Abstract
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.
Keywords: Cytology smear slide, endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA), next-generation sequencing (NGS), non-small cell lung cancer (NSCLC), nucleic acid yield
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Key findings
• Cytology smear slides obtained via endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) provided DNA and RNA in quantities adequate for next-generation sequencing (NGS) input in approximately 90% of cases.
• Among cases in which NGS using formalin-fixed paraffin-embedded (FFPE) specimens had failed or could not be performed, 76.5% met the sufficient nucleic acid amount for NGS input (SNA) criterion on smears, and 64.7% also met the required concentration; at the 10 ng threshold, these were 88.2% and 76.5%.
• No clinical or procedural factors were independently associated with SNA in multivariable analysis.
What is known and what is new?
• FFPE cell blocks obtained via EBUS-TBNA are widely used for NGS in advanced non-small cell lung cancer (NSCLC); however, inadequate nucleic acid quantity or quality can limit successful testing. Cytology smears can provide sufficient DNA for NGS, but evidence on combined DNA and RNA yield and on factors associated with adequacy remains limited, because prior studies were largely restricted to DNA.
• This study shows that surplus EBUS-TBNA cytology smears provided DNA and RNA meeting the predefined input threshold in most cases.
What is the implication, and what should change now?
• Cytology smears may serve as a complementary source for NGS, particularly when FFPE cell blocks are inadequate or suboptimal.
• Because this study assessed nucleic acid quantity rather than sequencing performance or quality, prospective studies with actual NGS, tumor cellularity assessment, and nucleic acid quality metrics are needed to validate these findings and optimize their clinical implementation.
Introduction
Background
Lung cancer is a major cause of cancer-related death worldwide, and in patients with non-small cell lung cancer (NSCLC), approximately half present with advanced-stage disease at diagnosis (1,2). Notably, molecular targeted therapies have significantly improved outcomes in patients with NSCLC harboring actionable gene mutations (3,4), making molecular testing essential for guiding treatment strategies.
Among molecular testing techniques, next-generation sequencing (NGS) is recommended over single-gene tests (5). Although surgical specimens are generally preferred for NGS analysis, for patients with unresectable disease, less invasive sampling methods, such as bronchoscopy or transthoracic needle biopsy, are required for diagnosis and subsequent NGS (6). Among these techniques, endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) is a widely used bronchoscopic technique for sampling hilar and mediastinal lymph nodes. Notably, this technique exhibits excellent diagnostic accuracy for lung cancer, with a pooled sensitivity of 93% and specificity of 100% (7).
Rationale and knowledge gap
Formalin-fixed paraffin-embedded (FFPE) cell blocks obtained via EBUS-TBNA are commonly used for NGS (8-10). However, a meta-analysis has indicated that approximately 10–20% of these samples fail to meet quality criteria for successful NGS analysis (11), resulting in testing failure. While many NGS panels primarily utilize DNA, recent evidence indicates that RNA sequencing offers superior sensitivity for detecting gene fusions compared to DNA sequencing (12). Specifically, RNA sequencing can successfully identify actionable gene fusions that may not be detectable by DNA sequencing alone, thus informing clinical management and therapeutic outcomes (12-14).
Cytology smears collected during EBUS-TBNA are routinely prepared for cytological assessment and rapid on-site evaluation (ROSE) (7). Notably, Fielding et al. reported that Diff-Quik-stained cytology smears from EBUS-TBNA yielded higher DNA yields and achieved greater NGS success rates than FFPE cell blocks (15,16). Although these previous studies demonstrated that cytology smears provide substantial DNA yield and high NGS success rates, they were limited to DNA-based analyses and did not explore RNA extraction or sequencing.
Objective
In the present study, we aimed to evaluate the nucleic acid yield, including both DNA and RNA, from surplus cytology smears obtained via EBUS-TBNA and investigate their adequacy for NGS analysis. We present this article in accordance with the STROBE reporting checklist (available at https://jtd.amegroups.com/article/view/10.21037/jtd-2026-1711/rc).
Methods
Study participants
Consecutive patients who underwent EBUS-TBNA for mediastinal or hilar lymph node metastases from NSCLC between March 2021 and October 2024 at our institution were retrospectively reviewed. Among them, patients who underwent DNA and RNA extraction from the surplus cytology smears were identified. This study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the Institutional Review Board of the National Cancer Center (No. 2018-090). Written informed consent for the clinical procedure and broad consent for the use of surplus specimens were obtained from all patients.
Procedures
All patients underwent bronchoscopy under moderate to deep sedation using a convex-probe ultrasound bronchoscope (BF-UC260FW or BF-UC290F; Olympus, Tokyo, Japan) combined with a dedicated ultrasound processor (EU-ME2 PREMIER PLUS or EU-ME3; Olympus), introduced via the oral route. Once a target lymph node was identified by EBUS, its B-mode features, vascular patterns, and elasticity were evaluated at the maximum diameter of the node. B-mode characteristics were categorized as follows: short diameter (>10 mm or ≤10 mm), shape (round or oval), echogenicity (heterogeneous or homogeneous), margin (distinct or indistinct), presence or absence of a central hilar structure, and presence or absence of a coagulation necrosis sign (17). Vascular patterns were evaluated using power and color Doppler modes and classified into grades 0–3, with grades 0–1 indicating likely benign nodes and grades 2–3 suggesting malignancy (18). Therefore, grades 0–1 and 2–3 were categorized as binary variables. EBUS elastographic images were categorized as blue, intermediate, or non-blue based on the dominant color of the lesion (19). Elastography was further dichotomized into blue and intermediate or non-blue, as previously described (20). The maximum short diameter of the target lesion was assessed using computed tomography (CT).
Following EBUS evaluation, either a 22- or 25-gauge needle was selected for TBNA by the operator. TBNA was typically performed 3–4 times per lesion, with approximately 20–50 needle agitations under negative pressure per puncture. The aspirated material was expelled onto a glass slide using a stylet or an air-filled syringe. Portions of the specimen were processed into cell block specimens, fixed in 10% neutral-buffered formalin, and embedded in paraffin. After removal of the material for cell block preparation, the remaining material on the slide was gently pressed and scraped against another glass slide to prepare a cytology smear. The smear was then either fixed with 95% alcohol for Papanicolaou staining or air-dried and stained with Diff-Quik stain for ROSE; ROSE was performed in all cases. If broad consent for the use of surplus specimens had been obtained and the target lesion was considered highly likely to represent metastasis from primary lung cancer based on ROSE findings or other assessments, one or two additional pairs of smear slides were fixed in alcohol in a separate container, based on the judgment of the operator. Only surplus specimens were utilized in this study, with no additional sampling performed.
Procedures for DNA and RNA extraction
A schematic diagram of the DNA and RNA extraction is presented in Figure 1. Cytology smear slides were fixed with 95% ethanol and stored at −20 ℃ until extraction. At the time of extraction, the specimens were scraped using a scalpel and cut into small fragments. To facilitate cell lysis and nuclease inactivation, the scraping materials were lysed using dissolution buffer (Buffer RLT; QIAGEN, Hilden, Germany). Both DNA and RNA were isolated using a spin column-based technique with the AllPrep DNA and RNA Mini Kit (QIAGEN) and automated using the QIAcube Connect system (QIAGEN). Briefly, Buffer RLT was added to the scraping materials to induce DNA release and inactivate nucleases. The lysate was then loaded onto the spin column of the AllPrep DNA and RNA Mini Kit, allowing the DNA to bind to the silica membrane. Following multiple washing and centrifugation steps with the QIAcube Connect, proteins and contaminants were removed. Finally, DNA was eluted with an elution buffer (Buffer EB; QIAGEN). RNA was similarly extracted using the RNeasy spin column, and RNA was eluted in RNase-free water (QIAGEN). Each specimen was adjusted to a total volume of 45 µL. The concentrations of nucleic acids were measured using the Qubit 4 Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA). The yield of DNA or RNA was calculated as follows:
Figure 1.
Schematic representation of DNA and RNA extraction from cytology smear slides.
Yield (ng) = concentration (ng/µL) × 45 (µL).
Statistical analysis
The DNA and RNA yield from each procedure and its suitability for NGS were assessed, with yields of 50 ng or more considered adequate for NGS. Cases in which both DNA and RNA yield met this threshold were defined as sufficient nucleic acid amount for NGS input (SNA). This criterion was established based on two comprehensive genomic assays, TruSight Tumor 170 (Illumina Inc., San Diego, CA, USA) and the TruSight Oncology 500 assay (Illumina Inc.), which require a minimum of 40 ng of DNA and 40 ng of RNA (21). As the TruSight Tumor 170 assay exhibits superior sensitivity for detecting gene mutations when the DNA or RNA input is 50 ng compared to 40 ng (22), 50 ng was defined as the threshold for the primary analysis, which was intended to assess adequacy for comprehensive hybrid-capture panels. In the present study, the paired FFPE specimens underwent NGS using the Oncomine Dx Target Test Multi-CDx System (Oncomine DxTT; Thermo Fisher Scientific), which requires substantially lower DNA and RNA input (10 ng each) (23). Therefore, we also evaluated nucleic acid adequacy using a threshold of 10 ng for both DNA and RNA as a sensitivity analysis. Because nucleic acid concentration depends on the elution volume, which was fixed at 45 µL, we additionally checked whether the measured concentrations met the minimum requirement of each assay: the TruSight assays specify minimum concentrations of 3.3 ng/µL for DNA and 4.7 ng/µL for RNA (24), and the Oncomine DxTT specifies minimum concentrations of 0.83 ng/µL for DNA and 1.43 ng/µL for RNA (23).
Continuous variables are presented as median (range), and categorical variables as number (%). The correlation between DNA and RNA yield, and between yield and the interval from slide preparation to extraction, was assessed using Spearman’s rank correlation coefficient. DNA and RNA yields were compared between categorized groups using the Wilcoxon rank-sum test. In addition, univariable and multivariable logistic regression analyses were performed to identify factors associated with the SNA threshold. Variables with P values ≤0.20 in the univariable analyses were included in the multivariable model. Furthermore, cases in which submission for NGS had been attempted using the corresponding FFPE specimens were analyzed. NGS was performed using the Oncomine DxTT. The association between NGS success or failure and attainment of the SNA threshold was also assessed. NGS failure was defined as unsuccessful sequencing of DNA, RNA, or both, or insufficient tumor cellularity on pathological evaluation. Statistical analyses were performed using JMP Pro 18.0.2 (SAS Institute Inc., Cary, NC, USA). All statistical analyses were two-sided, with a significance level of P<0.05.
Results
Baseline characteristics
A total of 223 patients with lymph node metastases from NSCLC were included in the cohort. The baseline characteristics of the included patients are summarized in Table 1. The median age of the patients was 68 (range, 26–87) years, and 140 patients (62.8%) were men and 83 patients (37.2%) were women. Adenocarcinoma accounted for 154 cases (69.0%) of the observed histological types. Samples were obtained from mediastinal lymph nodes in 156 patients (70.0%) and from hilar lymph nodes in 67 patients (30.0%). The median short diameter on CT was 14.0 (range, 4.2–41.0) mm, and the mediastinal and hilar lymph nodes measured 14.7 (range, 6.6–41.0) mm and 11.9 (range, 4.2–37.6) mm, respectively. A total of 175 patients (78.5%) showed a short diameter on CT larger than 10 mm. In terms of EBUS findings, 170 patients (76.2%) exhibited a short diameter on EBUS larger than 10 mm, 146 patients (65.4%) exhibited grade 0–1 vascular patterns, and 112 patients (50.1%) had non-blue or intermediate elasticity. The 22-gauge needles were selected in 198 patients (88.8%).
Table 1. Baseline characteristics and clinical factors (n=223).
| Variable | Value |
|---|---|
| Age (years), median (range) | 68 (26–87) |
| Sex, n (%) | |
| Male | 140 (62.8) |
| Female | 83 (37.2) |
| Histology, n (%) | |
| Adenocarcinoma | 154 (69.0) |
| Squamous cell carcinoma | 31 (13.9) |
| NSCLC others | 38 (17.1) |
| Target lesion, n (%) | |
| Mediastinal lymph node | 156 (70.0) |
| Hilar lymph node | 67 (30.0) |
| Needle size, n (%) | |
| 25-gauge | 25 (11.2) |
| 22-gauge | 198 (88.8) |
| Short diameter on CT (mm), median (range) | 14.0 (4.2–41.0) |
| ≤10 mm, n (%) | 48 (21.5) |
| >10 mm, n (%) | 175 (78.5) |
| Mediastinal lymph node | 14.7 (6.6–41.0) |
| Hilar lymph node | 11.9 (4.2–37.6) |
| EBUS findings | |
| Short diameter on EBUS, n (%) | |
| ≤10 mm | 53 (23.8) |
| >10 mm | 170 (76.2) |
| Shape, n (%) | |
| Oval | 120 (53.8) |
| Round | 103 (46.2) |
| Margin, n (%) | |
| Indistinct | 158 (70.9) |
| Distinct | 65 (29.1) |
| Echogenicity, n (%) | |
| Homogeneous | 4 (1.7) |
| Heterogeneous | 219 (98.3) |
| Central hilar structure, n (%) | |
| Present | 20 (9.0) |
| Absent | 203 (91.0) |
| Coagulation necrosis sign, n (%) | |
| Negative | 209 (93.7) |
| Positive | 14 (6.3) |
| Vascular patterns, n (%) | |
| Grade 0–1 | 146 (65.4) |
| Grade 2–3 | 77 (34.6) |
| Elasticity, n (%) | |
| Non-blue/intermediate | 112 (50.1) |
| Blue | 111 (49.9) |
CT, computed tomography; EBUS, endobronchial ultrasound; NSCLC, non-small cell lung cancer.
DNA and RNA yields
The median yields of DNA and RNA were 540 (range, 0–22,680) ng and 2,277 (0–26,910) ng, respectively (Table 2). Spearman’s rank correlation coefficient demonstrated a moderate positive correlation (ρ=0.551, P<0.001) (Figure 2). DNA yield was significantly associated with mediastinal lymph nodes (P=0.01), short diameter larger than 10 mm on both EBUS (P<0.001) and CT (P<0.001), use of a 22-gauge needle (P=0.04), and an indistinct margin on EBUS (P=0.01) (Figure 3). Furthermore, RNA yield was significantly associated with mediastinal lymph nodes (P=0.006) and short-axis diameter larger than 10 mm on EBUS (P=0.01) and CT (P<0.001) (Figure 4). The median interval between slide preparation and extraction was 13 (range, 1–68) days, and was not correlated with DNA (ρ=0.050, P=0.46) or RNA yield (ρ=−0.012, P=0.85).
Table 2. The nucleic acid yield and suitable proportion for NGS obtained from surplus smear slides.
| Variable | Value |
|---|---|
| Nucleic acid yield (ng), median (range) | |
| DNA | 540 (0–22,680) |
| RNA | 2,277 (0–26,910) |
| Suitability for NGS, n (%) | |
| ≥50 ng threshold | |
| DNA yield ≥50 ng | 204 (91.5) |
| RNA yield ≥50 ng | 217 (97.3) |
| SNA (Both DNA and RNA yield ≥50 ng) | 199 (89.2) |
| ≥10 ng threshold | |
| DNA yield ≥10 ng | 218 (97.8) |
| RNA yield ≥10 ng | 217 (97.3) |
| Both DNA and RNA yield ≥10 ng | 213 (95.5) |
NGS, next-generation sequencing; SNA, sufficient nucleic acid amount for NGS input.
Figure 2.
Correlation between DNA yield and RNA yield. A scatter plot depicting the relationship between DNA and RNA yields is presented.
Figure 3.
Distribution of DNA yield across clinical and procedural variables. DNA yield (log10-transformed) from cytology smears, stratified by the following factors: (A) target location, (B) needle size, (C) short-axis diameter on CT, and (D-K) endobronchial ultrasound findings, including (D) short-axis diameter, (E) shape, (F) margin, (G) echogenicity, (H) presence of a central hilar structure, (I) coagulation necrosis sign, (J) vascular pattern, and (K) elasticity. Box plots represent the median, interquartile range, and 1.5× interquartile range whiskers. Outliers are presented as individual dots. CT, computed tomography; EBUS, endobronchial ultrasound.
Figure 4.
Distribution of RNA yield across clinical and procedural variables. RNA yield (log10-transformed) from cytology smears, stratified by the following factors: (A) target location, (B) needle size, (C) short-axis diameter on computed tomography, and (D-K) endobronchial ultrasound findings, including (D) short-axis diameter, (E) shape, (F) margin, (G) echogenicity, (H) presence of a central hilar structure, (I) coagulation necrosis sign, (J) vascular pattern, and (K) elasticity. Box plots represent the median, interquartile range, and 1.5× interquartile range whiskers. Outliers are presented as individual dots. CT, computed tomography; EBUS, endobronchial ultrasound.
In terms of the adequacy of the smear samples for NGS, a DNA yield of ≥50 ng was achieved in 204/223 (91.5%) cases, while an RNA yield of ≥50 ng was achieved in 217/223 (97.3%) cases. Notably, SNA, defined as both DNA and RNA yield ≥50 ng, was successfully achieved in 199/223 cases (89.2%). Using the lower threshold of ≥10 ng, corresponding to the minimum nucleic acid input requirement of the Oncomine DxTT, 218/223 (97.8%) cases achieved a DNA yield ≥10 ng and 217/223 (97.3%) achieved an RNA yield ≥10 ng, and 213/223 (95.5%) met both criteria (Table 2). DNA was undetectable (0 ng) in three samples and RNA in six others; nine samples were effectively unusable; nevertheless, all samples were retained in the denominators of all analyses. The working concentrations required by the TruSight assays were met for both DNA and RNA input in 161/223 samples (72.2%), and 196/223 (87.9%) met both the input and concentration requirements of the Oncomine DxTT.
No variables were significantly associated with SNA in the univariable analysis, even after multivariable adjustment (Table 3).
Table 3. Factors associated with meeting DNA/RNA yield requirements for NGS.
| Factors | Category | No. of cases | Satisfied SNA (n=199) | Not satisfied SNA (n=24) | Univariable analysis | Multivariable analysis | ||
|---|---|---|---|---|---|---|---|---|
| Odds ratio (95% CI) | P value | Odds ratio (95% CI) | P value | |||||
| Target location | Hilar lymph node | 67 | 58 (86.6) | 9 (13.4) | (Reference) | |||
| Mediastinal lymph node | 156 | 141 (90.4) | 15 (9.6) | 1.45 (0.58–3.47) | 0.41 | |||
| Needle size | 25-gauge | 25 | 23 (92.0) | 2 (8.0) | (Reference) | |||
| 22-gauge | 198 | 176 (88.9) | 22 (11.1) | 0.70 (0.11–2.58) | 0.62 | |||
| Short diameter on CT (mm) | ≤10 | 48 | 41 (85.4) | 7 (14.6) | (Reference) | |||
| >10 | 175 | 158 (90.3) | 17 (9.7) | 1.58 (0.58–3.95) | 0.35 | |||
| Factors on EBUS findings | ||||||||
| Short diameter on EBUS (mm) | ≤10 | 53 | 44 (83.0) | 9 (17.0) | (Reference) | (Reference) | ||
| >10 | 170 | 155 (91.2) | 15 (8.8) | 2.11 (0.84–5.09) | 0.11 | 2.14 (0.84–5.18) | 0.11 | |
| Shape | Oval | 120 | 107 (89.2) | 13 (10.8) | (Reference) | |||
| Round | 103 | 92 (89.3) | 11 (10.7) | 1.02 (0.43–2.42) | 0.97 | |||
| Margin | Indistinct | 158 | 141 (89.2) | 17 (10.8) | (Reference) | |||
| Distinct | 65 | 58 (89.2) | 7 (10.8) | 1.00 (0.41–2.70) | 0.99 | |||
| Central hilar structure | Present | 20 | 18 (90.0) | 2 (10.0) | (Reference) | |||
| Absent | 203 | 181 (89.2) | 22 (10.8) | 0.91 (0.14–3.47) | 0.91 | |||
| Vascular patterns | Grade 0–1 | 146 | 132 (90.4) | 14 (9.6) | (Reference) | |||
| Grade 2–3 | 77 | 67 (87.0) | 10 (13.0) | 0.71 (0.30–1.73) | 0.44 | |||
| Elasticity | Non-blue/intermediate | 112 | 103 (92.0) | 9 (8.0) | (Reference) | (Reference) | ||
| Blue | 111 | 96 (86.5) | 15 (13.5) | 0.56 (0.23–1.32) | 0.19 | 0.52 (0.22–1.31) | 0.18 | |
Multivariable analysis was conducted using variables with P values ≤0.20 in the univariable analysis. Echogenicity and the coagulation necrosis sign were excluded from the regression analyses because complete separation rendered the estimates unstable. The values are represented as n (%), unless otherwise stated. CI, confidence interval; CT, computed tomography; EBUS, endobronchial ultrasound; NGS, next-generation sequencing; SNA, sufficient nucleic acid amount for NGS input.
SNA attainment and NGS success
Among 178 cases in which submission for NGS was attempted using the corresponding FFPE specimens, sequencing was successful in 161 (90.4%) and unsuccessful in 17 (9.6%). The SNA was achieved in 147/161 (91.3%) with successful sequencing and in 13/17 (76.5%) with sequencing failure (Table 4). When the TruSight concentration requirements were additionally applied, the proportions decreased slightly to 121/161 (75.2%) and 11/17 (64.7%), respectively.
Table 4. SNA achievement by NGS results in FFPE specimens.
| NGS result | SNA achieved, n (%) | SNA not achieved, n (%) | Total, n |
|---|---|---|---|
| Successful | 147 (91.3) | 14 (8.7) | 161 |
| Failed | 13 (76.5) | 4 (23.5) | 17 |
FFPE, formalin-fixed paraffin-embedded; NGS, next-generation sequencing; SNA, sufficient nucleic acid amount for NGS input.
Discussion
In the present study, we showed that DNA and RNA extracted from cytology smear slides obtained through EBUS-TBNA of lymph nodes met the predefined nucleic acid input threshold for NGS in 89.2% of cases. When the threshold was lowered to 10 ng for both DNA and RNA, consistent with the minimum input requirement of the Oncomine DxTT, 95.5% met both criteria. Because concentration is determined by the elution volume, which was fixed at 45 µL, 72.2% and 87.9% met the quantity and concentration requirements of the TruSight assays and the Oncomine DxTT, respectively. A previous meta-analysis reported an average DNA yield of 868.7 ng from EBUS-TBNA specimens, with 80.9–91.4% adequate for NGS (11), indicating that failure to meet NGS requirements remains common with current specimen handling.
The primary cause of NGS failure is insufficient quantity or poor quality of nucleic acids (25). Formalin fixation, in particular, compromises DNA integrity by inducing denaturation, fragmentation, and methylene bridge formation, thereby contributing significantly to NGS failure (26,27). RNA extraction is similarly impaired by incomplete lysis and the high RNase content in formalin-fixed tissues (28). Notably, approximately 25–40% of DNA samples extracted from FFPE specimens fail to meet the quality standards required for NGS. This failure results from DNA degradation or insufficient DNA quantity, and these factors are attributable, at least in part, to formalin fixation (29). In contrast, in this study, we employed ethanol fixation, as it preserves nucleic acids more effectively than formalin (26). Compared to formalin-based fixation, alcohol-based fixation has demonstrated significant benefits, including greater DNA yield and longer DNA fragment sizes (26). To compare surplus cytology smears with corresponding FFPE specimens, we investigated the relationship between DNA and RNA yield and NGS results obtained from corresponding FFPE specimens. In our cohort, 17/178 cases were classified as sequencing failures: NGS analysis failed in 10 cases owing to RNA quality control issues, whereas seven cases could not be submitted owing to insufficient tumor cellularity in the FFPE specimens. Notably, 13/17 cases (76.5%) met the criteria for SNA, and 11/17 (64.7%) met both the SNA (input amount) and the corresponding minimum concentration requirements of the TruSight assays. For the Oncomine DxTT, 15/17 cases (88.2%) met the minimum DNA and RNA input requirement (10 ng each), and 13/17 (76.5%) satisfied both the minimum input and concentration requirements. Although this finding suggests that cytology smears may represent a potential alternative source of nucleic acids, actual sequencing of these smear specimens was not performed. Therefore, whether smear specimens can successfully rescue cases in which FFPE specimens fail remains to be determined.
Several studies have demonstrated successful NGS utilizing cytology specimens. Cytology samples can broadly be classified into cytology smears and liquid-based cytology (LBC) samples, both of which exhibit a concordance rate of 94.9–99.5% with gene mutations detected in histology samples (30-34). In the evaluation of malignant cell quantity and cellularity for NGS, smear cytology may be more suitable than LBC. Specifically, smear cytology involves directly spreading cells onto slides, allowing a clear assessment of cellularity. In contrast, LBC concentrates cells through centrifugation or filtration techniques, producing uniform preparations with reduced background. Notably, smear cytology allows selection of slides enriched in tumor cells, thus optimizing the specimen for analysis (35). In this context, Fielding et al. reported that Diff-Quik-stained cytology smears from EBUS-TBNA yielded higher DNA yields and achieved greater NGS success rates than FFPE cell blocks (15,16).
Although previous studies demonstrated that cytology smears provided substantial DNA yield and high NGS success rates, they exclusively focused on DNA sequencing results, as RNA analysis was challenging with air-dried, Diff-Quik-stained specimens (16). In contrast, we utilized ethanol-fixed, unstained slides, which may have preserved RNA and enabled its analysis. Although DNA sequencing can identify gene fusion breakpoints without RNA purification (36), detection can be challenging when introns are too long to tile effectively or contain repetitive elements that are difficult to sequence accurately (12). Notably, RNA sequencing overcomes these limitations, as it analyzes fusions after intron removal (37). RNA sequencing can successfully identify gene fusions that remain undetected by DNA-based methods, allowing patients to receive targeted kinase inhibitors and demonstrate therapeutic responses (12-14). Therefore, incorporating RNA sequencing along with DNA sequencing can enhance the sensitivity for detecting genetic alterations, and this incorporation may be facilitated by using cytology smear samples.
In the present study, the short diameter on EBUS and CT, target location, needle size, and indistinct margin on EBUS were significantly correlated with nucleic acid yield. Although previous studies have emphasized CT and EBUS findings for differentiating between benign and malignant lesions, no study has specifically addressed the relationship between these imaging findings and the quantity of tumor cells obtained from malignant lesions. A previous study demonstrated that a smaller lesion diameter was associated with a lower diagnostic yield of EBUS-TBNA (38). As EBUS-TBNA acquires samples by agitating the needle within the target lesion, obtaining sufficient material from a smaller lesion is generally considered difficult. This explains the positive correlation observed between lesion diameter and the yield of DNA and RNA in our study. Moreover, the median size of mediastinal lymph nodes on CT was significantly larger than that of hilar lymph nodes (14.7 vs. 11.9 mm, P<0.001), which is consistent with a previous study demonstrating that mediastinal lymph node metastases tend to be larger than hilar lymph node metastases in patients with lung cancer (39). This size difference accounts for the association observed between mediastinal nodes and elevated DNA and RNA yield in the present study. In terms of needle size, 22-gauge needles yielded significantly more DNA than 25-gauge needles. This finding is also consistent with a previous study indicating that the 22-gauge needle collects larger tissue samples containing a higher number of malignant cells than the 25-gauge needle (40). Nevertheless, these findings should be interpreted with caution because no adjustment for multiplicity was applied. Importantly, achieving SNA was not significantly associated with factors such as target location, lesion size, or needle size. These findings suggest that none of the evaluated clinical or procedural factors independently predicted nucleic acid adequacy in this cohort. Because only 24 cases did not meet the predefined nucleic acid adequacy criteria, the analysis may have been underpowered to detect associations.
This study has certain limitations. First, it was a retrospective, single-center study using surplus cytology smears. Additional ethanol-fixed smears were collected only when the target lesion was considered highly likely to represent metastatic disease based on ROSE findings or the operator’s judgment. The cohort is therefore enriched for aspirates judged likely to be tumor-bearing, and the adequacy rates reported here are likely to overestimate those achievable in an unselected population undergoing EBUS-TBNA; the proportion of cases in which additional smears were not banked was not recorded and could not be determined retrospectively. Second, tumor cellularity and smear area were not assessed, owing to the use of unstained slides fixed in ethanol. This limitation is potentially important, as successful NGS generally requires a pathologically confirmed minimum quantity of nucleic acid and adequate tumor cellularity (41). Although we did not confirm the cellularity of the specimen, it is unlikely that normal tissue was inadvertently sampled, given that EBUS-TBNA provides real-time confirmation of needle positioning within the target lesion (42). Moreover, previous research has demonstrated a strong correlation in tumor cellularity between paired fine-needle aspiration slides, one stained for ROSE and the other left unstained (43). Therefore, tumor cellularity can be reasonably inferred by assessing the stained slide as a reference for the unstained counterpart. Future studies should consider adopting this approach to enhance the evaluation of cytology smears for NGS. Third, although nucleic acids were extracted from EBUS-TBNA smear specimens in quantities meeting the predefined input thresholds, we did not perform actual NGS analysis, and the quality and integrity of the extracted DNA and RNA were not assessed. Current guidelines recommend appropriate preanalytical quality control for NGS specimens (41), and DNA integrity has been shown to significantly influence NGS success rates (44). In addition, RNA integrity is commonly assessed using a standardized quality metric such as the RNA Integrity Number, which provides a reliable measure of RNA quality (45). Therefore, future studies should incorporate actual NGS analysis together with comprehensive assessments of DNA and RNA integrity to further validate the suitability of cytology smear specimens for clinical sequencing. In addition, as the eluates were eluted in a fixed volume of 45 µL, some cases fell below the required concentration; future studies are therefore warranted to determine the optimal elution volume that ensures sufficient nucleic acid concentration while maintaining recovery. Fourth, this study evaluated only ethanol-fixed, unstained cytology smear slides. Because many institutions routinely retain air-dried Diff-Quik-stained slides for ROSE instead of ethanol-fixed unstained slides, the generalizability of our findings to other cytology preparation protocols may be limited. However, as ethanol-fixed unstained smears can be prepared from the later passes of the routine sampling sequence, once ROSE has confirmed the presence of malignant cells, this approach is unlikely to substantially complicate the routine EBUS-TBNA workflow.
Conclusions
In conclusion, we demonstrated that cytology smears obtained from EBUS-TBNA specimens can yield DNA and RNA in amounts meeting predefined input thresholds for NGS. Further studies involving actual NGS analyses, including assessments of tumor cellularity and nucleic acid quality, are warranted to validate and extend our findings.
Supplementary
The article’s supplementary files as
Acknowledgments
We would like to thank Takako Ishiyama and Sachiko Kurihara (Department of Molecular Pathology, National Cancer Center Hospital, Japan) for their technical support in DNA and RNA extraction and for their kind guidance on the extraction procedure. We also thank Editage (www.editage.jp) for English language editing.
Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. This study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the Institutional Review Board of the National Cancer Center (No. 2018-090). Written informed consent for the clinical procedure and broad consent for the use of surplus specimens were obtained from all patients.
Footnotes
Reporting Checklist: The authors have completed the STROBE reporting checklist. Available at https://jtd.amegroups.com/article/view/10.21037/jtd-2026-1711/rc
Funding: This article was supported by JSPS KAKENHI (Grant Number JP24K19099).
Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://jtd.amegroups.com/article/view/10.21037/jtd-2026-1711/coif). H.F. reports grants from Hitachi High-Tech Corporation and DNA Chip Research Inc.; and honoraria from DNA Chip Research Inc., Erbe Elektromedizin GmbH, and AstraZeneca. Y.M. reports grants from DNA Chip Research Inc. and Hitachi Ltd.; honoraria from Olympus Corporation, AstraZeneca PLC, FUJIFILM Corporation, Takeda Pharmaceutical Company, Novartis AG, AMCO Inc., DNA Chip Research Inc., Erbe Elektromedizin GmbH, Merck KGaA, Chugai Pharmaceutical Co., Ltd., and Johnson & Johnson MedTech; and payment for expert testimony from Olympus Corporation, Intuitive Surgical Inc., and FUJIFILM Corporation. T.T. reports grants from JSPS KAKENHI; honoraria from Nippon Medical School Foundation; and participation on a Data Safety Monitoring Board for Hamamatsu University School of Medicine. Y.Y. reports grants from Merck Biopharma, Chugai-pharma, Konica-Minolta REALM, and Optieum Biotechnologies; consulting fees from AstraZeneca, MSD, AbbVie, Novartis, Amgen, Daiichi-Sankyo, Janssen Pharma, and Konica-Minolta REALM; honoraria from MSD, Chugai-pharma, AstraZeneca, Merck Biopharma, Novartis, Amgen, Daiichi-Sankyo, and Thermo Fisher Scientific; and serves as an editorial board member of Cancer Science. The other authors have no conflicts of interest to declare.
Data Sharing Statement
Available at https://jtd.amegroups.com/article/view/10.21037/jtd-2026-1711/dss
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