Residual Liquid-based Cytology Specimens Research Articles

Using the DNA Integrity Number to Analyze DNA Quality in Specimens Collected from Liquid-Based Cytology after Fine-Needle Aspiration of Breast Tumors and Lesions

Introduction: Cancer genome analysis using next-generation sequencing requires adequate and high-quality DNA samples. Genomic analyses were conventionally performed using formalin-fixed paraffin-embedded sections rather than cytology samples such as cell block or smear specimens. Specimens collected from liquid-based cytology (LBC) have the potential to be sources of high-quality DNA suitable for genetic analysis even after long-term storage. Methods: We collected breast tumor/lesion fractions from 92 residual LBC specimens using fine-needle aspiration (FNA) biopsy, including breast carcinoma (1 invasive carcinoma and 4 ductal carcinomas in situ), papillomatous lesion (5 intraductal papillomas), and fibroepithelial lesion (19 phyllodes tumors and 53 fibroadenomas) samples, and others (1 ductal adenoma, 1 hamartoma, 1 fibrocystic disease, and 7 unknown). DNA was extracted from all samples and subjected to DNA integrity number (DIN) score analysis. Results: Average DIN score collected from 92 LBC specimens was significantly higher score. In addition, high-quality DNA with high DIN values (7.39 ± 0.80) was successfully extracted more than 12 months after storage of residual LBC specimens. Conclusion: Residual LBC specimens collected from FNA of the breast were verified to carry high-quality DNA and could serve as an alternate source for genetic analysis.

Cancer gene analysis of liquid-based cytology specimens using next-generation sequencing: A technical report of bimodal DNA- and RNA-based panel application.

As liquid-based cytology (LBC) specimens harbor high-quality DNA, genomic analysis using LBC specimens is beneficial for integrative diagnosis. This study aimed to clarify the feasibility of LBC specimens for a bimodal application of DNA- and RNA-based next-generation sequencing (NGS) panels. LBC specimens were prepared from cultured human cancer HEC59 cells using commercially available fixatives (Cellprep, CytoRich Red, and SurePath solutions), and were subjected to NGS for a feasibility study. Clinical LBC specimens of thyroid and salivary gland tumors were prepared using CytoRich Red solution. After DNA and RNA extraction, NGS analyses were performed in a single run using combined DNA- and RNA-based custom-made cancer panels for the detection of gene mutations and fusions. High-quality DNA and RNA were obtained, and the expected gene mutations and fusions were detected in HEC59 cells using all types of LBC fixatives. Most available clinical cases (18 out of 20) exhibited pathogenic gene mutations (15 cases) and fusion genes (3 cases) using the bimodal DNA- and RNA-based panels. Overall, 18 cases (90%) showed oncogenic mutations or fusion genes of diagnostic values. Simultaneous application of bimodal DNA- and RNA-based gene panels was useful in NGS analysis using residual LBC specimens for integrative diagnosis. Residual LBC specimens for genomic analysis, including fusion gene analysis, are particularly useful for obtaining genomic information before surgical resection.

Sequential evaluation of MUC promoter methylation using next-generation sequencing-based custom-made panels in liquid-based cytology specimens of pancreatic cancer.

As liquid-based cytology (LBC) specimens preserve high-quality DNA, clinical sequencing of LBC specimens using next-generation sequencing (NGS) is becoming a common strategy. This study aimed to evaluate the feasibility of NGS-based custom-made panels for evaluating MUC promoter methylation in LBC specimens. Thirty-one patients with pancreatic cancer were enrolled in the study. Cancer tissue samples were obtained using endoscopic ultrasound-guided fine-needle aspiration/biopsy (EUS-FNA/FNB). LBC, formalin-fixed paraffin-embedded (FFPE), and fresh frozen specimens were prepared for DNA extraction after pathological diagnosis. These specimens were then subjected to NGS analysis using custom-made cancer gene screening and methylation panels comprising 28 cancer-related genes and 13 gene promoter regions, including MUC1, MUC2, and MUC4. The success rate of NGS using the cancer gene panel was comparable among the LBC, FFPE, and frozen specimens, and the presence of cancer cell-derived somatic mutations in each specimen was confirmed. The specimens were then tested using a methylation panel that revealed the sequential methylation status of CpG islands located in the promoter regions of MUC genes. The methylation status results obtained from LBC specimens were almost comparable with those from FFPE and frozen specimens. MUC and other gene methylation analyses using an NGS-based panel were successfully performed in residual LBC specimens obtained by EUS-FNA/FNB. Therefore, this approach provides an alternative source of molecular tests for gene mutations and methylation, especially in the pancreatic cancers, which are often unresectable and unsuitable for obtaining FFPE specimens.

Role of immunocytochemistry in cervical cancer screening.

The cervical cancer screening has been based conventionally on cytologic analysis. With advances in understanding the role of human papillomavirus, cotesting has been applied. But most of the patients subjected to colposcopy did not benefit, except in cases with HSIL [high-grade squamous intraepithelial lesion] cytology. Because of this, a step to increase the sensitivity to detect cancers and pre-cancers but with higher specificity with minimal overdiagnosis leading to prevention of unindicated cervical biopsies is highly desired. Such triaging step in cases with abnormal screening results is expected to minimize invasive interventions because of low false positivity. With availability of methodologies leading to quantitatively and qualitatively enhanced cell-blocks from residual liquid based cytology specimens, immunostaining can be performed for multiple immunomarkers with potential objectivity to triage initial screening test results. This is enhanced further with inclusion of AV marker in the cell-blocks and application of SCIP (subtractive coordinate immunoreactivity pattern) approach. The cell-blocks are also resource for performing other ancillary studies including molecular pathology and proteomics/metabolomics as potential tests in future. This review explores application of residual liquid based cytology specimen for cell-blocking with application of ancillary studies in algorithmic manner as adjunct to ASCCP management guidelines for improved patient care.

Kras Gene Analysis Using Liquid-Based Cytology Specimens Predicts Therapeutic Responses and Prognosis in Patients with Pancreatic Cancer.

Simple SummaryNew therapeutic strategies are needed to improve the prognosis of pancreatic ductal adenocarcinoma (PDAC) and developing biomarkers that can guide individualized treatment decisions is an important part of these strategies. In this study, we found that unresectable PDAC patients harboring wild-type Kras had significantly longer progression-free survival (PFS) and overall survival (OS) than those harboring mutant Kras after undergoing first-line gemcitabine and nab-paclitaxel (GA) therapy and that wild-type Kras was a significant predictor of longer PFS and OS. This is the first report suggesting that Kras gene analysis has the potential to predict therapeutic responses to GA and the prognosis of unresectable PDAC.Background: Although several molecular analyses have shown that the Kras gene status is related to long-term survival of patients with pancreatic ductal adenocarcinoma (PDAC), the results remain controversial. Here, we examined the Kras gene status in a cohort of unresectable PDAC patients who underwent first-line therapy with gemcitabine and nab-paclitaxel (GA) and assessed differences in chemotherapy responses and survival. Methods: Patients with a histological diagnosis of PDAC (based on EUS-guided fine-needle aspiration) from 2017 to 2019 were enrolled. Tumor genomic DNA was extracted from residual liquid-based cytology specimens and Kras mutations were assessed using the quenching probe method. The relationships between the Kras status and progression-free survival (PFS) and overall survival (OS) were assessed. Results: Of the 110 patients analyzed, 15 had wild-type Kras. Those with the wild-type gene showed significantly longer PFS and OS than those with mutant Kras (6.9/5.3 months (p = 0.044) vs. 19.9/11.8 months (p = 0.037), respectively). Multivariate analyses identified wild-type Kras as a significant independent factor associated with longer PFS and OS (HR = 0.53 (p = 0.045) and HR = 0.35 (p = 0.007), respectively). Conclusions: The analysis of the Kras gene status could be used to predict therapeutic responses to GA and prognosis in unresectable PDAC patients.

Evaluation and diagnostic value of next-generation sequencing analysis of residual liquid-based cytology specimens of pancreatic masses.

BackgroundLiquid‐based cytology (LBC) is a widely used method for processing specimens obtained by endoscopic biopsy. This study evaluated next‐generation sequencing (NGS) analysis of LBC specimens to improve the diagnostic accuracy of pancreatic lesions.MethodsUpon the diagnosis of a suspected pancreatic mass, LBC residues were used retrospectively. The quantity and quality of DNA extracted from residual LBC samples were evaluated, and an NGS analysis targeting 6 genes (KRAS, GNAS, TP53, CDKN2A, SMAD4, and PIK3CA) was performed.ResultsThe library was prepared from LBC specimens taken from 52 cases: 44 were successful, and 8 preparations failed. An analysis of DNA quantity and quality suggested that the success or failure of NGS implementation depended on both properties. The final diagnosis was achieved by a combination of the pathological analysis of the surgical excision or biopsy material with clinical information. Among the 33 cases of pancreatic ductal adenocarcinoma (PDAC), KRAS, TP53, CDKN2A, and SMAD4 mutations were identified in 31 (94%), 16 (48%), 3 (9%), and 2 (6%), respectively. Among the 11 benign cases, only a KRAS mutation was identified in 1 case. On the basis of NGS results, 18 of 33 PDACs (55%) were classified as highly dysplastic or more, and 10 of 11 benign lesions were evaluated as nonmalignant, which was consistent with the final diagnosis.ConclusionsNGS analysis using LBC specimens from which DNA of appropriate quantity and quality has been extracted could contribute to improving the assessment of pancreatic tumor malignancies and the application of molecular‐targeted drugs.

Direct next-generation sequencing analysis using endometrial liquid-based cytology specimens for rapid cancer genomic profiling.

Genomic examination of cytology specimens is often performed on cell blocks or conventional smears rather than on liquid-based cytology (LBC) specimens. Since LBC specimens preserve high-quality DNA, cancer genome profiling using next-generation sequencing (NGS) is also attainable from residual LBC specimens. One of the advantages of using LBC specimens for NGS is that it allows direct extraction of DNA from residual specimens, avoiding a sacrifice of smear slides and minimizing genomic profiling processing time. Endometrial LBC specimens were subjected to NGS analysis to validate the practicality of rapid cancer genomic profiling in a pathology laboratory. The extracted DNA was subjected to NGS using a customized cancer gene panel comprising 56 genes and 17 microsatellite regions. The workflow strategy was defined, and the processing time estimated for specimen sampling, cell counting, NGS run, and genome profiling. NGS analysis of most LBC specimens revealed somatic mutations, tumor mutation burden, and microsatellite instability, which were almost identical to those obtained from formalin-fixed paraffin-embedded tissues. The processing time for direct NGS analysis and cancer genomic profiling of the residual LBC specimens was approximately 5 days. The residual LBC specimens collected using endometrial cytology were verified to carry a high tumor fraction for NGS analysis and could serve as an alternate source for rapid molecular classification and diagnosis of endometrial cancers, as a routine process in a pathology laboratory.

Next-generation sequencing in residual liquid-based cytology specimens for cancer genome analysis.

Cancer genome profiling of cytology specimens using next-generation sequencing (NGS) requires adequate and good-quality DNA. Genomic examination of cytology samples was conventionally performed on cell block (CB) or smear specimens than on residual liquid-based cytology (LBC) specimens, which are high-quality DNA sources even after long-term storage. We estimated tumor fractions of 37 residual LBC specimens, including 30 fine needle aspiration (FNA) samples from the thyroid (12 papillary thyroid carcinomas and two malignant lymphomas), lymph node (13 metastatic carcinomas and one malignant lymphoma), and breast cancer (one phyllodes tumor and one invasive ductal carcinoma), two pancreatic carcinoma samples, and five liquid (ascites, pleural effusion, and cerebrospinal fluid) samples. The DNA was extracted from all samples and subjected to NGS using a customized cancer gene panel comprising 28 cancer-related genes. NGS analysis revealed somatic mutations corresponding to pathological diagnosis with adequate variant allele frequency (VAF) in 24 LBC specimens, which had significantly higher tumor fraction (72.5% ± 4.9%). Ten cases, including the five fluid samples, had very small tumor fractions (7.5% ± 2.3%) to obtain sufficient VAF. Other two samples had high tumor fractions but showed very low VAF, indicating the presence of fusion genes. The remaining one sample yielded no DNA recovery. The residual LBC specimens collected by FNA from the thyroid gland and lymph node were verified to carry high tumor fraction and could serve as an alternate source for molecular testing to screen and diagnose cancers without the use of CB or smears.

Chlamydia Trachomatis Infection in High-Risk Human Papillomavirus Based on Cervical Cytology Specimen.

Objective:High-risk human papillomavirus (HR HPV) was associated with the development of cervical cancer. Asymptomatic Chlamydia trachomatis (C. trachomatis) infection is the most common bacterial, sexually-transmitted infection. This study aimed to investigate the association of C. trachomatis in positive HR HPV and the cytological results from liquid-based cytology (LBC). Methods:150 residual LBC specimens were collected; all of which had undergone cytology and HPV testing by Cobas. The samples were established as C. trachomatis using real-time PCR (RT-PCR) with Cryptic F/Cryptic R primers. Results:Of 150 positive HPV findings, the most common (72.7%, 109/150) were the 12 other HR HPVs (viz., 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68). The cervical cytology of those positive HR HPVs were mostly negative (70.0%, 105/150). The C. trachomatis infections in positive HR HPV were 16% (24/150) HPV. The analysis of the abnormal cytology revealed that 41.6% had C. trachomatis co-infection (C. trachomatis and HPV infection) viz., LSIL (20.8%), HSIL (12.5%), and ASC-US (8.3%). A comparison with positive HPV without C. trachomatis co-infection revealed that the highest prevalence was for LSIL, ASC-US, and HSIL (11.1%, 10.3%, and 6.4%, respectively). There was no difference between the abnormalities and negative cervical cytology with negative and positive C. trachomatis co-infection in HR HPV positive (p = 0.174). Conclusion: C. trachomatis infection was not significantly associated HR-HPV and abnormal cytology. This study confirms the increasing rate of C. trachomatis infection in asymptomatic women so routine screening for these infections has been suggested to (a) prevent complications such as the chronic pelvic pain associated with prolong infection and (b) reduce sexual transmission of the infection.

K-ras mutation analysis of residual liquid-based cytology specimens from endoscopic ultrasound-guided fine needle aspiration improves cell block diagnosis of pancreatic ductal adenocarcinoma.

BackgroundEndoscopic ultrasound-guided fine needle aspiration (EUS-FNA) technology is widely used for the diagnosis of pancreatic masses. However, in some cases, inadequate tissue volume or difficulty of morphological diagnosis are constraining factors for adequate cytopathological evaluation. K-ras mutation is the most frequently acquired genetic abnormality, occurring in approximately 90% of all patients with pancreatic ductal adenocarcinoma (PDAC). In the present study, the clinical utility of residual liquid-based cytology (LBC) specimens obtained using EUS-FNA for K-ras mutation analysis was evaluated.MethodsIn this study, 81 patients with pancreatic lesions were examined. The cell block (CB) specimens separated from EUS-FNA samples were morphologically evaluated by hematoxylin–eosin (HE) staining. Final diagnoses were confirmed by CB specimens, surgical resection specimens, diagnostic imaging, and clinical follow-up. Genomic DNA of residual LBC specimens stored at 4°C for several months were extracted and assessed for K-ras mutations using a fluorescence resonance energy transfer-based preferential homoduplex formation assay.ResultsK-ras mutation analysis using residual LBC samples was successful in all cases. The sensitivity, specificity, and accuracy of CB examination alone were 77.4%, 100%, and 81.3%, respectively, and those of the combination of CB examination and K-ras mutation analysis were 90.3%, 92.3%, and 90.7%, respectively. Furthermore, K-ras mutations were detected in 8 (57.1%) of 14 PDAC samples for which the CB results were inconclusive.ConclusionThese findings suggest that K-ras mutation analysis using residual LBC specimens improves the diagnostic accuracy of EUS-FNA.

Sorting role of p16(INK4a)/Ki-67 double immunostaining in the cervical cytology specimens of ASCUS and LSIL cases

Objective: To investigate the sorting effect of p16(INK4a)/Ki-67 double immunostaining method in patients with atypical squamous cells of undetermined significance (ASCUS) or low-grade squamous intraepithelial lesion (LSIL) cytology results. Methods: Four-hundred and twenty cases collected during April 2014 to February 2015 of cervical cytology of ASCUS (n=318) and LSIL (n=102) were selected, and residual liquid-based cytology specimens were used for p16(INK4a)/Ki-67 double immunostaining. The sensitivity and specificity of the detection of cervical precancerous lesions and cervical cancer were calculated, and the results were compared with high risk HPV. Taking histological follow-up as the gold standard, the test was considered positive when at least one cell exhibited p16(INK4a)/Ki-67 co-staining, without requirement of adjunct morphologic interpretation of positive cells. Results: Further screening CIN2+ in cytology ASCUS and LSIL group , the sensitivity of p16(INK4a)/Ki-67 double immunostaining was slightly lower than high risk HPV (84.2% vs. 94.7%), while the specificity was higher (84.0% vs. 53.9%). For ASCUS patients, the sensitivity of p16(INK4a)/Ki-67 double immunostaining and high risk HPV was 82.6% and 91.3%, and the specificity was 88.8% and 63.7%, respectively. For LSIL patients, the sensitivity of p16(INK4a)/Ki-67 double immunostaining and high risk HPV was 86.7% and 100.0%, and the specificity was 67.8% and 20.7%, respectively. For patients younger and older than 30 years, specificity of p16(INK4a)/Ki-67 double immunostaining was both higher than that of high risk HPV (80.8% vs. 42.3%; 84.6% vs. 56.9%). Conclusions: p16(INK4a)/Ki-67 double immunostaining can effectively identify the high risk population in ASCUS or LSIL, with higher specificity than high risk HPV test. p16(INK4a)/Ki-67 double immunostaining may benefit patients younger than 30 years of age as a preliminary or potential cytology-combining screening tool.

Clinical Role of the Detection of Human Telomerase RNA Component Gene Amplification by Fluorescence in situ Hybridization on Liquid-Based Cervical Samples: Comparison with Human Papillomavirus-DNA Testing and Histopathology

Objective: This study was designed to evaluate whether the adjunct of human telomerase RNA component (hTERC) fluorescence in situ hybridization (FISH) analysis to cytological diagnosis and human papillomavirus (HPV)-DNA testing may serve as a predictive marker for distinguishing cervical lesions destined to regress from those at high risk of progression towards invasive cancer. Study Design: hTERC FISH analysis was performed on 54 residual liquid-based cytology specimens obtained from women referred to colposcopy for the detection of atypical squamous cells of undetermined significance or worse (ASCUS+) lesions. Histological diagnosis was considered the gold standard and cervical intraepithelial neoplasia of grade 2 or worse (CIN2+) as the worst outcome. Results: Oncogenic HPV-DNA was found in 96.3% of the specimens. Among these, 38.5% revealed a CIN2+ diagnosis. hTERC gene amplification was detected in 37% of the cases; among these, 70% showed up as CIN2+. hTERC FISH analysis significantly improves the specificity and positive predictive value of HPV-DNA testing, thus differentiating patients with a CIN2+ diagnosis from those with a CIN2- diagnosis. Conclusions: Despite the limitation of a small study sample, our findings provide promising data, indicating the possible role of hTERC analysis in the assessment of the risk of developing cervical cancer. This approach would implement the specificity of DNA testing, avoiding overtreatment at the same time. Prospective follow-up studies are needed with the aim of introducing hTERC FISH into decision-making algorithms.

Abstract 4669: Prevalence of high risk human papillomavirus infection in different cervical cytological features among health examination of women in National Cancer Institute, Thailand

Abstract High-risk (HR) human paillomavirus (HPV) testing is important in cervical cancer screening for triage colposcopy. The objective of the study was to evaluate the prevalence of HPV HR infection in different cytological features among health examination women. DNA was extracted from 2896 residual liquid-based cytology specimens collected during routine liquid-based Papanicolaou tests at National Cancer Institute. Overall, HPV HR prevalence was 9.3% including 1.6% of HPV-16 and 0.4% of HPV-18. HPV prevalent was highest in women younger than 35 years of age, decreasing in women of older age. Among women with normal cytology, HPV HR positive were found in 6.7%. In abnormal cytology, HPV HR were found in 46.7% atypical squamous cells (ASC), 54.8% were low-grade squamous intraepithelial lesions (LSIL) and 80.0% were high-grade squamous intraepithelial lesions (HSIL). HPV-16 were detected in 8.6%, 6.5% and 12.0% of ASC, LSIL and HSIL, respectively.The results of this study provide baseline information on the HPV type distribution, which may useful for clinicians to decide who should be monitored or treated more aggressively. Note: This abstract was not presented at the meeting. Citation Format: Suleeporn Sangrajrang, Piyawat Laowahutanont, Anant Kalalak, Paphawin Jamsri. Prevalence of high risk human papillomavirus infection in different cervical cytological features among health examination of women in National Cancer Institute, Thailand. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4669. doi:10.1158/1538-7445.AM2014-4669

Immunostaining of p16INK4a/Ki-67 and L1 Capsid Protein on Liquid-based Cytology Specimens Obtained from ASC-H and LSIL-H Cases

Background: Atypical squamous cell cannot exclude high-grade squamous intraepithelial lesion (ASC-H) and low-grade intraepithelial lesion cannot exclude high-grade squamous intraepithelial lesion (LSIL-H) are ambiguous diagnostic entities for the prediction of high-grade cervical lesion. Objective and reproducible tests for predicting high-grade cervical lesions are needed to reduce unnecessary colposcopic referrals or follow-ups.Objective: We aimed to identify an adequate set of adjunctive markers to predict cervical intraepithelial neoplasia grade 2+ (CIN2+) in residual liquid-based cytology specimens (LBCS).Methods: We conducted p16 INK4a/Ki-67 and L1 capsid protein immunostaining and human papillomavirus (HPV) DNA typing on 56 LBCS diagnosed with ASC-H or LSIL-H, all of which were subjected to histologic confirmation or follow-up cytologic examination.Results: Positivity for p16 INK4a/Ki-67 was associated with a histology of CIN2+ (P=0.047) and CIN3+ (P=0.002). Negativity for L1 capsid protein was associated with CIN2+ confirmed at follow-up (P=0.02).Positivity for high-risk HPV (HR-HPV) was associated with CIN2+ confirmed at follow-up (P=0.036) and a histology of CIN2+ (P=0.037). The sensitivity, specificity, positive predictive value, and negative predictive value for predicting follow-up CIN2+ were 76.2%, 51.4%, 48.5%, and 78.3%, respectively, for p16 INK4a/Ki-67 immunostaining; 95.2%, 34.3%, 46.5%, and 92.3%, respectively, for L1 capsid protein; and 66.7%, 67.7%, 54.5%, and 77.8%, respectively, for HR-HPV. The classification and regression tree analysis showed that the combined results of p16 INK4a/Ki-67 andL1 capsid protein immunostaining and the HR-HPV test, conducted sequentially, correctly classified 81.8% of samples (27/33)in the prediction of a histology of CIN2 + in ASC-H or LSIL-H. For determination of the histology of cervical intraepithelial neoplasia grade 3+ (CIN3+)in ASC-H or LSIL-H, we found that the combined results of p16 INK4a/Ki-67 and L1 capsid protein immunostaining correctly classified 78.8% (26/33) of samples.Conclusions: p16INK4a/Ki-67 and L1 capsid protein immunostaining and HR-HPV testing of residual LBCS diagnosed with ASC-H or LSIL-H are useful objective biomarkers for predicting CIN2+. Immunostaining for p16INK4a/Ki-67 and L1 capsid protein are sufficient to predict CIN3+.

Promoter methylation of DAPK1, RAR-β and MGMT in exfoliated cervical cytology and its clinical application

To assess the correlation of promoter methylation of DAPK1, RAR-β and MGMT with cervical lesions from cytology to histology, and to reveal the clinical value of DNA methylation in diagnosis of cervical intraepithelial neoplasia (CIN). A total of 103 random-selected cervical samples were collected from residual liquid-based cytology specimens after clinical use in cytopathological diagnosis in outpatient clinic of obstetrics and gynecology, Peking Union Medical Collage Hospital from March 2010 to October 2010. Informed consent was obtained from each woman before the initiation of the study. The methylation sensitive-high resolution melt (MS-HRM) assay was used to evaluate promoter methylation of three genes (DAPK1, RAR-β and MGMT) in 103 biopsy-confirmed liquid-based cervical cytology samples. Methylation levels and high-risk HPV DNA loading (HCII values) were analyzed in relation to both cytological and histological diagnosis. The methylation level of all three genes showed significant difference among the different cytological groups (P = 0.000, 0.011 and 0.002, respectively). The methylation level of DAPK1 and RAR-β showed significant difference among the different histological groups (P = 0.000 and 0.021), while there was no significant difference for MGMT. DAPK1 methylation levels was 1.47% in the CINII/high-grade precancerous lesions group, and 20.98% in the normal/CINI groups (P = 0.000), but there was no significant difference between CINII/high-grade precancerous lesions and normal/CINI groups for RAR-β and MGMT. The combination of DAPK1/HR-HPV loading showed a sensitivity of 0.825 and an area under the receiver operating characteristic curve (ROC) curve (AUC) of 0.695 as diagnostic methods for detecting CINII/high-grade precancerous lesions. DNA methylation such as DAPK1 and RAR-β, in combination with HR-HPV detection, may serve as biomarkers to detect CINII/high-grade precancerous lesions. Detection of methylated DNA from liquid-based cervical cytology specimens is technically feasible with the MS-HRM assay.

Comparison of ThinPrep and SurePath liquid‐based cytology and subsequent human papillomavirus DNA testing in China

Liquid-based cytology (LBC) has been compared with conventional cytology in numerous studies. In the current study of 2 LBC systems, the accuracy, rates of unsatisfactory cytology, and sufficiency of residual LBC specimens for Hybrid Capture 2 (HC2) HPV DNA testing were compared. Eligible women ages 30 to 49 years were recruited for this cross-sectional population-based study in rural China. Women were assessed by visual inspection with acetic acid (VIA), LBC, and high-risk HPV HC2 DNA assay. Cervical specimens were preserved according to SurePath or ThinPrep protocols. LBC results were manually read. HC2 testing was performed on specimens with sufficient residual volume. Colposcopies and biopsies were performed on women who were VIA positive at the time of initial screening. Women with abnormal LBC or HC2 test results were called back for colposcopies and 4-quadrant cervical biopsies. Of 2005 eligible women, 972 were tested by SurePath and 1033 by ThinPrep. Compared with SurePath samples, ThinPrep samples had higher rates of unsatisfactory cytology (0.2% for SurePath and 1.5% for ThinPrep) and insufficient residual volume for HC2 (0.0% for SurePath and 18.2% for ThinPrep). SurePath samples yielded higher sensitivities and similar specificities for LBC and HC2 testing of residual specimens, but these differences were not determined to be significant by area-under-the-curve analysis (LBC performance: 0.89 for SurePath and 0.85 for ThinPrep; HC2 performance: 0.91 for SurePath and 0.89 for ThinPrep). Both methods yielded similar validity in detecting significant cervical lesions. However, SurePath samples yielded higher rates of satisfactory LBC slides and sufficient residual volume for HC2.

High-risk human papilloma virus DNA detection Kit(cervista HPV HR)should be highly validated clinically in cervical cancer screening programs

The aim of this study is to evaluate the clinical significance of High-risk Human Papilloma virus DNA Detection Kit (Cervista HPV HR) designed to the utilized in cervical cancer screening programs. The investigation for Cervista HPV HR test is designed to detect 437 residual liquid-based cytology specimens collected during routine liquid-based Pap tests at standard care vistis and to identify the presence of HR HPV. We compared Cervista HPV HR Test against standard PCR, in order to examine the performance of Cervista HPV HR Test in populations with cervical intraepithelial neoplasia grade 2+ (CIN 2, CIN 3 and Cancer, CIN 2+), and the capabilities of A5/A6, A7, A9 oligonucleotides of Cervista for predicting CIN2+. The accuracy of Cervista compared to PCR with bi-directional sequencing was 88.26%. The positive percent of Cervista HPV HR Test and PCR were 38.96% and 29.08%, respectively. The sensitivity, specificity, negative predictive value (NPV) and positive predictive value (PPV) of Cervista HPV HR Test for the detection of CIN2+ were 98.46%, 58.49%, 99.54% and 29.68%, respectively. The A9 oligonucleotides positivity percent was significantly higher in CIN2 + (odds ratio: 24.037, 95% CI: 10.086 - 57.283). The Cervista HPV HR test can be clinically used for detecting HR HPV types during routine cervical cancer screening. A9 oligonucleotides were also strongly associated with CIN2+ diagnosis, which is improtant in cervical cancer screening for triage to colposcopy.

Clinical Validation of the Cervista HPV HR and 16/18 Genotyping Tests for Use in Women With ASC-US Cytology

Abstract Objective High-risk (HR) human papillomavirus (HPV) testing is important in cervical cancer screening for triage to colposcopy. This study evaluated the clinical performance of the Cervista® HPV HR and 16/18 genotyping tests for detection of HPV in cervical cytology specimens. Methods The tests were prospectively evaluated in a multicenter clinical study. DNA was extracted from approximately 4000 residual liquid-based cytology specimens collected during routine liquid-based Papanicolaou tests at standard of care visits and was assessed for the presence of HR HPV and/or HPV types 16 and 18. All women with cytology results of atypical squamous cells of undetermined significance (ASC-US) or greater underwent colposcopic examination and biopsies were collected. Test results were compared with local colposcopy and histology results from a central pathology review panel. Results There were 1347 subjects with complete data sets of cytology, HR HPV, colposcopy, and histology included in the analysis of the HPV HR test. Sensitivity of the HPV HR test for detection of cervical intraepithelial neoplasia (CIN) 2+ among women with ASC-US cytology was 92.8% (95% confidence interval [CI]: 84.1–96.9) and the negative predictive value (NPV) was 99.1% (95% CI: 98.1–99.6). Sensitivity for detection of ≥CIN 3 in women with ASC-US was 100% (95% CI: 85.1–100) and the NPV was 100% (95% CI: 99.4–100). The specificity of the test for detection of ≥CIN 2 and ≥CIN 3 was 44.2% (95% CI: 41.5–46.9) and 43% (95% CI: 40.3–45.7), respectively. The HPV 16/18 genotyping test also performed as expected in women with ASC-US cytology who were positive for HR HPV. Conclusion The Cervista® HPV HR test can be clinically used for detecting HR HPV types in conjunction with cervical cytology for use in triage of women with ASC-US cytology during routine cervical cancer screening.

P16INK4a immunocytochemistry on cell blocks as an adjunct to cervical cytology: Potential reflex testing on specially prepared cell blocks from residual liquid-based cytology specimens

Background:p16 INK4a (p16) is a well-recognized surrogate molecular marker for human papilloma virus (HPV) related squamous dysplasia. Our hypothesis is that the invasive interventions and related morbidities could be avoided by objective stratification of positive cytologic interpretations by p16 immunostaining of cell block sections of cytology specimens.Materials and Methods:Nuclear immunoreactivity for p16 was evaluated in cell block sections in 133 adequate cases [20 negative for intraepithelial lesion or malignancy, 28 high-grade squamous intraepithelial lesion (HSIL), 50 low-grade squamous intraepithelial lesion (LSIL), 21 atypical squamous cells, cannot exclude HSIL (ASC-H), and 14 atypical squamous cells of undetermined significance (ASCUS)] and analyzed with cervical biopsy results.Results:(a) HSIL cytology (28): 21 (75%) were p16 positive (11 biopsies available — 92% were positive for cervical intraepithelial neoplasia (CIN) 1 and above) and 7 (25%) were p16 negative (3 biopsies available — all showed only HPV with small atypical parakeratotic cells). (b) LSIL cytology (50): 13 (26%) cases were p16 positive (12 biopsies available — all were CIN1 or above) and 37 (74%) were p16 negative (12 biopsies available — all negative for dysplasia. However, 9 (75%) of these biopsies showed HPV). (c) ASC-H cytology (21): 14 (67%) were p16 positive (6 biopsies available — 5 showed CIN 3/Carcinoma in situ/Ca and 1 showed CIN 1 with possibility of under-sampling. Cytomorphologic re-review favored HSIL) and 7 (33%) were p16 negative (5 biopsies available — 3 negative for dysplasia. Remaining 2 cases — 1 positive for CIN 3 and 1 showed CIN 1 with scant ASC-H cells on cytomorphologic re-review with possibility under-sampling in cytology specimen). (d) ASCUS cytology (14): All (100%) were p16 negative on cell block sections of cervical cytology specimen. HPV testing performed in last 6 months in 7 cases was positive in 3 (43%) cases.Conclusion:p16 immunostaining on cell block sections of cervical cytology specimens showed distinct correlation patterns with biopsy results. Reflex p16 immunostaining of cell blocks based on the algorithmic approach to be evaluated by a multiinstitutional comprehensive prospective study is proposed.

Small cell carcinoma of the cervix in liquid‐based Pap test: Utilization of split‐sample immunocytochemical and molecular analysis

Small cell (neuroendocrine) carcinoma of the uterine cervix (SMCC) is a rare, highly aggressive malignant neoplasm. Both conventional and liquid-based cytology (LBC) cervical smears have low sensitivity in diagnosing SMCC, requiring immunocytochemical (ICH) confirmation. We present the first series of SMCC primarily diagnosed in cytology specimens, and ICH studies performed on the residual LBC specimens with subsequent confirmation of the diagnosis on surgical pathology specimens. Immunocytochemical stains for keratin, p16INK4, and neuroendocrine markers (synaptophysin, chromogranin, CD56) were performed on additional ThinPrep slides. HPV test used chromogenic in situ hybridization high risk HPV DNA probe. The Pap smears in all three specimens were highly cellular with a mixture of squamous cells and numerous well-preserved single or small cohesive clusters of malignant epithelial cells. Tumor cells were small, monomorphic with minimal cytoplasm and high nuclear/cytoplasmic ratio. There was significant nuclear overlap, but no nuclear molding, or smudging of nuclear chromatin. The chromatin pattern was stippled. A background tumor diathesis was prominent. Atypical squamous cells of undetermined significance (ASCUS) were noted in one case, and markedly abnormal squamous cells were seen in another case. The main cytology differential diagnoses included high-grade squamous intraepithelial lesion and an endometrial adenocarcinoma. Immunocytochemical positivity for the neuroendocrine markers supported the diagnoses of SMCC in all three cases. The morphologic features of the concurrent surgical pathology specimens were typical of SMCC. The tissue diagnoses were also confirmed by immunohistochemistry. Our study allows us to conclude that SMCC can be primarily diagnosed in LBC specimens using a panel of immunocytochemical stains.