FNA smears as a potential source of DNA for targeted next‐generation sequencing of lung adenocarcinomas

Abstract

Background Diff-Quik stained fine-needle aspiration (FNA) smears and touch preparations from biopsies represent alternative specimens for molecular testing when cell block or biopsy material is insufficient. We describe the use of these samples for targeted next-generation sequencing (NGS) of primary and metastatic lung adenocarcinoma and report DNA quality and success rates of FNA smears compared with other specimens from one year of clinical use. Methods A validation set of 10 slides from 9 patients with prior clinical EGFR Sanger sequencing and KRAS pyrosequencing (5 KRAS +/EGFR−, 4 KRAS/EGFR−) underwent DNA extraction, quality assessment, and targeted NGS. Subseqently, lung adenocarcinoma specimens submitted for NGS solid tumor mutation panel testing in one calendar year (60 biopsies, 57 resections, 33 FNA cell blocks, 12 FNA smears, 10 body fluid cell blocks) were reviewed for specimen adequacy, sequencing success, and DNA quality. Results All 10 validation samples met the DNA quality threshold (ΔCT threshold <8, range −2.2 to 4.9) and yielded 0.5 to 22 μg of DNA. KRAS and EGFR mutation status from FNA smears by NGS were concordant with previous clinical testing for all 10 samples. In the one year review, FNA smears were 100% successful, suggesting performance equivalent to or better than established specimen types, including FNA cell blocks. DNA quality by ΔCT was significantly better from FNA smears than from biopsies, resections, and FNA cell blocks. Conclusions We conclude that FNA smears of lung adenocarcinomas are a high quality alternative specimen for a targeted NGS panel with a high success rate in clinical practice.

Diff-Quik Cytology Smears from Endobronchial Ultrasound Transbronchial Needle Aspiration Lymph Node Specimens as a Source of DNA for Next-Generation Sequencing Instead of Cell Blocks

“…Fifteen of our cases yielded > 1 µg of DNA. These DNA yields were consistent with recent retrospective data of lymph node FNA smears from 3 adenocarcinoma patients preselected for tumour fraction ≥50%, which reported mean total slide DNA yields of 3.9 µg [50]. …”

“…Other studies compared the DNA integrity for genomic profiling from Diff-Quik- and PAP-stained slides and showed that PAP processing caused appreciable DNA degradation [26, 45]. Treece et al [50] showed significantly higher amplifiable DNA yields from FNA smears than from biopsies and FNA cell blocks. Kanagal-Shamanna et al [52] studied FNA samples from 61 cases of solid organ tumours.…”

“…The use of cytopathology specimens for DNA isolation also has procedural considerations with potential medicolegal consequences [17, 26, 47-49], which we have addressed. Good diagnostic concordance between digital scans and original slides was an important requisite for slides to progress to DNA extraction [50]. The digital scan not only provided a permanent record of the slide, but also an exact record of what cells were subjected to sequencing.…”

Lung Carcinoma Predictive Biomarker Testing by Immunoperoxidase Stains in Cytology and Small Biopsy Specimens: Advantages and Limitations

“…Direct smears, cell monolayers, and cell block sections can all be used for IPOX and molecular tests including next-generation sequencing. 7,[13][14][15][16][17] IPOX TESTING Similar to core biopsies, low tumor content or DNA quality may be encountered in cytologic specimens despite safeguards in tissue acquisition, preparation, and use. 8 Immunoperoxidase ameliorates this limitation, as it detects signals in specimens with low cellularity.…”

The use of endobronchial ultrasound guided transbronchial needle aspiration specimens for next generation sequencing in non-small cell lung cancer: a clinical perspective

“…Although there are studies published on the suitableness of formalin fixed paraffin embedded (FFPE) cell block samples for NGS testing (12,13), cytology smear preservation methods provide a superior quality DNA for testing (14)(15)(16). Moreover, an additional advantage of cytology slides lies in the ability for rapid onsite examination (ROSE) to assess smears for cellularity and adequacy in real time in the course of the procedure.…”