical cytology for human papilloma virus (HPV) infection using biospectroscopy and variable selection techniques

Cervical cancer is the second most common cancer in women worldwide. We set out to determine whether attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy combined with principal component analysis–linear discriminant analysis (PCA–LDA) or, variable selection techniques employing successive projection algorithm or genetic algorithm (GA) could classify cervical cytology according to human papilloma virus (HPV) infection [high-risk (hr) vs. low-risk (lr)]. Histopathological categories for squamous intraepithelial lesion (SIL) were segregated into grades (low-grade vs. high-grade) of cervical intraepithelial neoplasia (CIN) expressing different HPV infection (16/18, 31/35 or HPV Others). Risk assessment for HPV infection was investigated using age (≤29 years vs. >30 years) as the distinguishing factor. Liquid-based cytology (LBC) samples (n = 350) were collected and interrogated employing ATR-FTIR spectroscopy. Accuracy test results including sensitivity and specificity were determined. Sensitivity in hrHPV category was high (≈87%) using a GA–LDA model with 28 wavenumbers. Sensitivity and specificity results for >30 years for HPV, using 28 wavenumbers by GA–LDA, were 70% and 67%, respectively. For normal cervical cytology, accuracy results for ≤29 years and >30 years were high (up to 81%) using a GA–LDA model with 27 variables. For the low-grade cervical cytology dataset, 83% specificity for ≤29 years was achieved using a GA–LDA model with 33 wavenumbers. HPV16/18 vs. HPV31/35 vs. HPV Others were segregated with 85% sensitivity employing a GA–LDA model with 33 wavenumbers. We show that ATR-FTIR spectroscopy of cervical cytology combined with variable selection techniques is a powerful tool for HPV classification, which would have important implications for the triaging of patients.

Molecular epidemiology of HPV infection using a clinical array methodology in 2952 women in Greece

The molecular epidemiology of human papillomavirus (HPV) infection in a sample of Greek women (n = 2952, mean age 42.2 ± 13.3 years) was examined. HPV prevalence was 50.7% (95% confidence interval, 48.8-52.6). The most frequent HPV types were HPV 53, 51 and 66 (10.2%, 9.4% and 9.3%, respectively). HPV positivity was associated with age, age of sexual debut, number of sexual partners and duration of sexual relationship, while marriage or multiparity protected against infection (all p <0.001). Follow-up of this cohort will assist in predicting the effect of vaccination with the new HPV vaccines on future screening with HPV-based tests for cervical cancer.

Personalised management of women with cervical abnormalities using a clinical decision support scoring system

OBJECTIVES To develop a clinical decision support scoring system (DSSS) based on artificial neural networks (ANN) for personalised management of women with cervical abnormalities. METHODS We recruited women with cervical abnormalities and healthy controls that attended for opportunistic screening between 2006 and 2014 in 3 University Hospitals. We prospectively collected detailed patient characteristics, the colposcopic impression and performed a series of biomarkers using a liquid-based cytology sample. These included HPV DNA typing, E6&E7 mRNA by NASBA or flow cytometry and p16INK4a immunostaining. We used ANNs to combine the cytology and biomarker results and develop a clinical DSSS with the aim to improve the diagnostic accuracy of tests and quantify the individuals risk for different histological diagnoses. We used histology as the gold standard. RESULTS We analysed data from 2267 women that had complete or partial dataset of clinical and molecular data during their initial or followup visits (N=3565). Accuracy parameters (sensitivity, specificity, positive and negative predictive values) were assessed for the cytological result and/or HPV status and for the DSSS. The ANN predicted with higher accuracy the chances of high-grade (CIN2+), low grade (HPV/CIN1) and normal histology than cytology with or without HPV test. The sensitivity for prediction of CIN2 or worse was 93.0%, specificity 99.2% with high positive (93.3%) and negative (99.2%) predictive values. CONCLUSIONS The DSSS based on an ANN of multilayer perceptron (MLP) type, can predict with the highest accuracy the histological diagnosis in women with abnormalities at cytology when compared with the use of tests alone. A user-friendly software based on this technology could be used to guide clinician decision making towards a more personalised care.

Expression of HPV-related biomarkers and grade of cervical intraepithelial lesion at treatment.

New human papillomavirus (HPV)‐related biomarkers may allow better identification of clinically significant lesions that warrant excision and, conversely, identification of the false positive cases that have been overreported by traditional techniques. The aim of this study was to investigate how the expression of several HPV‐related biomarkers correlates to the severity of the lesion at treatment.

Cytokines of Cervical Mucosa and Human Papilloma Virus Infection of the Cervix: A Descriptive Study

Objective: The production of cytokines by various cervical cells can be triggered by antigenic stimuli, including human papilloma virus (HPV) infection. The aim of the present study was to evaluate differences in cytokine levels between various intraepithelial cervical lesions. Study Design: A total of 97 women participated in this descriptive study. Within our study population, 27 subjects presented with high-grade squamous intraepithelial lesions (HSIL), 48 with low-grade squamous intraepithelial lesion (LSIL) and 22 with a normal smear. Colposcopy and directed biopsy were performed as needed. To confirm the presence of HPV, an HPV-DNA test was performed using polymerase chain reaction. Using flow cytometry to ThinPrep cervical samples, we assessed the family of interleukins (ILs), including IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, tumor necrosis factor (TNF)-α and TNF-β and transforming growth factor-β1. Statistical analyses were performed using the Statistical Package for the Social Sciences (SPSS for Windows; version 20.0). Results: Significantly lower levels of IL-1α, IL-2, IL-4 and TNF-α were detected in cervical samples obtained from the LSIL group when compared to samples obtained from the HSIL group. Conclusion: Significant differences in tissue cytokine levels exist between intraepithelial cervical lesions obtained from patients presenting with HSIL versus LSIL.