Liquid-based Cytology Samples Research Articles

Clinical performance of DNA and RNA based HPV tests for test of cure (TOC) post treatment for cervical intraepithelial neoplasia (CIN) - a retrospective study.

HPV testing as a "test of cure" (TOC) of women treated for cervical high-grade lesions can support and inform appropriate clinical management pathways. However, there is a lack of studies that report the discrete performance of different HPV assays in this context, including HPV mRNA based assays. To address this, we performed an analysis of the clinical performance of two hrHPV assays in the (TOC) setting; the recently launched DNA based Alinity m HR HPV (Abbott Molecular) and RNA based Aptima HPV assay (Hologic). Using a retrospective case-control design, two panels of archived cervical liquid based cytology samples, originally taken as per routine TOC protocols in Scotland were assessed. Each panel contained 63 cases, where cervical intraepithelial neoplasia 2 or worse (CIN2+) was detected and 160 controls (women with no CIN2+ and two subsequent cytology negative results (minimum) 3 years apart or women who had histologically confirmed ≤CIN1). All samples were previously tested using the RealTime High Risk HPV assay (Abbott Molecular) as per national TOC protocol. Panel A and Panel B were tested using Alinity and Aptima assay respectively. Both assays showed similar performance to the original RealTime assay. Aptima had sensitivity for CIN2+ of 96.8% (95% CI: 89.0- 99.6) compared to RealTime (93.7% (95% CI: 84.5 - 98.2)). Alinity had sensitivity for CIN2+ of 92.1% (95% CI: 82.4- 97.4) compared to RealTime (98.4% (95% CI: 91.5- 99.95)). Both mRNA based and DNA based HPV tests show robust performance for the monitoring of residual disease post-treatment.

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

Use of the Biocartis Idylla™ Platform for the Detection of Epidermal Growth Factor Receptor, BRAF and KRAS Proto-Oncogene Mutations in Liquid-Based Cytology Specimens from Patients with Non-Small Cell Lung Carcinoma and Pancreatic Adenocarcinoma

The study aimed to demonstrate rapid and effective molecular testing on liquid-based cytology (LBC) samples for EGFR, KRAS and BRAF mutations using the Biocartis Idylla™. Rapid on-site evaluation (ROSE) LBC samples for patients with non-small cell lung carcinoma (NSCLC) or pancreatic ductal adenocarcinoma (PDAC) were tested for EGFR, KRAS and BRAF mutations based on the relevance to tumour subtype. The quantification values (Cq values) and mutation detection status were compared between LBC samples and routine formalin-fixed paraffin-embedded (FFPE) clot samples. ROSE LBC samples (n = 54) showed a higher yield of well-preserved tumour and wild type (WT) DNA, demonstrated by lower quantification cycles, no false positives or false negatives, and a higher sensitivity for low allele frequency mutations when compared with FFPE clot samples. The Biocartis Idylla™ provides highly sensitive, reliable and rapid testing for LBC samples for the detection of EFGR and KRAS mutations. BRAF mutations were not detected in the participant cohort; however, all LBC WT BRAF results correlated with the results from the FFPE clot samples. Access to rapid molecular testing using LBC samples can detect the most frequent driver mutations closer to the time of diagnosis, enabling the selection of the most effective first-line targeted therapy sooner, reducing delays or side effects from suboptimal treatments, patient anxiety and costs to healthcare systems, whilst improving patient outcomes.

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.

Diagnostic value of endometrial cytology and related technology.

Endometrial cytology is not much popular in the world, but is commonly used in a few countries. Although cytomorphological evaluation of endometrial cytology is complicating, recent advance in technology helps improve diagnostic accuracy. In addition, new reporting system, The Yokohama System, has been introduced as a standard reporting system resembling The Bethesda System of the uterine cervical cytology. Although sample standardization is one of the causes in diagnostic problem, it was solved by liquid-based cytology (LBC) technology. In addition, standardized diagnostic algorithm by cytomorphological assessment of LBC samples, the Osaki Study Group (OSG) method, was recently proposed as a reliable and reproducible method. LBC can be utilized for ancillary methods. Application of immunocytochemistry and molecular technology on endometrial cytology samples has been studied to improve diagnostic accuracy. Recent progress of molecular technology has revealed many driver gene mutations in endometrial cancer and its precursors. Surprisingly, many studies revealed that even normal endometrial tissue had driver gene mutations. Based on the recent advance in knowledge of molecular profile of endometrial lesions and normal endometrial tissue, endometrial cytology will gain much power in clinical usefulness.

Guided Needle Aspirations of Intra-Abdominal Masses: Analogy between Liquid-Based and Conventional Smear Preparation Methods

Objective: Image-guided fine needle aspiration cytology with conventional smear (CS) preparation offers onsite cellular adequacy evaluation; however, it still provides false negatives due to faulty smear preparations. Liquid-based cytology (LBC) can be advantageous in these scenarios. Hence, with an aim to investigate utility of LBC in these samples, we carried out the above study with objectives to find diagnostic accuracy of LBC and agreement of LBC with CS methods in guided aspiration samples from intra-abdominal masses. Methods: A prospective observational study, of 113 patients with clinical or radiological diagnosis of intra-abdominal masses, was carried out. SurePath BD™ was used for LBC smear preparation, and the standard protocol was used for CS preparation. Results: LBC alone was diagnostic in 80.8% of the cases, and CS alone was diagnostic in 71.2% cases (agreement was 83.7%, p = 0.03). Cellular morphology was better preserved in LBC; however, interpretation was easier in CS. Conclusion: CS may be complimented with LBC sample collection method to enhance the sensitivity of intra-abdominal FNA.

HIGH RISK HPV DETECTION IN PRECURSOR LESIONS OF CERVIX USING LBC SAMPLES.

Background: Liquid-based cytology (LBC) was developed as an alternative to conventional cytology screening to improve specimen adequacy and sensitivity in detecting cervical abnormalities. High risk HPV (HR HPV) types 16 and 18 are responsible for about 70% of all cervical cancer cases worldwide. Objectives: This study aimed (1) to compare the diagnostic efciency of liquid based cytology LBC and conventional tests in detecting cervical dysplasia and (2) detection of high risk HR HPV types 16 and 18 in cervical precursor lesions using liquid based cytology samples. Methods: A prospective comparative study was conducted from the year August 2014 – February 2019 at Basavatarakam Indoamerican Cancer Hospital and Research Institute, Hyderbad.A total of 200 women undergoing screening for cervical cancer were included in the study. HR-HPV 16 & 18 detection was done by Nested Multiplex Polymerase Chain Reaction (NMPCR). Results: The area under the ROC curve for conventional Pap smear was 0.725 and LBC was 0.974, showing that the diagnostic accuracy of LBC method in diagnosing precursor lesions and carcinoma of cervix is better than the conventional Pap smear method. HR-HPV 16/18 were positive in 43/102 (42.2%) of cases. HPV 16 alone was positive in 32/43(74.4%), HPV 18 alone in 6/43 (13.9%) and both HPV-16 and HPV-18 were positive in 5/43 (11.6%) of the total HR-HPV positive cases. Conclusion: The detection rate of epithelial abnormalities and infections in LBC preparation was better than CPS. LBC samples can be used for HR-HPV genotype detection to explore new paradigms of screening strategies.

Diagnostic accuracy of Thinprep® in cervical lymph node aspiration: Assessment according to the Sydney system.

Fine-needle aspiration cytology (FNAC) is a reliable technique that has been used for many years in lymphadenopathy diagnosis. Although conventional smear is the standard approach in FNAC, liquid-based cytology (LBC) is accepted as an alternative method. Reporting standardization is a significant gap in fine-needle aspiration cytology, leading to failure in pathologist-clinician dialog and interobserver variability. In 2020, an expert panel proposed the Sydney system for classifying and reporting lymph node aspiration cytology. This study aimed to evaluate the diagnostic accuracy of LBC in lymph node aspiration cytology under the guidance of the Sydney system. Five hundred-four LBC samples were reevaluated and classified according to the Sydney system. Of these, n= 24 were categorized as L1-inadequate/non-diagnostic, n= 283 as L2-benign, n= 36 as L3- atypical cells of undetermined significance/atypical lymphoid cells of uncertain significance (AUS/ALUS), n= 48 as L4-suspicious, and n= 113 as L5-malignant. Four hundred-one samples were histopathologically confirmed. The diagnostic accuracy of LBC and the risk of malignancy for each Sydney category were calculated. The results were as follows: sensitivity 98.97%; specificity 98.60%; positive predictive value 94.80%; negative predictive value 99.29%; and overall diagnostic accuracy 98.75%. The ROM was 16.6% for L1, 0.7% for L2, 88.8% for L3, and 100% for L4 and L5. LBC is suitable for use in lymph node aspiration under the guidance of the Sydney system and has high diagnostic accuracy. Future comprehensive studies will increase the applicability of the Sydney system and minimize interobserver variability.

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.

Targeted Next Generation Sequencing for Human Papillomavirus Genotyping in Cervical Liquid-Based Cytology Samples.

Simple SummaryTesting for Human Papillomavirus (HPV) is currently being implemented as part of cervical cancer (CC) screening in several countries. However, infections with all but one of the HPV types classified as possibly carcinogenic cannot be detected by the assays used for CC screening today. The aim of our study was to demonstrate the use of a targeted next generation sequencing (NGS) HPV panel for CC screening—both general practitioner-collected samples and self-samples. We here show that the targeted HPV panel can detect HPV with a sensitivity and specificity similar to commercial HPV assays, one of which is used for CC screening today. However, the targeted HPV panel possess several advantages compared to the screening assays, as it enables specific detection of all relevant HPV types and can identify viral integration, variants in the HPV genome, and dominant HPV types in multi-infected cases.At present, human papillomavirus (HPV) testing is replacing morphology-based cytology as the primary tool for cervical cancer screening in several countries. However, the HPV assays approved for screening lack detection for all but one of the possibly carcinogenic HPV types and do not genotype all included HPV types. This study demonstrates the use of a targeted HPV next generation sequencing (NGS) panel to detect and genotype all 25 carcinogenic, probably carcinogenic, and possibly carcinogenic HPV types as well as the low-risk types HPV6 and HPV11. The panel was validated using a cohort of 93 paired liquid-based cytology samples (general practitioner (GP)-collected cervical samples and cervico-vaginal self-samples (SS)). Overall, the targeted panel had a sensitivity (GP = 97.7%, SS = 92.1%) and specificity (GP = 98.0%, SS = 96.4%) similar to the commercial HPV assays, Cobas® 4800 HPV DNA test (Roche) and CLART® HPV4S assay (GENOMICA). Interestingly, of the samples that tested positive with the NGS panel, three (6.4%) of the GP-collected samples and four (9.1%) of the self-samples tested positive exclusively for HPV types only included in the NGS panel. Thus, targeted HPV sequencing has great potential to improve the HPV screening programs since, as shown here, it can identify additional HPV positive cases, cases with HPV integration, variants in the HPV genome, and which HPV type is dominant in multi-infected cases.

TUBB3 immunostaining improves the diagnostic accuracy of oral liquid-based cytology in squamous cell carcinoma.

Although class III beta-tubulin (TUBB3) is not expressed in normal epithelium, its expression in cancers of some organs has been reported. Herein, we investigated the expression pattern and expression levels of TUBB3 in oral squamous cell carcinoma (SCC), and assessed whether TUBB3 immunostaining could improve the diagnostic accuracy of oral scraping liquid-based cytology (LBC). Paraffin sections of biopsies from 107 patients with primary SCC and 30 patients with squamous papilloma of the tongue or gingiva were immunostained for TUBB3. In addition, 15 LBC samples obtained from the study participants with SCC were immunostained for TUBB3. Seven LBC samples were false-negative. The TUBB3 expression level in each sample was evaluated and classified as 3+, 2+, 1+, or 0. TUBB3 expression was confirmed in 91.6% of paraffin-embedded SCC specimens. Clear and diffuse positivity (2+ or above) was observed in 77.6% of the total cases. In the well-differentiated type, tumour cells in the middle layer of the parenchyma specifically expressed TUBB3. In almost LBC samples, cancerous intermediate cells showed immunopositivity similar to that of paraffin samples, even if cellular atypia was not clear in Papanicolaou staining. TUBB3 immunostaining is useful for diagnosing oral SCC in scraping LBC, especially when samples consist of intermediate cells with little morphological change. Moreover, TUBB3 immunostaining could improve the diagnostic accuracy of oral scraping LBC by reducing false-negatives.

Comparison of Visual Inspection with Acetic Acid (VIA) and Liquid Based Cytology (LBC) with Reflex HPV DNA Test in Cervical Cancer Screening

A cross-sectional study was conducted to examine the diagnostic outcome of visual inspection with acetic acid (VIA) and liquid based cytology (LBC) and see the association of VIA and LBC with HPV status, between March 2015 and June 2017. A total of 72 women attending the Out-Patient Department (OPD) of Gynaecology (Colposcopy Clinic) of Bangabandhu Sheikh Mujib Medical University (BSMMU) for VIA test participated in the study. All the participants underwent both VIA and LBC procedures first. Then HPV DNA test was done in all the cases by hybrid capture (hc2 High Risk HPA DNA test) from residual LBC samples. Thereafter, biopsies were taken from all the patients for histopathological confirmation. The mean age of the participants was 40.22±12.29 years, (age range 18-72). Cervical lesions of total 36 patients were VIA positive. Among them 2(5.6%) patients were HPV positive and 34(94.4%) were HPV negative. There was no significant association found between VIA positivity and HPV positivity (p>0.05). Based on LBC, ASCUS were found in 4 patients, NILM in 52 patients and LSIL in 2 patients; all were HPV negative. ASC-H were found in 4 cases among them 1(25.0%) was HPV positive and 3(75.0%) were HPV negative. Squamous cell carcinoma was found in 1, which was HPV positive. The association between two groups was statistically significant (p<0.05). We found the sensitivity, specificity, accuracy, positive predictive value and negative predictive value of (VIA), as compared against histopathology, were 75.0%, 40%, 45.8%, 20% and 88.9% respectively, while for liquid based cytology (LBC) those were found 75.0%, 90.6%, 88.9%, 50.0% and 96.6% respectively. Our data suggest that liquid based cytology (LBC) with reflex HPV testing gives better results than conventional screening through VIA test.
 CBMJ 2021 January: vol. 10 no. 01 P: 18-23

Immunohistochemistry Applied to Breast Cytological Material.

Fine-needle aspiration biopsies (FNABs) of the breast are minimally invasive procedures enabling the diagnosis of suspicious breast lesions. Unfortunately, they are often perceived as inferior to core-needle biopsies, namely because they are supposedly unable to differentiate between high-grade ductal carcinoma in situ and invasive carcinoma or provide material for ancillary testing. Several studies have shown, however, that FNAB samples, when handled properly, are indeed capable of providing sufficient and adequate material for ancillary testing, namely immunocytochemistry (ICC). We reviewed the published literature regarding the use of ICC for both diagnostic and theranostic uses in the different types of cytological samples obtained from FNABs of the breast, including smears, liquid-based cytology samples, and cellblocks. We found that p63 and 34βE12 show promise in aiding in the differential diagnosis between in situ and invasive lesions and that most other diagnostic markers may be used as in tissue. Regarding theranostic ICC markers, results vary between publications, but with care, these can successfully be performed in cytological samples. Air-dried smears should be avoided, and cellblocks are overall more versatile than cytology slides, enabling the evaluation of not only hormonal receptors and HER2 by ICC, but also of Ki-67. Particular attention should be paid to fixation and antigen retrieval procedures in all cases. We recommend that laboratories without experience perform short validation runs before adopting these techniques into clinical practice.

The Frequency and Typing of HPV Virus Among Suspected Women Referred to HPV Genotyping Test in Mazandaran, Northern Iran.

Human papilloma viruses are a group of the Papillomaviridae family (ds DNA viruses), which infect basal epithelial cells. So far, 228 types of HPV have been identified, in which about 40 types infect the genital mucosa. In Iran, cervical cancer has been reported as the second most common malignancy in women which is approximately 8.8% of all cancers in women. HPV genotypes are classified as high-risk and low-risk according to cervical cancer. According to previous reports, nearly 70% of cervical cancers occur by HPV genotypes 16 and 18, of which genotype 16 is known as the most prevalent type. The main goal of this study was determining the frequency of HPV virus and its genotypes in the female population of Mazandaran. This study was performed on 91 pathological samples. DNA was extracted from 500 μL of liquid-based cytology samples and PCR was performed for all of the samples. Genotyping step was performed based on strip assay method. HPV 39 (6.55%), 56 (3.27%), 51 (3.27%), and 68 (3.27%) were the most frequent types respectively. Also, HPV 11 (8.19%) and 6 (3.27%) show the most frequency among LR-HPV genotypes. HPV type 6 (16.39%), 56 (14.75%), 11 (14.75%), 16 (13.11%), and 66 (11.47%) were the four most common types seen in mixed infection samples. Differences among the types of HPV can be due to various geographical distributions of HPV. Our results revealed HPV 39 (6.55%) is the most common type among of HR-HPV followed by HPV 56, 51, 68 (3.27%); however, HPV 16 and HPV 18 were seen in just one case. HPV 11 (8.19%) and HPV 6 (3.27%) were the most common type among of LR-HPV.

The expression pattern of CD10 and CD31 identifies fine fibrovascular stroma of grade 1-endometrial endometrioid carcinomas in cytology.

The objective of this study was to assess the diagnostic utility of CD10 in the differential diagnosis of grade 1-endometrial endometrioid carcinoma (G1-EEC) and the metaplastic changes associated with the endometrial glandular and stromal breakdown (EGBD) on liquid-based cytological (LBC) samples. (1) The type and distribution of CD10-positive cells in EGBD and G1-EEC patients were evaluated. (2) Based on the results from (1), histological and cytological specimens were double-immunostained with CD31 and CD10 to confirm whether CD10-positive tubular-canalicular material found in (1) was represented by fine threads of endometrial-type fibrovascular stroma. (3) Based on the results from (2), additional immunostaining of histological specimens was performed for CD146 and αSMA as markers of perivascular cells. (1) CD10 positive cells showed two main patterns of expression: cytoplasmic immunoreactivity in the form of dense brown granules in EGBD and tubular-canalicular branching patterns in G1-EEC. (2) The tubular-canalicular material observed in cytological specimens of G1-EEC samples co-expressed CD10 and CD31, and was interpreted as representing fine threads of endometrial fibrovascular stroma in the corresponding histological samples. Conversely, metaplastic changes in EGBD cases, only a few CD31-positive signals were found inside the condensed stromal clusters with CD10-positive. (3) Cells surrounding the CD31-positive vascular endothelial cells expressed CD146 and αSMA; moreover, some of the thin CD10-positive fibrous stromal strands also co-expressed αSMA. CD10 is a very useful immunomarker for distinguishing between G1-EEC and the metaplastic changes of EGBD in LBC samples.

Human papillomavirus detected in sperm of Japanese infertile males affects reproductive parameters

The effects of human papillomavirus (HPV) infection on male reproductive parameters are currently a matter of controversy. In order to clarify the issue in Japanese infertile men, the prevalence and localization of HPV in semen, sperm parameters, and superoxide dismutase (SOD) activity in seminal plasma were examined in 216 Japanese infertile men. DNA was extracted from liquid-based cytological semen samples. The β-globin gene was amplified by polymerase chain reaction (PCR), and HPV-DNA was amplified using nested PCR with MY09/MY11 as outer primers and GP5+/GP6+ as inner primers. HPV genotyping was performed in the HPV-positive samples. In addition, SOD levels in seminal plasma were analysed quantitatively. In-situ hybridization (ISH) was performed to localize HPV-DNA in sperm from HPV-positive samples. Any-risk and high-risk prevalence rates of HPV in semen were 12.5% and 6.9%, respectively. No significant difference in the prevalence of HPV was observed between azoospermic and non-azoospermic subjects. Among non-azoospermic patients, those with HPV detected in semen had significantly lower sperm motility and concentration compared with subjects without HPV detected in semen. SOD levels in seminal plasma were significantly higher in HPV-positive patients compared with HPV-negative patients. ISH analysis of HPV-positive samples revealed that HPV-DNA was localized to the head and mid-piece of sperm. HPV-DNA was present in the sperm of young infertile men. HPV infection of sperm was associated with reduced sperm motility and concentration, and resulted in an increase in seminal SOD activity.

Predicting regression of cervical intraepithelial neoplasia grade 2 in women under 25 years

A number of retrospective and prospective studies have documented substantial rates of regression in cervical intraepithelial neoplasia grade 2 lesions in young women. Initial observational management of cervical intraepithelial neoplasia grade 2 is increasingly accepted as appropriate for women under 25 years of age with screen-detected abnormalities and is included in a number of clinical guidelines. However, there has been a paucity of large prospective studies on observational management with strict inclusion criteria. A number of important questions remain, specifically regarding the clinical variables that are associated with the risk of progression or persistence of disease. To investigate these factors and to ensure that young women with cervical intraepithelial neoplasia grade 2 undergoing observational management were being managed in a well-monitored and an appropriately informed fashion, we conducted a large, multicenter prospective study on observational management of cervical intraepithelial neoplasia grade 2 in women under 25 years. This study aimed to determine the regression rates and clinical, cytologic, and pathologic predictors of regression of cervical intraepithelial neoplasia grade 2 in women under 25 years undergoing observational management over 24 months. This study was a multicenter prospective study on observational management of cervical intraepithelial neoplasia grade 2 (ie, repeat colposcopy, cytology, and cervical biopsy every 6 months) for up to 24 months. A total of 615 consenting women under 25 years with newly-diagnosed, biopsy-proven cervical intraepithelial neoplasia grade 2 were recruited (from 2010 to 2016) through 16 hospital-based colposcopy units in New Zealand and Australia. At completion, 326 women had confirmed regression, 156 had persistent high-grade cervical intraepithelial neoplasia grade 2 or 3 or adenocarcinoma in situ, and 24 had unconfirmed regression (ie, first regression at the 24-month follow-up). A total of 109 women did not complete the protocol (41 because of delayed follow-up, 41 lost to follow-up, 22 elected treatment, 4 refused a biopsy, and 1 died of an unrelated cause). Confirmed regression was observed in 53% (326 of 615) of all women enrolled in the study and, when missing data were imputed, it was estimated that 64% of women (95% confidence interval, 60%-68%) would have experienced regression. Similarly, lesions regressed in 64% (326 of 506) of women who completed the observational protocol. Based on a multivariable analysis, detection of human papillomavirus 16 in a liquid-based cytology sample at the time of initial colposcopy decreased the chance of regression by 31% (risk ratio, 0.69; 95% confidence interval, 0.56-0.86; P<.001). In addition, at initial colposcopy, low-grade or normal colposcopic impression, later year of diagnosis, low-grade or normal cytology, and being a nonsmoker were all independently associated with an increased chance of regression. More than half of women under 25 years with cervical intraepithelial neoplasia grade 2 will regress to cervical intraepithelial neoplasia grade 1 or normal within 24 months without destructive treatment. The absence of human papillomavirus 16 is the most important predictor of regression.

Performance of a 50-gene next generation sequencing panel with post-centrifuge supernatant cytology fluid in non-small-cell lung cancer.

Liquid based cytology (LBC) specimens are increasingly utilized for molecular analysis, as results are comparable to molecular analysis performed on traditional specimens (biopsy or cell block). However, there are few studies demonstrating the long-term viability of DNA in LBC samples. In this study, a 50-gene next generation sequencing (NGS) panel was performed on DNA isolated from post-centrifuged supernatant LBC samples of cases of non-small-cell lung carcinoma. Comparison was made to results of an identical NGS panel performed on a concurrent clinical sample (biopsy or cell block). Quality parameters including DNA concentration, total reads, amplicons with reads under 450 and 350, and variant allele fraction were also compared. For a subset of LBC samples, DNA was isolated after being held for varying extended lengths of time after collection (up to 41 days) at 5°C and results compared. Results of NGS mutation analysis were concordant between LBC samples and clinical samples. DNA concentration was on average higher in the LBC samples compared to the clinical samples. The remaining metrics were more variable, but illustrated the adequacy of LBC samples for NGS testing. DNA isolated from LBC samples held for longer periods of time was of good concentration. NGS analysis was successfully performed on all samples, with concordance with results of clinical samples. DNA isolated directly from LBC fluid is suitable for NGS analysis. DNA is also stable in LBC preservative for extended periods of time before isolation and NGS analysis can subsequently be successfully performed.

SARS-CoV-2 RNA may rarely be present in a uterine cervix LBC sample at the asymptomatic early stage of COVID 19 disease.

ObjectiveCurrently, it is thought that uterine cervix mucosal samples present a low risk of SARS‐CoV‐2 exposure. So far, there is no evidence of SARS‐CoV‐2 detection in Papanicolaou (Pap) smears. Nevertheless, clinicians could be exposed unaware to the coronavirus while performing and handling a Pap smear. We aimed to retrospectively evaluate the presence of SARS‐CoV‐2 RNA in cervical liquid‐based cytology (LBC) samples in women who tested positive for a nasopharyngeal COVID‐19 PCR test.MethodsFrom our laboratory database, we identified patients with data on a cervical cancer screening LBC sample paired with a positive nasopharyngeal COVID‐19 PCR test. Relevant LBC samples taken within an incubation period of 14 days and post‐onset RNA shedding interval of 25 days were subsequently tested for SARS‐CoV‐2 RNA using RT‐PCR tests.ResultsThe study group consisted of 102 women. Of those, 23 LBC samples were tested. SARS‐CoV‐2 RNA was detected in one LBC sample from a 26‐year‐old asymptomatic woman taken six days before reporting headaches and knee arthralgia with a positive nasopharyngeal SARS‐CoV‐2 RT‐PCR test.ConclusionsIt is possible to detect SARS‐CoV‐2 RNA in cervical LBC samples at an early asymptomatic stage of COVID‐19. In general, this finding is infrequent in asymptomatic women who tested SARS‐CoV‐2 positive within an incubation of 14 days and a post‐onset RNA shedding period of 25 days. We fully support the current thinking that cervical LBC samples from asymptomatic women pose a low risk of SARS‐CoV‐2 exposure and can be handled in the frame of good microbiological practice and procedures.

Detection of HIV mRNA in routine liquid-based cytology specimens of HIV-infected women.

Human immunodeficiency virus-infected women have a high incidence of HPV infection, and HIV and HPV coinfection is associated with high incidence of cervical intraepithelial lesions and cervical cancer. This study investigated the ability to detect HIV mRNA in routine screening cervical liquid-based cytology (LBC) samples and its correlation with HPV coinfection and cervical intraepithelial lesions. Liquid-based cytology samples from 80 HIV-infected women under combined antiretroviral therapy (cART) were studied for detection of HIV and HPV mRNA using Aptima® tests and for cytology diagnosis according to the 2014 Bethesda System for Reporting Cervical Cytology. Peripheral blood (PB) HIV mRNAs were assessed by real-time polymerase chain reaction (RT-PCR). Statistical analysis used Fisher's exact or Chi-square test to compare frequencies among groups and the Mann-Whitney U test to compare continuous variables. Human immunodeficiency virus mRNA was present in 21.3% of routine LBC samples in HIV-infected women, 12.5% of which had no detectable PB viral load. Among 10 patients diagnosed with high-grade squamous intraepithelial lesion (HSIL), 50% had detectable HIV viral load. The occurrence of HSIL vs low-grade intraepithelial lesion/negative intraepithelial lesion or malignancy in LBC samples was significantly higher in women with detectable HIV viral load (P=.029). Human immunodeficiency virus mRNA was present in routine LBC samples in HIV-positive women under cART. Detection of HIV viral load in LBC is significantly associated with cervical HSIL. This suggests the relevance of HIV mRNA viral load assessment in routine LBC, to evaluate patients' infectious potential and monitor efficacy of the cART scheme.