Liquid-based Cervical Cytology Research Articles

Integrity of RNA in long-term-stored cervical liquid-based cytology samples: implications for biomarker research.

Biobanks of cervical screening (LBC) samples annotated with disease status are an invaluable resource to support the development of tools for the risk stratification of disease. Although there is growing interest in the assessment of RNA-based biomarkers, little is known on the suitability and durability of stored clinical samples (commonly used in cervical screening) to support RNA-based research. RNA was extracted from 260 stored LBC samples. Storage at -80°C or -25°C allowed isolation of sufficient RNA for further analysis. RNA was found to be substantially degraded according to Agilent Bioanalyser data. Despite this, RT-qPCR was successful in 95% samples tested. These data suggest that biobanked LBC samples are suitable for RNA-based assessment even if stored for up to 14years.

Effect of 5-aminolevulinic acid photodynamic therapy versus loop electrosurgical excision procedure for cervical intraepithelial neoplasia grade 2 treatment

BackgroundBoth the traditional loop electrosurgical excision procedure (LEEP) and the newly developed 5-aminolevulinic acid photodynamic therapy (ALA-PDT) are used to treat high-grade squamous intraepithelial lesions. However, the clinical efficacy and safety of these two therapies have rarely been compared. Thus, this study aimed to compare the clinical efficacy and safety of the two treatment regimens. MethodsOne hundred and twenty patients in two groups (60 + 60) with grade 2 cervical intraepithelial neoplasia (CIN2) were voluntary treated with photodynamic therapy or LEEP between June 2020 and December 2022. Follow-up was conducted at 3, 4–6, and 7–12 months after treatment. ResultsAlthough the total effective rate of LEEP was higher during the first 6 months after treatment, both the total effective rate of lesion degradation and the double-negative rate of high-risk HPV16/18 and liquid-based cervical cytology by ALA-PDT treatment increased with time and finally exceeded those of LEEP at 7–12 months. ConclusionsALA-PDT may be more promising than LEEP for treating patients with CIN2 because of the better CIN2 degradation and high-risk HPV negativity, less damage, and greater fertility conservation, especially after 6 months.

Evaluation of dual p16/Ki-67 immunostaining on anal cytology specimens.

Dual immunostaining for p16/Ki67 is FDA-approved for use on liquid-based cervical cytology specimens; however, the utility of dual staining in anal cytology especially for ASCUS risk stratification is not well established. We investigated dual staining performance on anal cytology specimens and correlated with subsequent cytologic interpretation, high-risk HPV status, and anal biopsy results. Dual staining for p16/Ki-67 was performed on all liquid-based anal cytology specimens from December 2021 to June 2022 (n = 43). Three patients had high grade squamous intraepithelial lesion (HSIL/AIN2-3) on biopsy; dual staining was positive in all three cases. All HR-HPV negative cases were negative for dual staining. Among the 12 ASCUS samples with subsequent anal biopsy results all also had HR-HPV testing. Due to small sample size of cases with squamous intraepithelial lesion (SIL) diagnosed on biopsy, the sensitivity and positive predictive value was not calculated. However, the specificity and negative predictive value of p16/Ki-67 dual staining for SIL of any grade on biopsy were 1 (95% CI: 0.66-1) and 0.9 (95% CI: 0.65-0.97) respectively, whereas the specificity and negative predictive value of HR-HPV testing for SIL of any grade on biopsy were 0.44 (95% CI: 0.14-0.79) and 0.8 (95% CI: 0.41-0.96) respectively. Dual p16/Ki-67 staining indicates transforming HPV infection and could help serve as an ancillary test for risk stratification for atypical anal cytology specimens. Among ASCUS samples, dual staining was specific for SIL of any grade with a high negative predictive value and therefore could be useful in clinical practices with limited availability for follow-up care.

Incidence and clearance of cervical and anal high-risk human papillomavirus in kidney transplant recipients: Results from a Danish prospective clinical study

This study investigates the incidence and clearance of cervical and anal high-risk human papillomavirus (hrHPV) infection in kidney transplant recipients (KTRs) compared to immunocompetent controls. During 2016-2017, we enrolled 125 female KTRs and 125 female controls. Liquid-based cervical and anal cytology samples collected at enrollment and follow-up were tested for human papillomavirus (HPV) DNA using the CLART HPV2 test. All participants answered a questionnaire on lifestyle and sexual behavior at both examinations. KTRs had an increased age-adjusted risk of incident cervical hrHPV infection compared to controls (hazard ratio [HR] = 3.6, 95% CI = 1.2-11.2). Probability of cervical hrHPV clearance at 18 months was lower among KTRs (8.3%) than controls (66.7%). There was no statistically significant difference in anal hrHPV incidence between KTRs and controls (HR = 0.9, 95% CI = 0.4-2.0). Clearance of anal hrHPV was similar between KTRs and controls at 18 months. During the total follow-up, a lower anal hrHPV clearance, although not statistically significant, was observed among KTRs (HR = 0.3, 95% CI = 0.06-1.2). KTRs had higher incidence of cervical hrHPV and lower probability of clearance, especially of cervical hrHPV infections, than controls. Our findings support that KTRs are at increased risk of HPV infection and point to the need for targeted HPV prevention strategies, such as cervical cancer screening.

Comparative analysis of different cervical screening methods with special reference to the WHO recommended human papillomavirus detection at a rural tertiary care center - A pilot study

Background: Cervical cancer (Ca Cx) the 4th common cancer has a high mortality rate. Human Papilloma Virus (HPV) is chiefly responsible for a long latent period to progress from preinvasive to invasive Ca Cx. So it can be prevented by screening procedures such as visual inspection with acetic acid (VIA), conventional cervical PAP smear (CP), liquid-based cytology (LBC), and high-risk (HPV-hr) detection along with vaccination. Aims and Objectives: The study aims to determine the socio-demographic profile of Ca Cx and the most appropriate cost-effective community screening method. Materials and Methods: A prospective observational study with 80 symptomatic women of reproductive and peri/postmenopausal age groups attending the Gynecology Department who underwent VIA, CP, and LBC and thereby detected positive along with screened negative but clinically suspicious women were tested for HPV-hr detection. Results were tabulated into an Excel sheet and screening methods were compared using Chi-square test, gold standard being cervical histopathology (P<0.5 significant). Statistical software used - Graphpad Prism 9 (San Diego, CA). Results: The mean age was 43.3 years. Multiparity and early marriage were seen in 80% and 45% respectively. Inflammatory lesions were best diagnosed by CP but epithelial cell abnormality and more satisfactory smears by LBC (P<0.05). Sensitivity was highest (93.75%) in VIA, positive predictive value, and specificity in CP (91.66%, 75%). negative predictive value was highest in VIA and HPV-hr DNA (50%). Conclusion: VIA with CP/LBC is an economically viable and effective screening method for Ca Cx in developing countries along with HPV vaccination.

DPD-Net: Dual-path Proposal Discriminative Network for abnormal cell detection in cervical cytology images

Cytology inspection is a basic examination for the prevention of cervical cancer, which is still done manually and is a labor-intensive and time-consuming process with high inter-observer variability. With the rapid development of deep learning, automatic cytology inspection methods have achieved gratifying results, but the research on the effective detection of abnormal cells remains insufficient. In this paper, we propose a novel Dual-path Proposal Discriminative detection Network (DPD-Net) for abnormal cell detection in cervical cytology images. Specifically, considering the distinctive characteristics of abnormal nuclei such as increased sizes and unclear boundaries, we first design a dual-path architecture, where the cell path acts as the primary detector and the nucleus path serves as the auxiliary detector to provide supplementary information. In addition, the proposal information isolation may result in ambiguous classification boundaries. To transcend individual features and delve deeper into cellular relationships as implicit features, the proposal relation modules are added to explore relation information and refine features adaptively. Finally, to tackle problems of intra-class variation and inter-class similarity, we calculate the proposal contrastive losses in two paths respectively to guide a better distinction between the features of normal and abnormal cells. The proposed method is evaluated on our in-house cervical liquid-based cytology dataset (CLBC) and a public pap smear cytology dataset (CRIC) and gains satisfactory results, outperforming other state-of-the-art methods. Comprehensive experiments demonstrate the superiority of the proposed DPD-Net and the effectiveness of each proposed component. Our code is available at https://github.com/chaisiyii/DPD-Net.

Liquid-Based Cervical Cytology: Monitoring the Laboratory Quality Indicators.

Many developments in cervical cancer screening have happened in the past century, helping women in earlier detection of cervical cancer and its precursors. Cytology still holds the fort as being a specific test, though it suffers in sensitivity. As a part of the quality control program, the aim of the study is to determine the total number of abnormal liquid-based cervical cytology (LBC) at our center and correlate the abnormal LBC with histology and human papillomavirus (HPV) DNA test results. Retrospective analysis of 4286 LBC screening cases was carried out over a period of 5 years. For cytology-histology correlation, cervical biopsy and cytology test results were analyzed. The two-tier grading system for biopsy interpretation was used. HPV DNA test results wherever available were correlated. Of the 4286 LBC cases, 157 samples (3.7%) were unsatisfactory for evaluation, 3915 samples (91.3%) were negative for intra-epithelial lesion or malignancy, and 214 samples (5%) showed epithelial cell abnormality. ASC-US was reported in 60 cases (1.4%), ASC-H in 35 cases (0.8%), LSIL in 47 cases (1.1%), HSIL in 41 cases (1.0%), squamous cell carcinoma in a single case (0.02%), and atypical glandular cells in 30 cases (0.7%). The ASC/SIL ratio was 1.07:1. The CHC major discrepancy was calculated as 16.2%. The concordance of HSIL on cytology and biopsy as a measure of PPV is 94.4%. Of the epithelial cell abnormalities, 24 cases were positive for high-risk HPV (hrHPV). Molecular test results of 2737 samples showed HPV detected in 50 cases, of which 24 cases were positive for hrHPV. The study helped us to analyze the quality parameters of our cytopathology laboratory which are within the acceptable limits.

Clinical performance of the novel full-genotyping OncoPredict HPV Quantitative Typing assay using the VALGENT framework.

Clinical validation of human papillomavirus (HPV) assays according to international criteria is prerequisite for their implementation in cervical cancer screening. OncoPredict HPV Quantitative Typing (QT) assay (Hiantis Srl, Milan, Italy) is a novel full-genotyping multiplex real-time PCR quantitative assay targeting E6/E7 genes, allowing individual viral load determination of 12 high-risk (HR) HPV types. Quality controls for sample adequacy, efficiency of nucleic acid extraction and PCR inhibition are included in the assay. Clinical performance of OncoPredict HPV QT test was assessed as part of the "Validation of HPV Genotyping Tests" (VALGENT-2) framework, consisting of 1300 cervical liquid-based cytology (LBC) samples of women aged between 20 and 60 years who had originally attended for routine cervical screening in Scotland. The clinical accuracy of the OncoPredict HPV QT (index test) for the detection of CIN2+ was assessed relative to the GP5+/6+ Enzyme ImmunoAssay (GP5+/6+ EIA) (comparator test), using noninferiority criteria. Intra- and interlaboratory reproducibility of the assay was assessed on a subpopulation, comprising 526 samples. The relative sensitivity and specificity for OncoPredict HPV QT vs GP5+/6+-PCR-EIA were 1.01 (95% CI: 0.99-1.03) and 1.03 (95% CI: 1.0-1.06) respectively. The P-values for noninferiority were ≤0.001. The intra- and inter-laboratory reproducibility demonstrated a high concordance (>98.7%) with kappas for individual types ranging from 0.66 to 1.00. OncoPredict HPV QT fulfills the international validation criteria for the use of HPV tests in cervical cancer screening.

Exploring the prevalence of Human Papillomavirus (HPV) genotypes in PAP smear samples of women in northern region of United Arab Emirates (UAE): HPV Direct Flow CHIP system-based pilot study.

The aim of this study was to explore the prevalence of low and high-risk HPV genotypes in PAP smear samples of women in northern region of the UAE using HPV direct flow CHIP method. A cross-sectional retrospective study was conducted between September 2021 to April 2022. A total of 104 liquid-based cervical cytology samples were obtained from women aged 20-59 years attending the Gynaecology out-patient department of Thumbay University Hospital and other hospitals of Northern Emirates of UAE, processed for the routine cytological examination to identify and differentiate morphological changes of the PAP smear samples. HPV genotyping was performed using HPV direct flow CHIP method. In total, 112 HPV genotypes were detected in 63 women (60.57%) included 18 abnormal cytological and 45 normal epithelial samples. 63 LR and 49 HR HPV genotypes were identified in all the 63 positive samples. Highest rate of infection with multiple LR and HR HPV genotypes were detected in women aged 40-49 years (25.9%) and 20-29 years (23.5%). Infection by HPV6 (13.46%), HPV11 (9.61%), HPV16 (9.61%), HPV62/81 (7.69%) and HPV45 (7.69%) were the most common genotypes. A moderate increase than expected incidence of HPV45 and 62/81 (7.69%) were detected. Co-infection with multiple low and high-risk genotypes is present in 20.2% cases; in that, HPV6 (15.9%) was the most common followed by HPV62/81 (12.7%) and HPV16 (11.11%). The prevalence of HPV18 was found to be 1.6%. The genotypes 6, 45, 16, 11, 67, 62/81 were the most common HPV infections in the women between the age group of 21 and 59-years-old. A moderate increase of HPV45, 62/81 and much less prevalence of HPV18 were detected in the study population. 43.27% of the normal epithelia were positive to different low and high-risk HPV genotypes. This finding highlights the importance of molecular genotyping of HPV to emphasize the cervical screening triage.

Bethesda system reporting rates, histological follow-up and HPV genotypes distribution of abnormal cytology in Anhui province of China: Analysis of 93,168 cervical liquid-based cytology test reports.

To analyze the Bethesda System reporting rates, histological follow-up, and HPV genotypes distribution of abnormal cytology in Anhui province of China. According to the Bethesda Reporting System of Cervical Cytology (2014), a retrospective analysis of the cervical liquid-based cytology (LBC) results, abnormal cytology with concurrent HPV genotype testing, and immediate histological follow-up. HPV genotype testing was performed for 15 High-risk types and 6 Low-risk types. Immediate histological correlation results within 6 months after the LBC and HPV results. 6.70% of women with abnormal LBC results, and ASC/SIL was 1.42. The severe histological results in abnormal cytology were ASC-US (18.58%), ASC-H (53.76%), LSIL (16.62%), HSIL (82.07%), SCC/ACa (100.00%), AGC (63.77%). The total HPV-positive rate in abnormal cytology was 70.29%, of which ASC-US, ASC-H, LSIL, HSIL, SCC/ACa, and AGC were 60.78%, 80.83%, 83.05%, 84.93%, 84.51%, 33.33%. The top three detected genotypes were HR HPV 16, 52, and 58. The most commonly detected genotype in HSIL and SCC/ACa was HPV 16. Of the 91 AGC patients, 34.78% were cervical lesions, and 42.03% were endometrial lesions. The HPV-positive rate in the group of AGC-FN was highest and lowest in the group of AGC-EM. The Bethesda System reporting rates of cervical cytology were all within the benchmark range of the CAP laboratory. HPV 16, 52, and 58 were the most common genotypes in our population, and HPV 16 infection has a higher degree of malignancy of cervical lesions. Among patients with ASC-US results, HPV positive patients had a higher rate of biopsy-detected CIN2+ than HPV negative patients.

Spindle-nucleated cells in cervical liquid-based cytology: Difficulties in distinguishing between epithelial and non-epithelial tumours based on morphology.

Uterine tumours resembling ovarian sex cord tumours of the uterine cervix are highly sporadic. Cervical liquid-based cytology revealed two cell patterns: spindle-nucleated cells and polygonal cells.

Prevalence of cervical human papillomavirus and the risk of anal co-infection in kidney transplant recipients: Results from a Danish clinical study.

Kidney transplant recipients (KTRs) have increased risk of human papillomavirus (HPV)-related cancers, including cervical and anal cancer. In this cross-sectional clinical study, we investigated the prevalence of cervical high-risk HPV (hrHPV) and low-risk (lrHPV), risk factors for cervical hrHPV infection, and the prevalence of cervical and anal hrHPV co-infection in KTRs and immunocompetent controls. During 2016-2017, we recruited 125 female KTRs and 125 female immunocompetent controls from one dermatology department (KTRs and controls) and five nephrology departments (KTRs) in Denmark. Liquid-based cervical and anal cytology samples were tested for HPV DNA using the INNO-LiPA test and participants answered a questionnaire on lifestyle. Odds ratios (ORs) were estimated using logistic regression, adjusting for age, lifetime sexual partners, smoking, and (in models concerning anal HPV) receptive anal sex. KTRs had higher prevalence of cervical hrHPV than controls (35.5%vs. 18.2; ORadjusted = 2.9, 95% CI, 1.5-5.5). In contrast, the prevalence of lrHPV was similar in KTRs and controls (25.6%vs. 23.1; ORadjusted = 1.2, 95% CI, 0.7-2.3). KTRs were more likely than controls to have cervical and anal hrHPV co-infection (27.3%vs. 6.6%, ORadjusted = 6.3, 95% CI, 2.7-15.0). Female KTRs had high prevalence of cervical hrHPV, and co-infection with anal and cervical hrHPV was common. Our results underline that KTRs are an important target group for preventive efforts against HPV-related diseases.

The WID-EC test for the detection and risk prediction of endometrial cancer.

The incidence of endometrial cancer is rising. Measures to identify women at risk and to detect endometrial cancer earlier are required to reduce the morbidity triggered by the aggressive treatment required for advanced endometrial cancer. We developed the WID-EC (Women's cancer risk IDentification-Endometrial Cancer) test, which is based on DNA methylation at 500 CpG sites, in a discovery set of cervical liquid-based cytology samples from 1086 women with and without an endometrial cancer (217 cancer cases and 869 healthy controls) with a worse prognosis (grade 3 or ≥stage IB). We validated the WID-EC test in an independent external validation set of 64 endometrial cancer cases and 225 controls. We further validated the test in 150 healthy women (prospective set) who provided a cervical sample as part of the routine Swedish cervical screening programme, 54 of whom developed endometrial cancer within 3 years of sample collection. The WID-EC test identified women with endometrial cancer with a receiver operator characteristic area under the curve (AUC) of 0.92 (95% CI: 0.88-0.97) in the external set and of 0.82 (95% CI: 0.74-0.89) in the prospective validation set. Using an optimal cutoff, cancer cases were detected with a sensitivity of 86% and a specificity of 90% in the external validation set, and a sensitivity and specificity of 52% and 98% respectively in the prospective validation set. The WID-EC test can identify women with or at risk of endometrial cancer.

Expert consensus on label criterion and quality control of artificial intelligence-assisted liquid-based cervical cytology diagnosis dataset (2022 version)

随着数字化病理切片扫描和人工智能技术的迅速发展，宫颈癌智能辅助筛查有望减轻病理医师的工作量，提升宫颈癌筛查效率。用于宫颈癌智能辅助筛查算法研发的液基制片数据集不可或缺，因此标准化数据集建设迫在眉睫。为解决当前宫颈癌人工智能辅助筛查产品缺乏标准化标注数据集且难以开展规范化质量可控的算法训练和评估问题，中华医学会病理学分会细胞病理学组联合北京大学工学院有关专家经反复讨论，对数据集数据质量、数据采集、数据处理、数据标注等形成共识。.

The WID-CIN test identifies women with, and at risk of, cervical intraepithelial neoplasia grade 3 and invasive cervical cancer

BackgroundCervical screening is transitioning from primary cytology to primary human papillomavirus (HPV) testing. HPV testing is highly sensitive but there is currently no high-specificity triage method for colposcopy referral to detect cervical intraepithelial neoplasia grade 3 or above (CIN3+) in women positive for high-risk (hr) HPV subtypes. An objective, automatable test that could accurately perform triage, independently of sample heterogeneity and age, is urgently required.MethodsWe analyzed DNA methylation at ~850,000 CpG sites across the genome in a total of 1254 cervical liquid-based cytology (LBC) samples from cases of screen-detected histologically verified CIN1-3+ (98% hrHPV-positive) and population-based control women free from any cervical disease (100% hrHPV-positive). Samples were provided by a state-of-the-art population-based cohort biobank and consisted of (i) a discovery set of 170 CIN3+ cases and 202 hrHPV-positive/cytology-negative controls; (ii) a diagnostic validation set of 87 CIN3+, 90 CIN2, 166 CIN1, and 111 hrHPV-positive/cytology-negative controls; and (iii) a predictive validation set of 428 cytology-negative samples (418 hrHPV-positive) of which 210 were diagnosed with CIN3+ in the upcoming 1–4 years and 218 remained disease-free.ResultsWe developed the WID-CIN (Women’s cancer risk IDentification-Cervical Intraepithelial Neoplasia) test, a DNA methylation signature consisting of 5000 CpG sites. The receiver operating characteristic area under the curve (AUC) in the independent diagnostic validation set was 0.92 (95% CI 0.88–0.96). At 75% specificity (≤CIN1), the overall sensitivity to detect CIN3+ is 89.7% (83.3–96.1) in all and 92.7% (85.9–99.6) and 65.6% (49.2–82.1) in women aged ≥30 and <30. In hrHPV-positive/cytology-negative samples in the predictive validation set, the WID-CIN detected 54.8% (48.0–61.5) cases developing 1–4 years after sample donation in all ages or 56.9% (47.6–66.2) and 53.5% (43.7–63.2) in ≥30 and <30-year-old women, at a specificity of 75%.ConclusionsThe WID-CIN test identifies the vast majority of hrHPV-positive women with current CIN3+ lesions. In the absence of cytologic abnormalities, a positive WID-CIN test result is likely to indicate a significantly increased risk of developing CIN3+ in the near future.

Predictive Value of HPV E6/E7 mRNA Detection on the Outcome of Cervical LSIL.

Objective. To assess the predictive worth of HPV E6/E7 mRNA detection in the outcome of the cervical low-grade squamous intraepithelial lesion (LSIL). From September 2017 to early September 2019, patients screened for high-risk HPV positive or abnormal cervical liquid-based cytology were retrospectively analyzed and diagnosed with LSIL by cervical biopsy were recruited. The independent influencing factors of the regression of LSIL lesions after follow-up were analyzed, and the outcome of LSIL was calculated. The results of the initial colposcopy in this study were CIN I, CIN II/P16-negative, CIN II/P16-positive, and CIN III. At the time of re-examination, LSIL patients had three outcomes: regression, persistence, and progression. In the two follow-ups, 330 patients were finally included, including 276 CIN I patients (group A) and 54 CIN II/P16-negative patients (group B). The positive rates of HPV E6/E7 mRNA in each group were 66.67% and 70.37% for A and B, respectively. The total positive rate of E6/E7 mRNA was 67.27%, and there was no significant difference between the two groups (P > 0.05). After 1 year follow-up, whether HPV E6/E7 mRNA regressed or was negative was associated with the outcome of LSIL-related lesions (P < 0.05). The regression or negative rate of HPV E6/E7 mRNA was 1.57 times higher than the progression rate of HPV E6/E7 mRNA-positive diagnosis of LSIL lesions. Univariate logistic regression analysis showed that age at first sexual intercourse, HPV E6/E7 mRNA results, and lesion type were statistically significant (P < 0.05). Whether HPV E6/E7 mRNA was negative (OR = 2.420, P=0.001) and age at first sexual intercourse ≥20 years (OR = 0.420, P=0.002) were independent influencing factors associated with LSIL regression. Multivariate logistic analysis showed that age of first sexual intercourse ≥20 years (OR = 0.420, P=0.002) and HPV E6/E7 mRNA-negative (OR = 2.420, P=0.001) were independent factors associated with LSIL. HPV E6/E7 mRNA detection can be used for predicting the outcome of LSIL and has a good application value.

Effects of Menstrual Cycle on Various Morphologies of High-Grade Squamous Intraepithelial Lesions in SurePath™ Liquid-Based Cervical Cytology

Introduction: The morphology of high-grade squamous intraepithelial lesion (HSIL) on Papanicolaou (Pap) smears widely varied, including syncytial aggregates, sheets, and scattered single cells, and no particular cellular pattern is consistently observed. Therefore, this study aimed to determine whether the menstrual cycle affects the cellular pattern of HSILs, an effort to avoid false negatives due to the oversight of scattered small single HSIL cells in the cytological triage of human papillomavirus-positive women. Methods: A total of 147 HSIL samples of liquid-based cytology (LBC) in patients with cervical intraepithelial neoplasia grade 2 or 3 were obtained, and then, the relationship between cellular patterns, such as single-cell-like and syncytial aggregates, and menstrual cycles classified into six phases was analyzed. If a syncytial aggregate was present, the number of cells constituting the aggregate was visually counted under the microscope. Results: HSILs in scattered single cells and small sheets of <6 on LBC samples accounted for 43% (23/54) during the late proliferative phase of the menstrual cycle. A moderately strong statistically significant association was observed between cellular patterns and menstrual cycles (χ² [3] = 9.423, p < 0.05) (Cramer’s V = 0.253). The value of adjusted residuals showed a statistically significant increased proportion of single-cell-like patterns during the late proliferative phase (p < 0.01). Conclusions: The present study demonstrated that HSIL cells in Pap smears in the late proliferation phase have a high frequency of single-cell-like patterns. In human papillomavirus-positive Pap smears with a clean background and predominantly superficial cells, careful microscopic observation by targeting single HSIL cells can potentially reduce false negatives.

Clinical validation of full HR-HPV genotyping HPV Selfy assay according to the international guidelines for HPV test requirements for cervical cancer screening on clinician-collected and self-collected samples

BackgroundAccording to international guidelines, Human Papillomavirus (HPV) DNA tests represent a valid alternative to Pap Test for primary cervical cancer screening, provided that they guarantee balanced clinical sensitivity and specificity for cervical intraepithelial neoplasia grade 2 or more (CIN2+) lesions. The study aimed to assess whether HPV Selfy (Ulisse BioMed – Trieste, Italy), a full-genotyping HPV DNA test that detects and differentiates 14 high-risk HPV (HR-HPV) types, meets the criteria for primary cervical cancer screening described in the international guidelines, on clinician-collected as well as on self-collected samples.MethodsFor each participant woman, consecutively referring to Azienda Sanitaria Universitaria Giuliano Isontina (Trieste, Italy) and CRO—National Cancer Institute (Aviano, Italy) for the cervical cancer screening program, the following samples were tested: (a) a clinician-collected cervical specimen, analyzed with the reference test (Hybrid Capture®2 test, HC2) and HPV Selfy; and (b) a self-collected vaginal sample, analyzed with HPV Selfy. Enrolled women were also asked to fulfill a questionnaire about self-sampling acceptability. As required by guidelines, a non-inferiority test was conducted to compare the clinical performance of the test under evaluation with its reference test.ResultsHPV Selfy clinical sensitivity and specificity resulted non-inferior to those of HC2. By analysis of a total of 889 cervical liquid-based cytology samples from a screening population, of which 98 were from women with CIN2+, HPV Selfy showed relative sensitivity and specificity for CIN2+ of 0.98 and 1.00 respectively (non-inferiority score test: P = 0.01747 and P = 0.00414, respectively); the test reached adequate intra- and inter-laboratory reproducibility. Moreover, we demonstrated that the performance of HPV Selfy on self-collected vaginal samples was non-inferior to the performance obtained on clinician-collected cervical specimen (0.92 relative sensitivity and 0.97 relative specificity). Finally, through HPV Selfy genotyping, we were able to describe HPV types prevalence in the study population.ConclusionsHPV Selfy fulfills all the requirements of the international Meijer’s guidelines and has been clinically validated for primary cervical cancer screening purposes. Moreover, HPV Selfy has also been validated for self-sampling according to VALHUDES guidelines. Therefore, at date, HPV Selfy is the only full-genotyping test validated both for screening purposes and for self-sampling.Trial registration ASUGI Trieste n. 16008/2018; CRO Aviano n.17149/2018

Development and Validation of a Raman Spectroscopic Classification Model for Cervical Intraepithelial Neoplasia (CIN).

Simple SummaryThere is an unmet clinical need for new methods to aid clinicians in the early detection of cervical cancer and precancer. Spectroscopic methods such as Raman spectroscopy can provide a rapid, label-free and nondestructive measurement of the biochemical fingerprint of cells, tissues and biofluids. This study aims to demonstrate the clinical utility of Raman spectroscopy for the identification of cervical precancer. Raman spectra were recorded from cervical smear samples (n = 662) and a classifier was developed based on histology. A classification accuracy of 91.3% was achieved in an independent blinded test set (n = 69), demonstrating the potential clinical utility of Raman spectroscopy.The mortality associated with cervical cancer can be reduced if detected at the precancer stage, but current methods are limited in terms of subjectivity, cost and time. Optical spectroscopic methods such as Raman spectroscopy can provide a rapid, label-free and nondestructive measurement of the biochemical fingerprint of a cell, tissue or biofluid. Previous studies have shown the potential of Raman spectroscopy for cervical cancer diagnosis, but most were pilot studies with small sample sizes. The aim of this study is to show the clinical utility of Raman spectroscopy for identifying cervical precancer in a large sample set with validation in an independent test set. Liquid-based cervical cytology samples (n = 662) (326 negative, 200 cervical intraepithelial neoplasia (CIN)1 and 136 CIN2+) were obtained as a training set. Raman spectra were recorded from single-cell nuclei and subjected to a partial least squares discriminant analysis (PLSDA). In addition, the PLSDA classification model was validated using a blinded independent test set (n = 69). A classification accuracy of 91.3% was achieved with only six of the blinded samples misclassified. This study showed the potential clinical utility of Raman spectroscopy with a good classification of negative, CIN1 and CIN2+ achieved in an independent test set.

The WID-BC-index identifies women with primary poor prognostic breast cancer based on DNA methylation in cervical samples

Genetic and non-genetic factors contribute to breast cancer development. An epigenome-based signature capturing these components in easily accessible samples could identify women at risk. Here, we analyse the DNA methylome in 2,818 cervical, 357 and 227 matched buccal and blood samples respectively, and 42 breast tissue samples from women with and without breast cancer. Utilising cervical liquid-based cytology samples, we develop the DNA methylation-based Women’s risk IDentification for Breast Cancer index (WID-BC-index) that identifies women with breast cancer with an AUROC (Area Under the Receiver Operator Characteristic) of 0.84 (95% CI: 0.80–0.88) and 0.81 (95% CI: 0.76–0.86) in internal and external validation sets, respectively. CpGs at progesterone receptor binding sites hypomethylated in normal breast tissue of women with breast cancer or in BRCA mutation carriers are also hypomethylated in cervical samples of women with poor prognostic breast cancer. Our data indicate that a systemic epigenetic programming defect is highly prevalent in women who develop breast cancer. Further studies validating the WID-BC-index may enable clinical implementation for monitoring breast cancer risk.