Evaluation of Endobronchial Ultrasound-Guided Transbronchial Needle Aspiration (EBUS-TBNA) Samples from Advanced Non-Small Cell Lung Cancer for Whole Genome, Whole Exome and Comprehensive Panel Sequencing.

Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) is often the only source of tumor tissue from patients with advanced, inoperable lung cancer. EBUS-TBNA aspirates are used for the diagnosis, staging, and genomic testing to inform therapy options. Here we extracted DNA and RNA from 220 EBUS-TBNA aspirates to evaluate their suitability for whole genome (WGS), whole exome (WES), and comprehensive panel sequencing. For a subset of 40 cases, the same nucleic acid extraction was sequenced using WGS, WES, and the TruSight Oncology 500 assay. Genomic features were compared between sequencing platforms and compared with those reported by clinical testing. A total of 204 aspirates (92.7%) had sufficient DNA (100 ng) for comprehensive panel sequencing, and 109 aspirates (49.5%) had sufficient material for WGS. Comprehensive sequencing platforms detected all seven clinically reported tier 1 actionable mutations, an additional three (7%) tier 1 mutations, six (15%) tier 2-3 mutations, and biomarkers of potential immunotherapy benefit (tumor mutation burden and microsatellite instability). As expected, WGS was more suited for the detection and discovery of emerging novel biomarkers of treatment response. WGS could be performed in half of all EBUS-TBNA aspirates, which points to the enormous potential of EBUS-TBNA as source material for large, well-curated discovery-based studies for novel and more effective predictors of treatment response. Comprehensive panel sequencing is possible in the vast majority of fresh EBUS-TBNA aspirates and enhances the detection of actionable mutations over current clinical testing.

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

Introduction: Tumour Mutation Burden (TMB) is a potential biomarker for immune cancer therapies. Here we investigated parameters that might affect TMB using duplicate cytology smears obtained from endobronchial ultrasound transbronchial needle aspiration (EBUS TBNA)-sampled malignant lymph nodes. Methods: Individual Diff-Quik cytology smears were prepared for each needle pass. DNA extracted from each smear underwent sequencing using large gene panel (TruSight Oncology 500 (TSO500 - Illumina)). TMB was estimated using the TSO500 Local App v. 2.0 (Illumina). Results: Twenty patients had two or more Diff-Quik smears (total 45 smears) which passed sequencing quality control. Average smear TMB was 8.7 ± 5.0 mutations per megabase (Mb). Sixteen of the 20 patients had paired samples with minimal differences in TMB score (average difference 1.3 ± 0.85). Paired samples from 13 patients had concordant TMB (scores below or above a threshold of 10 mutations/Mb). Markedly discrepant TMB was observed in four cases, with an average difference of 11.3 ± 2.7 mutations/Mb. Factors affecting TMB calling included sample tumour content, the amount of DNA used in sequencing, and bone fide heterogeneity of node tumour between paired samples. Conclusion: TMB assessment is feasible from EBUS-TBNA smears from a single needle pass. Repeated samples of a lymph node station have minimal variation in TMB in most cases. However, this novel data shows how tumour content and minor change in site of node sampling can impact TMB. Further study is needed on whether all node aspirates should be combined in 1 sample, or whether testing independent nodes using smears is needed.

Whole Genome Sequencing in Advanced Lung Cancer can be Performed Using Diff-Quik Cytology Smears Derived from Endobronchial Ultrasound, Transbronchial Needle Aspiration (EBUS TBNA).

INTRODUCTION: Maximising alternative sample types for genomics in advanced lung cancer is important because bronchoscopic samples may sometimes be insufficient for this purpose. Further, the clinical applications of comprehensive molecular analysis such as whole genome sequencing (WGS) are rapidly developing. Diff-Quik cytology smears from EBUS TBNA is an alternative source of DNA, but its feasibility for WGS has not been previously demonstrated. METHODS: Diff-Quik smears were collected along with research cell pellets. RESULTS: Tumour content of smears were compared to research cell pellets from 42 patients, which showed good correlation (Spearman correlation 0.85, P < 0.0001). A subset of eight smears underwent WGS, which presented similar mutation profiles to WGS of the matched cell pellet. DNA yield was predicted using a regression equation of the smears cytology features, which correctly predicted DNA yield > 1500 ng in 7 out of 8 smears. CONCLUSIONS: WGS of commonly collected Diff-Quik slides is feasible and their DNA yield can be predicted.

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

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.

Prospective Optimization of Endobronchial Ultrasound-Guided Transbronchial Needle Aspiration Lymph Node Assessment for Lung Cancer: Three Needle Agitations Are Noninferior to 10 Agitations for Adequate Tumor Cell and DNA Yield.

Introduction: Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS TBNA) is an important means of obtaining a tissue for advanced lung cancer. Optimizing the EBUS TBNA needling technique is important to maintain procedural simplicity and maximize sample quality for emerging molecular diagnostics. Methods: We prospectively explored three versus 10 agitations of the needle in sequential passes into the lymph node using separate needles. Resulting Diff-Quik cytology smears were quantitatively assessed using microscopic (tumor cell cellularity, abundance scores, erythrocyte contamination) and DNA yields. Microscopy was reported by two cytopathologists, and an inter-rater assessment was made by four additional cytopathologists. Results: In 86 patients confirmed as having malignant disease by EBUS TBNA (45 males, 41 females), a mean of 5.3 smears were made per patient with a total of 459 smears scored by pathologists and 168 paired smears extracted for DNA. There was no significant difference between three versus 10 agitations for smear cellularity (p = 0.44), DNA yield (p = 0.84), or DNA integrity (p = 0.20), but there was significantly less contamination by erythrocytes from three agitations (chi-square p = 0.008). There was significantly more DNA in the first pass into the node using three agitations than with other passes and with 10 agitations (pass × agitations interaction, p = 0.031). Reviewing pathologists correctly classified smears as more than or equal to 25% cellularity 86.3% of the time (κ = 0.63 [95% confidence interval: 0.55-0.71]). Conclusions: Three agitations are noninferior to 10 agitations for overall abundance of malignant cells and DNA content on smears. A smear with adequate DNA for panel sequencing could almost always be made with the first needle pass using three agitations.

First Human Use of a New Robotic-Assisted Fiber Optic Sensing Navigation System for Small Peripheral Pulmonary Nodules.

BACKGROUND: We tested a new, investigational robotic-assisted bronchoscope system with a remotely controlled catheter to access small peripheral bronchi with real-time driving under live visualization and distal tip articulation of the catheter. The unique catheter remains stationary once located at the biopsy position. OBJECTIVES: The primary objectives of this study were to evaluate the safety and feasibility of a new shape-sensing robotic bronchoscope system to bronchoscopically approach and facilitate the sampling of small peripheral pulmonary nodules of 1-3 cm. Secondary objectives included evaluating procedural characteristics and early performance trends associated with the use of the new robotic bronchoscope system. METHODS: Subjects were enrolled according to study eligibility criteria at a single center. Navigation pathways were semi-automatically created using pre-procedure CT scans. Simultaneous (real-time) viewing of actual and virtual bronchi was used real time during navigation to the displayed target. An endobronchial ultrasound mini-probe was used to confirm lesion location. Flexible 19- to 23-G needles specifically designed to accommodate tight bend radii in transbronchial needle aspiration were used along with conventional biopsy tools. Enrolled subjects completed follow-up visits up to 6 months after the procedure. RESULTS: The study included 29 subjects with a mean lesion size of 12.2 ± 4.2, 12.3 ± 3.3, and 11.7 ± 4.1 mm in the axial, coronal, and sagittal planes, respectively. The CT bronchus sign was absent in 41.4% of cases. In 96.6% of cases, the target was reached, and samples were obtained. No device-related adverse events and no instances of pneumothorax or excessive bleeding were observed during the procedure. Early performance trends demonstrated an overall diagnostic yield of 79.3% and a diagnostic yield for malignancy of 88%. CONCLUSION: This new robotic-assisted bronchoscope system safely navigated to very small peripheral airways under continuous visualization, and through maintenance of a static position, it provides a unique sampling capability for the biopsy of small solitary pulmonary nodules.