Cytology smears for DNA extraction: Practical approach for selecting the best slide.

BACKGROUND: Next generation sequencing (NGS) to detect actionable genetic abnormalities is standard of care in advanced stage lung adenocarcinoma. Many studies have shown that the molecular results obtained from fine needle aspiration cytology material are comparable to those obtained from formalin-fixed tissue samples. We undertook this study to validate DNA extraction from cytology material for molecular studies and to find any correlation between DNA yield, pattern of tumour cells and tumour fraction. METHODS: DNA was extracted from 34 cytology slides of pulmonary adenocarcinoma cases with predetermined EGFR mutation status. Cytology slides were reviewed for pattern of tumour distribution and tumour fraction. NGS was performed on five slides with variable DNA and compared with original results. RESULTS: There were 14 alcohol-fixed and 20 air-dried smears. The mean DNA yield was 1.74 µg and median of 0.4 µg (range, 0.02-21 µg). Tumour fractions varied from 10% to 90%. No correlation was found between tumour fraction and DNA yield (P = 0.14). The mean DNA yield was high in slides with tumour throughout the slide (sheets or scattered clusters) as compared to rare scattered clusters and/or single cells. EGFR mutation was found in four of the five cases sent for NGS lung panel while one case revealed BRAF mutation. CONCLUSIONS: DNA with good quantity and quality can be extracted from the cytology slides for NGS irrespective of type of fixation. DNA yield has better correlation with distribution pattern of tumour cells on the slides rather than tumour fraction.

Analysis of the Cytomorphological Features in Atypical Urine Specimens following Application of The Paris System for Reporting Urinary Cytology.

BACKGROUND: This study investigates the use of The Paris System (TPS) for Reporting Urinary Cytopathology and examines the performance of individual and combined morphological features in atypical urine cytologies. METHODS: We reviewed 118 atypical cytologies with subsequent bladder biopsies for the presence of several morphological features and reclassified them into Paris System categories. The sensitivity and specificity of individual and combined features were calculated along with the risk of malignancy. RESULTS: An elevated nuclear-to-cytoplasmic ratio was only predictive of malignancy if seen in single cells, while irregular nuclear borders, hyperchromasia, and coarse granular chromatin were predictive in single cells and in groups. Identification of coarse chromatin alone yielded a malignancy risk comparable to 2-feature combinations. The use of TPS criteria identified the specimens at a higher risk of malignancy. CONCLUSION: Our findings support the use of TPS criteria, suggesting that the presence of coarse chromatin is more specific than other individual features, and confirming that cytologic atypia is more worrisome in single cells than in groups.

The impact of subdividing the "atypical" category for urinary cytology on patient management.

INTRODUCTION: The purpose of the study is to determine the impact of subdividing the "atypical" cytology interpretation into two groups: Atypical urothelial cells of uncertain significance (AUC-US) and Atypical urothelial cells suspicious for high-grade urothelial carcinoma (AUC-H/SHGUC), on management of patients with no prior history of UC. MATERIALS AND METHOD: This is a retrospective study of "atypical" urine cytology with subsequent tissue examination occurring within six months. Cytology reports with "atypical" interpretation were reclassified into AUS-UC and AUC-H based on morphologic features identified by the Johns Hopkins system and the Paris system for urine cytology. Follow-up and categorical outcomes were compared between the reclassified AUC-US and AUC-H groups. RESULTS: There was no significant difference (P < 0.4539) in the rate of cytology follow-up, the follow-up cytology result (P < 0.1845), or time between follow-up cytologies (P < 0.0869) between the reclassified atypical group of AUC-H and AUC-US. There was a significant association (P < 0.0001) of rate of malignancy with the reclassified AUC-H (87.18%) compared to the AUC-US (58.68%) groups. CONCLUSION: There was no difference in follow-up between the AUC-H and AUC-US, however there was a difference in the rates of malignancy in the two groups. The AUC-H group is similar to the SHGUC group of the Paris system and can be considered as such, whereas the AUC-US group should continue to be considered atypical. We conclude that reclassification of the "atypical" category into AUC-US and AUC-H/SHGUC can reduce the rate of atypia and help in focused follow-up and targeted management. Diagn. Cytopathol. 2016;44:477-482. © 2016 Wiley Periodicals, Inc.

Two-tiered subdivision of atypia on urine cytology can improve patient follow-up and optimize the utility of UroVysion.

BACKGROUND: The annual incidence of urothelial carcinoma continues to increase, and it is projected that greater than 70,000 new cases will occur in the year 2015. However, as much as 23% of cytologic specimens will demonstrate some degree of atypia without meeting the criteria for urothelial carcinoma and thus will be reported as atypical. METHODS: The authors conducted 2 laboratory information searches and 1 survey. In total, 311 patients who had atypical cytology-biopsy pairs available were identified from the initial data search. The second data search identified 942 patients who had fluorescence in situ hybridization (FISH) results available. RESULTS: There was fair agreement between FISH results and cytology results (κ = 0.34; 95% confidence interval, 0.27-0.41). The analysis did not reveal any benefits of using additional atypical subcategories beyond the 2 suggested in the literature. It was determined that 2 strategies would provide an optimal balance: standardizing patient management and facilitating the adoption of universally recognized templates. CONCLUSIONS: When combining cytology and the 2-tiered atypical classification system with FISH testing, a marked increase in sensitivity and an accompanying decrease in specificity were observed compared with either test individually. Thus, highly sensitive FISH testing may help to identify high-risk patients among those in the group with uncertain atypical findings.

Accuracy and risk of malignancy for diagnostic categories in urine cytology at a large tertiary institution.

BACKGROUND: At a high-volume center, it became necessary to provide benchmarks for the accuracy and risk of malignancy per urine cytology diagnostic category. The additive sensitivity for the determination of the residual risk of disease was calculated with the goal of determining the performance of cytology and optimal triage, including the number of urine samples, before the detection of malignancy in surveillance patients. METHODS: A 2-year laboratory information system-based search was conducted, and it yielded 587 subjects (695 biopsy and cytology pairs) with histological follow-up. The sensitivity and specificity of cytology for urothelial malignancy, the risk of malignancy per diagnostic category, the additive sensitivity, and the time for conversion from a negative initial cytology result to a positive cytology result were examined. RESULTS: The overall average sensitivity and specificity of cytology were 48.9% and 83.0%, respectively. The additive sensitivity increased with each subsequent cytology and peaked with the third cytology. A median conversion time of 22.2 months from a negative initial cytology result to a positive cytology result and a decline in predictive positive cytology after the fourth cytology were noted. Subcategorization of the atypical category failed to show statistical significance in predicting outcomes of biopsy. Surveillance subjects, as compared to primary subjects, showed a higher sensitivity for the detection of high and low grade cancers. CONCLUSIONS: The findings suggest that atypia favoring malignancy is being appropriately flagged. However, further definition of the atypical category is needed to increase specificity with a better qualitative or quantitative morphological algorithm. This study provides a risk of malignancy for each category for benchmarking and clinical triage. The data suggest that follow-up should include at least 4 consecutive urine specimens over a period of 22.2 months.