Evaluating Diff‐Quik cytology smears for large‐panel mutation testing in lung cancer—Predicting DNA content and success with low‐malignant‐cellularity samples

Abstract

Background Cytology smears are commonly collected during endobronchial ultrasound–guided transbronchial needle aspiration (EBUS TBNA) procedures but are rarely used for molecular testing. Studies are needed to demonstrate their great potential, in particular for the prediction of malignant cell DNA content and for utility in molecular diagnostics using large gene panels. Methods A prospective study was performed on samples from 66 patients with malignant lymph nodes who underwent EBUS TBNA. All patients had air‐dried, Diff‐Quik cytology smears and formalin‐fixed, paraffin‐embedded cell blocks collected for cytopathology and molecular testing. One hundred eighty‐five smears were evaluated by microscopy to estimate malignant cell percentage and abundance and to calculate smear size and were subjected to DNA extraction. DNA from 56 smears from 27 patients was sequenced with the TruSight Oncology 500 assay (Illumina). Results Each microscopy parameter had a significant effect on the DNA yield. An algorithm was developed that predicted a >50‐ng DNA yield of a smear with an area under the curve of 0.86. Fifty DNA samples (89%) with varying malignant yields were successfully sequenced. Low‐malignant‐cell content (<25%) and smear area (<15%) were the main reasons for failure. All standard‐of‐care mutations were detected in replicate smears from individual patients, regardless of malignant cell content. Tier 1/2 mutations were discovered in two cases where standard‐of‐care specimens were inadequate for sequencing. Smears were scored for tumor mutation burden. Conclusions Microscopy of Diff‐Quik smears can triage samples for comprehensive panel sequencing, which highlights smears as an excellent alternative to traditional testing with cell blocks.

Discrepancies in tumor mutation burden reporting from sequential endobronchial ultrasound transbronchial needle aspiration samples within single lymph node stations - brief report

“…Sequencing sensitivity was therefore compromised, with few somatic coding mutations being detected (9 versus 51) and used in TMB scores (5 versus 24) compared to its paired slide ( Table 1 ; Supplementary Table 1 ). Our previous study has reported a higher failure rate of sequencing metrics in samples with <25% malignant cells ( 7 ). These samples might allow detection of actionable mutations but need to be carefully considered for TMB estimation.…”

“…Methods and data related to the microscopic estimation of tumour content on the Diff-Quik slide (involving estimating percent malignant cells to non-malignant cells, the approximate overall abundance of malignant cells and area of the glass slide covered by the smear), DNA extraction and sequencing by (TruSight Oncology 500 (TSO500 - Illumina)and the description of somatic mutations and TMB was previously reported ( 7 , 8 ). The sequence data was processed and analysed using the TruSight Oncology 500 Local App version 2.0 (Illumina), which estimates TMB using eligible variants/effective panel size (coding region with >50x coverage).…”

“…Recently we studied the feasibility of using Diff-Quik cytology smears routinely produced during the EBUS TBNA procedure as an alternative source of tumour material for genomic testing ( 6 ). We used EBUS TBNA aspirates deposited on Diff-Quik stained cytology smears from individual passes into mediastinal nodes as a way to directly compare tumour content and DNA yield and the subsequent success of sequencing using the TSO500 assay for identifying actionable mutations ( 7 ). The sequencing of multiple smears per EBUS TBNA procedure enabled us to further explore the issue of reliability of these specimens for TMB assessment.…”

Performance of non‐formalin fixed paraffin embedded samples in hybrid capture and amplicon next‐generation sequencing panels

“…The analysis pipeline of sequenced TSO500 libraries generates variant calls and also quality metrics for each sample and guideline quality thresholds. The use of TSO500 has been recently reported in non‐FFPE tissue, 20 however, studies aimed at comparing the performance of this type of materials to other FFPE materials generally preferred for NGS are still lacking in the academic literature, either regarding the yield of nucleic acid extractions or regarding the library quality metrics.…”

Molecular profiling and feasibility using a comprehensive hybrid capture panel on a consecutive series of non-small-cell lung cancer patients from a single centre

“… 30 A recent study assessed the feasibility of the TSO500 panel using Diff-Quick cytology smears from 27 patients with NSCLC, but sequencing failed in 5 cases (1 due to library preparation failure and 4 due to low median coverage). 31 …”