Evaluation of Cytologic Sample Preparations for Compatibility With Nucleic Acid Analysis

Abstract

Objectives In this study, the influence of several key elements of the cytologic sample workflow on DNA and RNA content was evaluated. Methods The A549 cell line, patient-derived organoids, and pleural effusions were used to investigate the effect of (1) several collection media and delayed time to processing; (2) cytology specimens; (3) cytologic staining; and (4) formalin-fixed, paraffin-embedded (FFPE) cell block processing on nucleic acid quality and quantity as determined by fragment analyzer, Qubit analysis (Thermo Fisher Scientific), and quantitative polymerase chain reaction–based analysis on the Idylla platform (Biocartis). Results Alcohol-based collection media (CytoRich Red [Thermo Fisher Scientific] and EtOH95%) displayed high DNA and RNA preservation capacity, while phosphate-buffered saline and, to a lesser extent, formalin were associated with high RNA quality. Cytospin and smear cytology specimens were subject to DNA and RNA loss. Cytologic staining had no further impact on sample quality, hence destaining is not necessary. Both H&E-stained and unstained FFPE sections are compatible with nucleic acid analysis, despite a strong decrease in DNA and RNA quality. Conclusions Although several key elements of the cytologic sample workflow have an influence on DNA and RNA quality and quantity, the selection of these elements is also dependent on the downstream (ancillary) testing methods.

Detection of clinically actionable gene fusions by next‐generation sequencing‐based RNA sequencing of non–small cell lung cancer cytology specimens: A single‐center experience with comparison to fluorescence in situ hybridization

“…Our findings are consistent with previous studies that have evaluated the clinical utility of RNA-seq in detecting gene fusions in NSCLC tissue specimens. 37,45 In this study, clinically relevant oncogenic alterations detected by RNA-based NGS were concordant with corresponding FISH results in 84% (n = 16) cases, with three cases (two with EML4::ALK and one with SLC34A2::ROS1) that were negative by FISH. Although these discrepancies may be attributable to presence of a low level fusion or a fusion formed by an insertion that is beyond the detection of a FISH break-apart probe, they highlight the value of different testing methods, each with unique advantages and limitations, that can be helpful in identifying patients with oncogenic driver mutations.…”

“…Because a large fraction of NSCLC patients present with metastatic disease with only cytology specimens available for diagnosis, these samples may be the sole specimens available for biomarker assays, including gene fusion testing. 36,37 In this study, we evaluated a retrospective cohort of NSCLC cytology specimens that underwent RNA-based NGS and concurrent testing using FISH to compare the performance of the two gene fusion detection techniques.…”

“…However, the clinical utility of RNA‐based sequencing for detecting oncogenic alterations in NSCLC cytology specimens has not been extensively studied. Because a large fraction of NSCLC patients present with metastatic disease with only cytology specimens available for diagnosis, these samples may be the sole specimens available for biomarker assays, including gene fusion testing 36,37 …”

Semi-automated preparation of fine-needle aspiration samples for rapid on-site evaluation

“…24 Our chosen Giemsa staining procedure is compatible with genetic analysis. 25 Therefore, samples prepared with our device could be further used by scrapping material from the glass slide for ThinPrep® preparation or molecular analysis via DNA/RNA extraction, following common procedure. 26…”

Predictive immunocytochemistry in non-small cell lung cancer cytological samples

“…13,19,21,23,26,27 The main reason why cytology remains an underutilized resource for predictive molecular ICC in NSCLC patients is not the insufficient quality of noncell block samples but the challenging translation of biomarker assays validated for FFPE tissue to cytological samples. 22,28 Multigene single assay testing is considered sample saving and less time consuming due to the possibility of analysing numerus biomarkers in only one run. 2,20,22 However, obtaining the biopsy sample of optimal quantity and quality for multigene single assay testing is of great challenge in everyday clinical practice.…”