Cytology smears from endobronchial ultrasound-guided transbronchial needle aspiration as a source of nucleic acids with adequate yield for next-generation sequencing.

Background: Formalin-fixed paraffin-embedded (FFPE) specimens obtained via endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) are commonly used for next-generation sequencing (NGS), which has become integral to guiding treatment strategies in advanced non-small cell lung cancer (NSCLC). However, the DNA and RNA extracted from these samples often exhibit suboptimal quality, leading to test failure. Although cytology smears are routinely prepared during the processing of EBUS-TBNA specimens, their utility for DNA and RNA extraction remains insufficiently explored. Therefore, in the present study, we aimed to evaluate the DNA and RNA yield and adequacy of cytology smears for NGS analysis. Methods: We retrospectively reviewed data from consecutive patients diagnosed with NSCLC via EBUS-TBNA of mediastinal or hilar lymph nodes who had surplus cytology smear slides prepared at our institution between March 2021 and October 2024. We quantified DNA and RNA yields, defining cases with ≥50 ng of both as sufficient nucleic acid amount for NGS input (SNA). We also performed univariable and multivariable logistic regression analyses to identify factors associated with the SNA threshold. Results: Among 223 patients, the median DNA and RNA yields were 540 (range, 0-22,680) ng and 2,277 (range, 0-26,910) ng, respectively. Adequate yields (≥50 ng) were achieved in 91.5% for DNA and 97.3% for RNA, whereas 89.2% of cases were SNA. Moreover, DNA yield was associated with mediastinal lymph nodes, a larger lesion size on both EBUS and computed tomography (CT), use of a 22-gauge needle, and indistinct margins on EBUS. RNA yield was associated with mediastinal lymph nodes and larger lesion size on EBUS and CT. However, in the univariable and multivariable analyses, no variable was significantly associated with SNA. Conclusions: EBUS-TBNA-derived cytology smears provided sufficient DNA and RNA yield to meet predefined NGS input thresholds. These findings suggest that cytology smears may represent a potentially useful source for NGS, pending validation by sequencing.