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

Fielding, David; Dalley, Andrew J.; Bashirzadeh, Farzad; Singh, Mahendra; Nandakumar, Lakshmy; Reed, Amy E. McCart; Black, Debra; Kazakoff, Stephen H.; Pearson, John V.; Nones, Kátia; Waddell, Nicola; Lakhani, Sunil R.; Simpson, Peter T.

doi:10.1159/000495661

Cited by 26 publications

(28 citation statements)

References 55 publications

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“…In our series, the adequacy of EBUS-TBNA cytologies for genetic testing was high, above 90% for both NGS and nCounter, and was in line with other previously published studies ( Supplementary Table S3 [ 24 , 25 , 26 , 27 , 28 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 , 43 ]). Molecular results in cytology fully agreed with that of tissue testing, including some EGFR , BRAF , and MET ∆ex14 alterations, endorsing cytologies as an optimal source of material for genetic testing.…”

Section: Discussionsupporting

confidence: 92%

“…However, they can be a rich source of genetic material as they can be preserved without FFPE, avoiding the risk of fading of PD-L1 IHC expression or DNA quality found with samples with an extended storage time [ 5 , 32 ]. Fielding and colleagues investigated the potential of using DNA extracted from diff-1uik cytology smears versus cell blocks from EBUS-TBNA procedure and observed that smears have a better yield and therefore success in triaging samples to sequencing [ 33 ].…”

Section: Discussionmentioning

confidence: 99%

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EBUS-TBNA Cytological Samples for Comprehensive Molecular Testing in Non–Small Cell Lung Cancer

Martín-Deleon

Teixidó

Lucena

et al. 2021

Cancers

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Clinical guidelines promote the identification of several targetable biomarkers to drive treatment decisions in advanced non-small cell lung cancer (NSCLC), but half of all patients do not have a viable biopsy. Specimens from endobronchial-ultrasound transbronchial needle aspiration (EBUS-TBNA) are an alternative source of material for the initial diagnosis of NSCLC, however their usefulness for a complete molecular characterization remains controversial. EBUS-TBNA samples were prospectively tested for several biomarkers by next-generation sequencing (NGS), nCounter, and immunohistochemistry (PD-L1). The primary objectives were to assess the sensitivity of EBUS-TBNA samples for a comprehensive molecular characterization and to compare its performance to the reference standard of biopsy samples. Seventy-two EBUS-TBNA procedures were performed, and 42 NSCLC patients were diagnosed. Among all cytological samples, 92.9% were successfully genotyped by NGS, 95.2% by nCounter, and 100% by immunohistochemistry. There were 29 paired biopsy samples; 79.3% samples had enough tumor material for genomic genotyping, and 96.6% for PD-L1 immunohistochemistry. A good concordance was found between both sources of material: 88.9% for PD-L1, 100% for NGS and nCounter. EBUS-TBNA is a feasible alternative source of material for NSCLC genotyping and allows the identification of patient candidates for personalized therapies with high concordance when compared with biopsy.

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Section: Discussionsupporting

confidence: 92%

Section: Discussionmentioning

confidence: 99%

EBUS-TBNA Cytological Samples for Comprehensive Molecular Testing in Non–Small Cell Lung Cancer

Martín-Deleon

Teixidó

Lucena

et al. 2021

Cancers

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“…Furthermore, comprehensive genome analysis of LBC samples has been attempted recently in the fields of pathology and laboratory medicine [13, 14]. Studies have reported that DNA extracted from conventional cytological or LBC samples could be applied to NGS analysis [15–18], but these studies did not always include detailed descriptions of their fixation and DNA extraction methods. At present, therefore, an efficient and suitable method for DNA extraction to acquire high-quality of DNA for NGS from LBC samples has not been introduced.…”

Section: Introductionmentioning

confidence: 99%

Comprehensive validation of liquid-based cytology specimens for next-generation sequencing in cancer genome analysis

et al. 2019

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In addition to conventional cytology, liquid-based cytology (LBC) is also used for immunocytochemistry and gene analysis. However, an appropriate method to obtain high quality DNA for next-generation sequencing (NGS) using LBC specimens remains controversial. We determined the optimal conditions for fixation with an alcohol-based fixative for LBC and DNA extraction using cultured cancer cell lines and clinical specimens. The extracted DNA was processed for NGS after the DNA quality was confirmed based on the DNA concentration and degree of degradation. The optimal conditions for cultured cells to obtain high quality DNA were to fix the cells at a density of 6 × 10 3 or 2 × 10 4 cells/mL and to use the magnetic bead-based DNA extraction method. Even after storing the fixed cells for 90 days, DNA extracted using the above and other extraction kits, including membrane-based methods, did not undergo degradation. Furthermore, 5-year-old residual LBC samples demonstrated high DNA quality that was suitable for NGS. Furthermore, a cancer genome panel analysis was successfully performed with DNA extracted from cultured cells fixed at 6 × 10 3 cells/mL for 90 days, and with DNA from residual LBC samples even after 1 year of storage. Residual LBC samples may be a useful source of DNA for clinical NGS to promote genome-based cancer medicine.

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“…The data in the literature, 6‐15 show a high concordance between ROSE cytology and a final diagnosis. However, only a few papers have demonstrated that ROSE increases yields for molecular testing, 20‐27 but this study was conducted exclusively on TBNA. A correlation between ROSE on TIC and/or on brushing and endobronchial biopsy for immunohistochemical and molecular predictive testing has not yet specifically been addressed.…”

Section: Discussionmentioning

confidence: 99%

The role of rapid on site evaluation on touch imprint cytology and brushing during conventional bronchoscopy

Botticella

Summa

Cisternino

et al. 2021

Diagnostic Cytopathology

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Background The increase in immunohistochemical and molecular predictive tests in lung cancer requires new strategies for managing small samples taken during bronchoscopic procedures. The value of Rapid On Site Evaluation (ROSE) during conventional bronchoscopic procedures on endobronchial neoplasms in optimizing small biopsies and cytologlogical tissue specimens for diagnostic testing, and ancillary studies was evaluated. Method ROSE on touch imprint cytology (TIC) and brushing was performed on 690 consecutive cases of patients undergoing biopsies, using fiber optic bronchoscopy. Immunohistochemical assay for PD‐L1, ALK, and ROS1 and molecular testing, via next generation technique for EGFR, KRAS, and BRAF, were performed. Results The concordance between ROSE and final diagnoses was almost perfect for brushing (sensitivity: 0.84; specificity: 0.96), and less so for touch preparations (sensitivity: 0.77; specificity: 0.89). Immunohistochemical assay for PD‐L1 was evaluated on 256 bioptic cases with only six unsuitable samples. Material available for immunohistochemistry for ALK was sufficient in 151 biopsies with no inadequate cases. ROS1 was evaluated in 132 biopsies, with only two unsuitable samples. Molecular analysis was performed on 128 biopsies, 29 TIC, and 17 brushing. Out of these, only ten were considered to be unsuitable. Conclusions ROSE is an effective procedure for monitoring the quality and quantity of material taken during conventional bronchoscopic procedures for evaluating the suitability of small samples that must undergo immunohistochemical and molecular assay.

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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

Cited by 26 publications

References 55 publications

EBUS-TBNA Cytological Samples for Comprehensive Molecular Testing in Non–Small Cell Lung Cancer

EBUS-TBNA Cytological Samples for Comprehensive Molecular Testing in Non–Small Cell Lung Cancer

Comprehensive validation of liquid-based cytology specimens for next-generation sequencing in cancer genome analysis

The role of rapid on site evaluation on touch imprint cytology and brushing during conventional bronchoscopy

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