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.

Malignant pleural effusion and pericardial effusion, an unusual presentation of metastatic adenoid cystic carcinoma of the floor of mouth: A case report with cyto-histopathological correlation.

Adenoid cystic carcinoma (ACC) is an uncommon tumour that represents 5%-10% of salivary gland tumours and 1% of all head and neck malignancies. It is characterised by a protracted clinical course with late metastasis and poor long-term prognosis. We report the case of a 38-year-old woman presenting with pulmonary and pleural metastases, on the background of ACC of the floor of mouth, which had been treated 4 years ago with surgical excision and radiotherapy. Cytological evaluation of the pleural effusion showed exfoliated ACC tumour cells. Despite palliative chemotherapy, the patient developed disease progression including metastatic spread to the pericardium, and died of disease within a year. This case illustrates an unusual presentation of ACC and highlights the importance of considering this entity when encountering a basaloid neoplasm in extra-salivary locations.

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.

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.

BACKGROUND: Next-generation sequencing (NGS) in lung cancer specimens from endobronchial ultrasound transbronchial needle aspiration (EBUS-TBNA) is usually performed on formalin-fixed paraffin-embedded cell block material. OBJECTIVES: Since DNA can be damaged by this process, we investigated the potential of using DNA extracted from Diff-Quik cytology smears made for rapid on-site evaluation during EBUS-TBNA. METHODS: In a prospective study, 67 patients undergoing diagnostic EBUS-TBNA were ana-lysed. We compared cell blocks and smears for DNA yields and sequencing (TruSeq Amplicon Cancer Panel) outcomes. Smears were also evaluated for tumour cell fraction and overall cellularity (cell count). RESULTS: Primary lung cancer was diagnosed in 64 patients and metastatic malignancy in 3 patients. The DNA yield from smears was significantly higher than that obtained from matched cell blocks (mean 1,740 vs. 434 ng; p = 0.001). For 33 cases with matched smears and cell blocks the mutation profiles were similar. Smears with abundant malignant cells (using a cut-off of > 25% tumour cell fraction and > 1,000 cells) accurately predicted high (> 50 ng) DNA yield and therefore success in triaging samples to sequencing. In terms of tissue workflow, using only smears as source DNA for sequencing was an improvement in the use of only cell blocks (54/67 [80.6%] vs. 41/67 [61.2%]); however, the use of cell blocks when smears were not available or did not yield sufficient DNA further improved the success rate to 62/67 (92.5%) cases. CONCLUSION: We recommend smears in laboratory workflows as the primary source of DNA for NGS following an EBUS procedure.

EBUS-TBNA Cytology Specimens are Predictive of Occupational Dust Exposure in Patients With Bilateral Mediastinal and Hilar Lymphadenopathy.

OBJECTIVES: The aim of this study was to describe the cytological features of patients with significant occupational dust exposure presenting with benign bilateral mediastinal and hilar lymphadenopathy (BHL). METHODS: A retrospective cohort study including patients undergoing EBUS-TBNA for investigation of benign BHL. Patient characteristics, dust exposure history, radiology, and cytology samples from EBUS-TBNA were assessed. RESULTS: EBUS-TBNA cytology in patients with exposure showed a significant increase in the presence of birefringent fibers (60.7% vs 19.2%, P = 0.001) and intracellular carbon pigment (75.0% vs 28.9%, P = 0.001) compared with patients without exposure. The presence of these two features together yielded a sensitivity of 53.6% and a specificity of 88.5%. CONCLUSION: In patients with BHL and a history of occupational dust exposure, the presence of birefringent fibers and intracellular carbon pigment in EBUS-TBNA cytology samples may assist in a diagnosis of lymphadenopathy due to occupational dust exposure.

Respiratory bronchiolitis-associated interstitial lung disease secondary to electronic nicotine delivery system use confirmed with open lung biopsy.

As a modern phenomenon, there is currently limited understanding of the possible toxic effects and broader implications of electronic nicotine delivery systems (ENDS). Large volumes of aerosolized particles are inhaled during "vaping" and there are now an increasing number of case reports demonstrating toxic effects of ENDS, as well as human studies demonstrating impaired lung function in users. This article presents a case of respiratory bronchiolitis interstitial lung disease (RB-ILD) precipitated by vaping in a 33-year-old male with 10 pack years of traditional cigarette and prior treatment for mixed germ cell tumour. The patient had started vaping 10-15 times per day while continuing to smoke 10 traditional cigarettes per day. After 3 months of exposure to e-cigarette vapour, chest computed tomography demonstrated multiple new poorly defined pulmonary nodules with fluffy parenchyma opacification centred along the terminal bronchovascular units. Video-assisted thoracoscopy with lung biopsy of the right upper and right middle lobes was undertaken. The microscopic findings were overall consistent with RB-ILD. This case demonstrates toxicity with use of ENDS on open lung biopsy with resolution of radiographic findings on cessation. We believe that this is the first case where open lung biopsy has demonstrated this and our findings are consistent with RB-ILD.