Optimizing Workflows and Processing of Cytologic Samples for Comprehensive Analysis by Next-Generation Sequencing: Memorial Sloan Kettering Cancer Center Experience

Abstract

The value and suitability of cytology specimens for molecular diagnosis has been demonstrated by numerous studies. In practice, however, the success rates vary widely across institutions depending on the disease setting, institutional practices of acquisition, handling/processing, and testing methodologies. As the number of clinically relevant biomarkers continues to increase, more laboratories are turning to next-generation sequencing platforms for testing. Although amplicon-based next-generation sequencing assays, interrogating a limited genomic territory, can be performed with minimal input material, broader-based next-generation sequencing assays have higher DNA input requirements that may not be met if the small tissue samples are not acquired and handled appropriately. We briefly describe some of the process changes we have instituted in our laboratories when handling cytologic material to maximize the tissue available for broad hybrid-capture-based next-generation sequencing assays. Among the key changes established were the consolidation and preservation of previously discarded supernatant material in cytologic samples, the introduction of mineral oil for deparaffinization of cell blocks, and adjustments in the molecular laboratory process and bioinformatics pipelines. We emphasize that even minimal changes can have broad implications for test performance, highlighting the importance of a cohesive group-based approach among clinical, cytopathology, surgical pathology, molecular, and bioinformatics teams.

Comparison of endoscopic ultrasound tissue acquisition methods for genomic analysis of pancreatic cancer

“…Liquid cytology samples, including FNA rinse material, have been demonstrated to be adequate for genomic testing in institutions that have validated these types of samples . In a prior study from our institution, adequate material was obtained for genomic testing from residual FNA rinse or FNA‐acquired body fluid specimens in all 17 cases when tumor cellularity was ≥ 10% in the aspirate .…”

Molecular Testing of Non–Small Cell Lung Carcinoma Diagnosed by Endobronchial Ultrasound–Guided Transbronchial Fine-Needle Aspiration: The Cleveland Clinic Experience

“…Given the diversity of cytopathologic specimen types and the different methods of fixation and processing available, options for procuring DNA from cytologic specimens for molecular analysis vary; material can be successfully obtained from cell blocks, smears, cytospins, and liquid-based preparations. 7,9,11,12,14,[19][20][21][22][23][24][25] Most commonly, cell blocks are used for molecular testing on cytopathologic specimens. 21 The cell block closely approximates surgical pathology material; tissue fragments from cytopathologic specimens are embedded in paraffin and are sectioned akin to surgical specimens.…”

“…29 Although liquid-based cytology specimens are not commonly used for molecular testing of lung adenocarcinomas, 21 a few groups have reported their utility. 7,12,14,25 In our laboratory, we have found that the liquid-based cytology specimen is an excellent resource for EGFR mutation analysis and ALK FISH. We have implemented this method of molecular testing for all of our liquid-based cytology specimen types, including EBUS-TBNA, computed tomography-guided transthoracic fine-needle aspiration, peripheral fine-needle aspiration, and effusion cytology specimens.…”

“…Other groups have reported success with NGS on cytology specimens, with material obtained from either smears or cell blocks. 8,11,14 In our laboratory, the AmpliSeq Cancer Hotspot panel v2 (Thermo Fischer Scientific, Waltham, Massachusetts) is used with the Ion Torrent Personal Genome Machine (Life Technologies, Grand Island, New York) to detect mutations in several hot spot regions in 50 oncogenes and tumor suppressor genes; currently, we only report mutations in EGFR, Kirsten rat sarcoma viral oncogene homolog (KRAS), and B-raf proto oncogene (BRAF) on clinical samples ( Figure 3). Next-generation sequencing testing on liquidbased cytologic samples was validated in our laboratory, and the results were reported in a recent publication by Reynolds et al 12 In that study, EGFR mutation detection by NGS performed on the residual cell pellet of 20 EBUS-TBNA samples was successful and showed 100% concordance with results previously obtained by real-time PCR.…”

Molecular testing on endobronchial ultrasound (EBUS) fine needle aspirates (FNA): Impact of triage

“…moderate-to-high tumor content (all from Group B) on smears/TP, which if triaged differently, would have theoretically resulted in greater cellularity of cell blocks and improved the overall results of Group B.Specific components of triage are often overlooked and underaddressed with limited reports calling attention to proper specimen acquisition, triage, and management 12,36. These are significant elements of ROSE that could increase MT adequacy 37. Having >1 person (e.g., Group A) from cytology participate in ROSE at start of procedure was advantageous for a few reasons.…”