Optimizing schools of cytology: Discussions from the 2022 ASC/IAC Cytology Education Symposium, North American Strategies, and European Symbiosis.

This report highlights information and outcomes from the November 2022 ASC/IAC joint Cytology Education Symposium, an annual conference organized by the Cytology Programs Review Committee. The manuscript provides information on shared educational opportunities and practices for cytology students and other learners in anatomic pathology, discusses recruitment strategies for schools of cytology, conveys teaching resources, introduces perspectives on virtual microscopy and online learning, and transmits information about wellness of students in schools of cytology.

Impact of serous fluid volume on next-generation sequencing: a significant step forward for optimization of serous fluid sample collection.

Current literature lacks data regarding the influence of serous fluid volume (SFV) on next-generation sequencing (NGS) performed on malignant cases. In this study, we highlight the impact of SFV and other parameters influencing the outcome of NGS analysis. We evaluated 827 samples of serous fluids from 607 patients. Of these, 72 samples underwent NGS analysis. Effusion volume, tumor cellularity, DNA, and RNA quality metrics, as well as clinicopathologic and molecular data were evaluated. Pleural fluid accounted for 56.3% of the fluid samples collected. The most common primary tumor site was gastrointestinal/pancreatobiliary, adenocarcinoma was the most common histologic type. Overall mean volume was 293 mL. The mean Qubit DNA of the 72 effusion samples that underwent NGS analysis was 14.3 ng/µL and mean Qubit RNA was 28.2 ng/µL. The mean Qubit DNA concentration increases in SFV up to 100 mL only. No correlation exists between SFV and mean tumor cellularity. In addition, 74.6% (50 of 67) of sequenced samples showed oncogenic drivers; KRAS was the most common driver followed by EGFR. Three cases displayed ALK fusions, and 1 case displayed NTRK1 fusion. The DNA yield is higher in SFV of 100 mL as a cutoff. Beyond 100 mL, there is no impact of SFV on DNA yield. SFV does not impact RNA yield and mean tumor cellularity. Effusion samples should be submitted for molecular testing despite low tumor cellularity. Our results as a pilot study are important in optimization of SFV for both diagnosis as well as NGS analysis for improving management.

Editorial: Optimizing Schools of Cytology: Discussions from the 2022 ASC/IAC Cytology Education Symposium, North American Strategies, and European Symbiosis.

This report highlights information and outcomes from the November 2022 ASC/IAC joint Cytology Education Symposium, an annual conference organized by the Cytology Programs Review Committee. The manuscript provides information on shared educational opportunities and practices for cytology students and other learners in anatomic pathology, discusses recruitment strategies for schools of cytology, conveys teaching resources, introduces perspectives on virtual microscopy and online learning, and transmits information about wellness of students in schools of cytology.

Optimal fluid volume for detecting malignancy in serous effusions: a single institution experience.

INTRODUCTION: Detection of malignant cells in serous fluids is an indicator of advanced stage of malignancy and is critical in clinical management decisions and prompt treatment initiation. The minimum volume which is ideal for detecting malignancy in serous fluid is not well established. In this study, we aim to identify optimal volume that will be ideal for adequate cytopathological diagnosis. MATERIALS AND METHODS: A total of 1597 samples of serous fluids from 1134 patients were included in the study. Samples were diagnosed based on International System for Reporting Serous Fluid Cytopathology (ISRSFC). Clinicopathologic results from different diagnostic groups were compared and statistically analyzed. RESULTS: Pleural fluids comprised 890 (55.7%) specimens, followed by 456 (28.6%) peritoneal, 128 (8%) ascites, and 123 (7.7%) pericardial fluid specimens. The majority were negative for malignancy (1138, 71.3%), followed by malignant (376, 23.5%), atypical (59, 3.7%), and suspicious for malignancy (24, 1.5%). Malignancy was identified in sample with volumes from 5 mL to 5000 mL. Rate of detection of malignant cells increased significantly with higher sample volumes. For malignancy detection the optimal volume for overall serous fluid is 70 mL. Pericardial fluid is an exception, with lower mean volume and significantly lower proportion of cases with malignant diagnosis. CONCLUSIONS: Our study indicates that higher fluid volumes have a higher rate of malignancy detection and a low false-negative rate. We recommend a minimum of 70 mL of serous fluid for optimal cytopathologic examination and malignancy detection. Pericardial fluid is an exception, with lower mean volume and thus lower requirement.

Evaluating the role of Z-stack to improve the morphologic evaluation of urine cytology whole slide images for high-grade urothelial carcinoma: Results and review of a pilot study.

BACKGROUND: Whole slide imaging (WSI) adoption has been slower in cytopathology due, in part, to challenges in multifocal plane scanning on 3-dimensional cell clusters. ThinPrep and other liquid-based preparations may alleviate the issue by reducing clusters in a concentrated area. This study investigates the use of Z-stacked images for diagnostic assessment and the experience of evaluating urine ThinPrep WSI. METHODS: Thirty ThinPrep urine cases of high-grade urothelial carcinoma (n = 22) and cases of negative for high-grade urothelial carcinoma (n = 8) were included. Slides were scanned at 40× magnification without Z-stack and with Z-stack at 3 layers, 1 µm each. Six cytopathologists and 1 cytotechnologist evaluated the cases in 2 rounds with a 2-week wash-out period in a blinded manner. A Cohen's Kappa (CK) calculated concordance rates. A survey after each round evaluated participant experience. RESULTS: CK with the original report ranged from 0.606 to 1.0 (P < .05) without Z-stack and 0.533 to 1.0 (P < .05) with Z-stack both indicating substantial-to-perfect concordance. For both rounds, interobserver CK was moderate-to-perfect (0.417-1.0, P < .05). Intraobserver CK was 0.697-1.0 (P < 0.05), indicating substantial to perfect concordance. The average scan time and file size for slides without Z-stack and with Z-stack are 6.27 minute/0.827 GB and 14.06 minute/2.650 GB, respectively. Surveys demonstrated a range in comfort and use with slightly more favorable opinions for Z-stacked cases. CONCLUSIONS: Z-stack images provide minimal diagnostic benefit for urine ThinPrep WSI. In addition, Z-stacked urine WSI does not justify the prolonged scan times and larger storage needs compared to those without Z-stack.

Combining molecular testing and the Bethesda category III:VI ratio for thyroid fine-needle aspirates: A quality-assurance metric for evaluating diagnostic performance in a cytopathology laboratory.

BACKGROUND: Molecular testing (MT) of thyroid fine-needle aspiration (FNA)-derived genetic material is commonly used to assess malignancy risk for indeterminate cases. The Bethesda System for Reporting Thyroid Cytopathology (TBS) provides limited guidance for the appropriate use of category III (atypia of undetermined significance [AUS]). The authors combined MT with cytomorphology to monitor AUS diagnoses in a cytopathology laboratory. METHODS: Neoplasia-associated genetic alterations (NGAs) were determined by MT of preoperative FNA biopsies or resected malignancies and were categorized as BRAF V600E mutations, RAS-like mutations (HRAS, NRAS, or KRAS mutations or non-V600E BRAF mutations), or other mutations. RESULTS: Among 7382 thyroid FNA biopsies, the AUS rate was 9.3% overall and ranged from 4.3% to 24.2% among 6 cytopathologists (CPs) who evaluated >150 cases. The ratio of specimens falling into TBS category III to specimens falling into category VI (malignant) (the III:VI ratio) was 2.4 overall (range, 1.1-8.1), and the ratio of specimens falling into TBS categories III and IV (follicular neoplasm or suspicious for follicular neoplasm) combined (III+IV) to specimens falling into category VI (the [III+IV]:VI ratio) was 2.9 overall (range, 1.4-9.5). MT was performed on 588 cases from 560 patients (79% women) with a median age of 56 years (range, 8-89 years). BRAF V600E mutation was the most common (76% of cases) in TBS category VI and was rare (3%) in category III. RAS-like mutations were most common in TBS categories III (13%), IV (25%), and V (suspicious for malignancy) (17.5%). The NGA rate in AUS cases fell between 5% and 20% for 5 of 6 CPs and did not correlate with the III:VI ratio or the (III+IV):VI ratio. CONCLUSIONS: Lack of correlation between the NGA rate and easily calculable diagnostic ratios enables the calibration of diagnostic thresholds, even for CPs who have normal metrics. Specifically, calculation of the NGA rate and the III:VI ratio may allow individual CPs to determine whether they are overcalling or undercalling cases that other CPs might otherwise recategorize.

Standardizing a volume benchmark for cerebrospinal fluids for optimal diagnostic accuracy.

INTRODUCTION: Cerebral spinal fluid (CSF) cytomorphologic analysis remains the gold standard in the evaluation of malignant leptomeningeal involvement. However, collection of optimal volumes for adequate cytomorphologic evaluation is not standardized. Our study investigated optimal CSF volumes that result in a significant diagnostic result. METHODS: A total of 4114 samples were retrospectively identified from 2014 to 2018, and 2557 samples had concurrent flow cytometry (FC) study. Each specimen was grouped as unsatisfactory, negative, atypical, or positive. Positive samples were grouped as either solid tumors, leukemia, or lymphoma by the type of malignancy detected. Demographic data as well as CSF source was recorded. Specimens with FC were separated by detection on cytology and/or FC. A t-test and ANOVA test were used to compare the average volumes for each group. RESULTS: Average volumes for negative, atypical, and positive samples are 7.48 mL (95% CI: 7.33, 7.63), 7.97 mL (95% CI: 7.37, 8.57), and 8.44 mL (95% CI: 7.46, 9.43), respectively. Average volumes for solid tumors, leukemia, and lymphoma positive samples are 12.0 mL (95% CI: 9.11, 14.89), 6.73 mL (95% CI: 5.94, 7.53), and 8.44 mL (95% CI: 6.78, 10.09). For cases with FC, the volumes are 10.11 mL (95% CI: 9.28, 10.96), 7.28 mL (95% CI: 6.87, 7.70), and 6.86 mL (95% CI: 6.25, 7.49) for positive cytology only, positive cytology/FC, and negative for both, respectively. CONCLUSIONS: Our results suggest that higher volumes produce better results for analysis. We recommend an optimal volume of 8.44 mL for cytologic work-up of malignancies. However, optimal volumes may differ based upon malignancy type and utilization of flow cytometry.

Underrecognized Patterns of High-Grade Squamous Intraepithelial Lesion on ThinPrep Preparations.

OBJECTIVES: To identify less readily identifiable patterns of high-grade squamous intraepithelial lesions (HSIL) in negative human papillomavirus (HPV)-positive Papanicolaou (Pap) tests on ThinPrep preparations. METHODS: Of all HPV-positive Pap tests that were negative for intraepithelial lesion or malignancy (NILM) from July 2013 to June 2018, those with HSIL on subsequent histology within 6 months were identified. ThinPrep slides from the latter group (group 1) and from NILM HPV-negative Pap tests with negative follow-up (group 2) were reviewed independently by 4 participants. Group 1 cases were then reviewed together for consensus and with the ThinPrep Imaging System (TIS). Any discrepancies from the original interpretation were recorded. RESULTS: The study cohort included 57 cases each in groups 1 and 2. On final review of group 1 cases, 17 (29.8%) were classified as NILM or unsatisfactory. Of the remaining, 4 cases revealed rare abnormal cells not flagged by the TIS in the fields of view. In the 36 cases (63.1%) with screening or interpretative errors, the key cytologic findings accounting for major discrepancies included atypical metaplastic cells, atypical repair, rare syncytial groups, and atypical immature metaplastic cells. CONCLUSIONS: There are 3 main underrecognized patterns of HSIL in cervical cytology: atypical metaplastic cells, atypical repair, and rare syncytial groups.

Next-Generation Sequencing of Cell-Free DNA Extracted From Pleural Effusion Supernatant: Applications and Challenges.

Cell-free DNA (cfDNA) extracted from diverse specimen types has emerged as a high quality substrate for molecular tumor profiling. Analytical and pre-analytical challenges in the utilization of cfDNA extracted from pleural effusion supernatant (PES) are herein characterized in patients with metastatic non-small cell lung carcinoma (NSCLC). Pleural effusion specimens containing metastatic NSCLC were collected prospectively. After ThinPrep® (TP) and cell block (CB) preparation, DNA was extracted from residual PES and analyzed by gel electrophoresis for quality and quantity. Libraries were prepared and sequenced with a targeted next-generation sequencing (NGS) platform and panel clinically validated for plasma specimens. Results were compared with DNA extracted from corresponding FFPE samples that were sequenced using institutional targeted NGS assays clinically validated for solid tumor FFPE samples. Tumor (TC) and overall cellularity (OC) were evaluated. Fourteen specimens were collected from 13 patients. Median specimen volume was 180 mL (range, 35-1,400 mL). Median TC and OC on TP slides and CB sections were comparable. Median extracted DNA concentration was 7.4 ng/µL (range, 0.1-58.0 ng/µL), with >5 ng/µL DNA extracted from 10/14 specimens (71%). Mutations were identified in 10/14 specimens, including 1/3 specimens with median molecular coverage <1,000 reads. The minimal detected allelic fraction was 0.6%. NGS was falsely negative for the presence of one driver mutation. No correlation was identified between sample volume or OC, quality or quantity of extracted DNA, or mutation detection. Despite analytical and pre-analytical challenges, PES represents a robust source of DNA for NGS.

Effects of Implementing the Dual Papanicolaou Test Interpretation of ASC-H and LSIL Following Bethesda 2014.

OBJECTIVES: To evaluate the impact of implementing the dual interpretation of atypical squamous cells, cannot exclude high-grade squamous intraepithelial lesion (ASC-H) and low-grade squamous intraepithelial lesion (LSIL) after the Bethesda System 2014 and to compare it with other indeterminate interpretations. METHODS: Rates of high-risk human papillomavirus (HPV) positivity and histologic follow-up and the proportion of women with high-grade squamous intraepithelial lesion on histologic follow-up were compared for the combined interpretation of ASC-H and LSIL (ASCHL) and the categories of LSIL, cannot exclude high-grade squamous intraepithelial lesion (LSIL-H) and ASC-H. RESULTS: The percentage of ASCHL HPV-positive cases (86.0%) was similar to that of LSIL-H but significantly higher in comparison to that of ASC-H. The rates of cervical intraepithelial neoplasia grade 2 or higher (CIN 2+) and CIN 3+ for ASCHL (29.6% and 3.6%, respectively) were similar to those of LSIL-H and ASC-H. When stratified by HPV test results, the proportions of patients with CIN 2+ and CIN 3+ remained statistically similar to those with ASCHL and with LSIL-H and ASC-H. CONCLUSIONS: Considering the similar risks of CIN 2+ and CIN 3+ for ASCHL and ASC-H, having a separate category of ASCHL for reporting cervical cytology appears to be redundant.

Preanalytic variables in quality and quantity of nucleic acids extracted from FNA specimens of thyroid gland nodules collected in CytoLyt: Cellularity and storage time.

BACKGROUND: Morphologic and genetic analysis of thyroid nodules may be performed from a single vial. Preanalytic variables that affect nucleic acid extracted from a single vial are evaluated. METHODS: Thyroid fine-needle aspiration (FNA) specimens collected in CytoLyt were evaluated. A ThinPrep slide was prepared. Extracted nucleic acids were analyzed using Oncomine Comprehensive Panel, version 2, after Ion AmpliSeq library preparation. A pathologist and a cytotechnologist enumerated specimen cellularity. RESULTS: Fifty-six samples were collected from 55 nodules in 53 patients. Bethesda category correlated with cellularity (P = .01), and storage time (median, 43 days; range, 7-77 days) was longer for specimens in categories II and III than for those in categories IV and VI (P = .01). The mean specimen DNA concentration was 4.5 ng/µL (range, 0-23.8 ng/µL), and 25 (45%) had concentrations >3.3 ng/µL. The mean specimen RNA concentration was 4.8 ng/µL (range, 0-42.4 ng/µL), and 31 (55%) had concentrations >1.4 ng/µL. Nucleic acid quantity increased with epithelial cellularity. Storage time weakly correlated with the quantity of extracted DNA, independent of cellularity, but not extracted RNA. Greater proportions of cell-free DNA and lesser proportions of long, intact RNA fragments were extracted from a subset of samples with longer storage time. Among 15 single nucleotide variants, the median mutant allelic fraction was 15.1%. One false-negative result was identified. Five specimens subsequently determined to harbor a genetic alteration failed quality metrics. CONCLUSIONS: Cellularity and storage time affect the quantity and quality of nucleic acid extracted from thyroid FNA specimens collected in CytoLyt. Further investigation will serve to quantify the magnitude of such effects and to elucidate other contributing factors.

Cytologic diagnosis of adenocarcinoma on bile duct brushings in the presence of stent associated changes: A retrospective analysis.

BACKGROUND: Bile duct brushing (BDB) cytology, for the characterization of bile duct strictures, can be challenging to interpret when associated with a stent. Our study aims to identify the cytologic criteria for the diagnosis of adenocarcinoma in BDBs in the presence of a stent. METHODS: A database search (January 2010-December 2015) identified three groups of BDBs-negative with stent, malignant with stent, malignant without stent. All malignant cases had histologic and/or cytologic evidence of malignancy within 1 month of the brushing sample. All reactive cases had ≥6 months of benign clinical follow-up. ThinPrep slides were reviewed by two cytopathologists and cytologic features were recorded. Statistical analysis was performed using Fisher's exact test. RESULTS: The study cohort included 12 reactive cases with stent, 17 malignant cases with stent and 32 malignant cases without stent. Among the stented cases, the cytologic features that reached statistical significance were 3D architecture, anisonucleosis to the extent of ≥1:6, coarse chromatin distribution and the presence of single atypical cells in the malignant group in contrast to the benign group. Cases that were diagnosed malignant in the presence of a stent revealed a spectrum of cell populations more frequently as compared with the malignant cases without stent (76% vs 16%). CONCLUSION: Our findings reveal that the cytologic features of malignancy in non-stented BDBs mostly hold true for stented specimens as well. Application of these criteria in the presence of a stent can improve diagnostic accuracy and thereby patient care.

HPV test result monitoring of different Bethesda categories in gynaecologic cytology: A valuable quality assurance measure.

BACKGROUND: High-risk human papillomavirus (HPV) test ordering has evolved since the 2006 ASCCP guidelines. In light of the availability of the HPV test results for most women ≥30 y, regardless of the Pap test diagnosis; we examined their value in assessing the overall performance of cytopathologists (CPs). METHODS: Data were derived for six CPs for Pap test interpretations over 4 y. HPV positivity rates for atypical squamous cells of undetermined significance (ASC-US) and for patients ≥30 y for negative for intraepithelial lesion or malignancy (NILM) and squamous intraepithelial lesion (SIL) (inclusive of low grade SIL (LSIL), high grade SIL (HSIL), and carcinoma) categories were retrieved for individual CPs. ASC/SIL ratios were analysed overall and separately for patient groups <30 y and ≥30 y. Pearson correlation coefficient was calculated to assess correlation between HPV positivity rates for ASC-US, NILM and SIL, and ASC/SIL ratios. RESULTS: The overall ASC-US HPV positivity rate was 41%-49% for patients <30 y, 32% for patients ≥30 y. Stratifying by patient age group, ASC-US HPV positivity rate, and ASC/SIL ratio showed a negative correlation. Excluding an outlier, the NILM HPV positivity rate and ASC/SIL ratio showed a strong negative correlation. CONCLUSION: Our study shows that ASC-US HPV positivity rate is dependent on the age of the population that is tested. Monitoring of the HPV positivity rates for NILM and SIL categories can serve as an additional objective measure to assess the performance of CPs. Based on the patient population, the laboratory can establish an initial baseline for these rates and use it to adjust interpretive thresholds in ensuring the diagnostic sensitivity of the test and the quality of the interpretation.

Diagnostic yield of cytopathology in evaluating pericardial effusions: Clinicopathologic analysis of 419 specimens.

BACKGROUND: Pericardial effusions can cause considerable morbidity and potentially may lead to mortality. Malignant pericardial effusions are uncommon, and data on malignancies encountered in pericardial effusion cytology specimens are limited. METHODS: Relevant records of all pericardial effusions from January 2008 to September 2014 were examined and compared with pericardial biopsy results when performed. Discrepant cases were reviewed to determine the cause of the disagreement. RESULTS: In total, 419 pericardial effusion specimens obtained from 364 patients were examined. Cytologic diagnostic categories included: negative for malignancy (332 specimens; 79%), equivocal (25 specimens; 6%), and positive (62 specimens from 51 patients; 15%). Forty-seven patients who had positive effusions were known to have malignancy. The most common primary malignancies were breast (39.3%) and lung (39.3%) cancers in women and lung cancer (47.4%) in men. A concurrent pericardial biopsy was performed in 46% of patients. Excluding equivocal cytologic diagnoses, cytology and biopsy were concordant in 153 of 173 paired samples (88.4%). The sensitivity of cytology in diagnosing malignancy was 92.1% compared with 55.3% for pericardial biopsy. CONCLUSIONS: Cytologic examination has significant diagnostic utility in the evaluation of pericardial effusions and exhibits a lower false-negative rate compared with pericardial biopsy. Submission of pericardial biopsy alongside effusion cytology is associated with increased sensitivity for detecting malignancy and may be especially useful in the setting of low-volume pericardial effusion. Cancer Cytopathol 2017;125:128-137. © 2016 American Cancer Society.

Negative computer-imaged ThinPrep Pap test and positive hybrid capture2 HPV co-testing results: A quality assurance review.

Women ≥30 years of age with negative (-) Pap tests and positive (+) HPV co-test results have a higher prevalence and cumulative risk of developing high-grade cervical intraepithelial neoplasia (CIN 2+). Thus, the current management in these women is to repeat co-test in 12 months or immediate reflex genotyping for HPV16 or HPV 16/18. If genotyping is not an option, timely quality assurance (QA) rescreen of such Pap tests may be a valuable alternative. All ThinPrep Pap tests (TPPT) interpreted as negative for intra epithelial lesion (NILM) or NILM with reactive cellular changes (NILM/RCC) and a (+) high-risk HPV [Hybrid Capture 2 (HC2), Qiagen, Hilden, Germany] co-test result over a 45-month period (10/2009-06/2013) underwent monthly QA review. The TPPT were screened by the TP Imaging System [TIS, Hologic Inc., Bedford, MA]. Twenty five thousand six hundred and seventy five (18%) NILM and NILM/RCC TPPT of a total of 141,548 TPPT underwent HPV co-test. HPV test was (+) in 2,300 (8.9%) TPPT cases. HPV (+) cases by age group were <30 years, 486 (21%), and ≥30 years, 1,814 (79%). Upon QA review, 10 cases (0.4%) were reclassified, with significant findings in three cases in ≥30 years. Two cases showed high-grade squamous intraepithelial lesion (HSIL) on repeat Pap, and one case showed endocervical adenocarcinoma in situ (AIS) on biopsy. Timely QA review of HPV (+) Pap (-) co-tests is a valuable monitor. Ninety percentage of reclassified cases were in ≥30 age group and 70% were originally signed out by using TIS 22 Field of View (FOV) only. Three reclassified cases had significant findings on follow up (F/U).