Anupama Sharma, MD, MHA
Tatjana Antic, MD
Jordan P. Reynolds, MD
August 2024—Case. A 58-year-old female patient undergoes robotic endobronchial ultrasound-guided fine-needle aspiration for a peripherally located right upper lobe stellate-shaped lung lesion with ground-glass opacification. Rapid onsite evaluation (ROSE) assessments were adequate on four Diff-Quik (DQ) smears that were prepared for evaluation. One slide was cover-slipped for diagnostic purposes and the remaining slides were retained, uncovered. The cytopathologist requested additional material for further ancillary studies. The needle was rinsed in CytoLyt solution used to prepare a cell block from the cell pellet, and the residual supernatant was stored (at -80°C) for possible future molecular studies. The cell block cellularity was adequate, and the malignant cell population represented 50 percent of the sampled cells. A minimal immunohistochemistry panel including TTF-1, p40, and PD-L1 was performed. The in-house molecular laboratory performed the limited National Comprehensive Cancer Network recommended panel from the uncovered DQ slides using cell scrapings. Subsequently, an additional request was received for an expanded next-generation sequencing panel. The reference molecular laboratory was provided with approximately 15 unstained cell block slides meeting the threshold of at least 25 percent tumor representation.
Discussion. The preceding case exemplifies the expanded scope of ROSE in cytology, not just in adequacy and diagnostic triage but also as the primary checkpoint for judicious deployment of tissue for ancillary studies, including PCR-based assays and expanded NGS panels for therapeutic decision-making.
Biomarker testing has become the mainstay for prognostication and therapeutic decision-making in cancer diagnostics. Current NCCN guidelines for lung recommend a broad panel of biomarker analysis, including EGFR, KRAS, ALK1, ROS, ERBB2, NTRK, RET, and PDL1, at a minimum.1 Advances in interventional diagnostics complemented by increasing sensitivity and accuracy of nucleic acid-based assays has made cytologic sampling the initial choice for tissue acquisition. Nucleic acid-based techniques have also evolved with increased sensitivity and significantly lower cellularity requirements than those in earlier versions of the same platforms. “Liquid biopsy” techniques that use free fragments of DNA in the plasma have sparked an interest in similar applications in “liquid” exfoliative samples and residual supernatant samples that are typically discarded in cytology laboratories. A growing number of publications have validated different cytological preparations, such as scraping and cell lifting techniques from DQ and Pap-stained smears, and liquid-based cytology ThinPrep and cytospin preps for viability and DNA content geared for molecular studies. Cell blocks are being used broadly for immunocytochemical analysis and are treated at par with biopsies in their usefulness in nucleic acid-based assays. The underlying theme in these studies is to extract viable, robust DNA from limited samples.
This article highlights each cytological preparation type for its cyto-molecular yield in terms of ROSE, diagnosis, immunocytochemical testing, and molecular testing. Using this brief, case-based discussion, we hope to exemplify the advantages of a presumptive approach to a malignant diagnosis and a proactive and judicious triage of the sampled material.
Aspirate smears. Smeared slides prepared for ROSE cytomorphological analysis can also be used as a tissue source for molecular testing by simply scraping material from uncovered modified Giemsa-stained slides. Air-dried and DQ-stained smears from aspirate and bronchial brush specimens yield higher-quality nucleic acids as these are minimally processed and yield whole and preserved cellular and nuclear material. The evolving role of the operating cytopathologist is not only to preserve slides with representative morphology for diagnosis but also to proactively set aside air-dried material rich in tumor representation with minimal contamination for future molecular studies. Most commercially available molecular tests do not accept these samples due to lack of a uniformly applicable cellularity requirement. However, in-house molecular laboratories can perform validation studies to use this high-yield sample type with minimally preserved neoplastic cells for testing. Studies have reported reliable and robust DNA extractions at varying cellularity levels, ranging from 500 to 5,000 scraped neoplastic cells from air-dried slides.
Touch imprints. Touch imprints of core biopsies can be performed on slides and allow for ROSE to help assess the adequacy of the biopsy samples during the procedure for histological, ancillary, and molecular testing. Core biopsy samples are widely accepted as the sample of choice for immunohistochemistry, in situ hybridization, and other ancillary tests. The larger tissue volume allows for multiple tests to be conducted such as IHC for subtyping, PD-L1, and molecular testing. In addition, commercially available NGS testing and other advanced molecular techniques used in clinical trials can be performed to identify genetic mutations and guide targeted therapy. In this context, the touch prep air-dried and DQ-stained smear is an often overlooked resource for accessing minimally processed tumor DNA, similar to air-dried aspirate smears, particularly where the core biopsy material is exhausted due to extensive IHC workup for diagnostic confirmation.
Needle rinse fluid pellet for cell block and post-centrifuged supernatant. Cell-block preparation involves processing the residual sample from FNA needle rinse or other cytological methods into a paraffin-embedded block. This technique provides a paraffin-embedded pellet with a solid tissue-like section that allows for detailed histopathological examination and serves as a tissue source for IHC and molecular testing. In many cases, a cell block offers sufficient material for DNA and RNA extraction, enabling the detection of actionable genetic alterations through techniques such as polymerase chain reaction and NGS. However, in some cases, the DNA yield may be lower due to prolonged exposure to formaldehyde. In cases in which a cell block is paucicellular or the nucleic acids are poorly preserved, the post-centrifuged supernatant could be harnessed for free-floating or cell-free nucleic acids. Recent articles have shown a great value for this residual supernatant, particularly in cases where limited tissue availability precluded even a primary diagnostic categorization.2,3 In these instances, despite the indeterminate evidence of diagnostic tumor cells, the free-floating supernatant nucleic acids could provide key information on targetable mutations.
Liquid-based cytologic preparations including ThinPrep, SurePath, and Cytospin. Smears from brush samples and effusion and body fluid samples can similarly be used for limited ancillary and molecular testing. Recently, a triple-needle brush with a three-pronged end has been used to trap more cellular material to improve the cellular yield. The liquid-based preparations of these samples can also provide scraped cells that can be used for molecular testing, irrespective of the cell enrichment platform used (ThinPrep and SurePath) for sample preparations. The inherent preanalytical variable introduced here is the alcohol fixation, requiring additional validation steps; however, the distinct advantage is the ease of calculating cellular density (cells/mm2 ). This benefit allows for establishing minimal cellularity requirements with ease across multiple platforms.
Guidelines and validation. The CAP, International Association for the Study of Lung Cancer, and Association for Molecular Pathology updated and revised their 2013 evidence-based molecular testing guideline for the selection of lung cancer patients for EGFR and ALK tyrosine kinase inhibitors to include, in their recommendations, any cytology sample with adequate cancer content, as opposed to the prior recommendation that supported only cell blocks as a tissue source for DNA in such samples.4,5 Consequently, irrespective of the source, CAP guidelines require a robust validation of any testing platform prior to deploying it for patient diagnostic purposes. This process includes independent validation of all possible preanalytical variables to ensure robust and reproducible results that are internally validated and controlled for quantity and quality of the extracted sample nucleic acid. This includes controlling for variations in sample types, supernatant or minimally processed cell blocks, cell scraping from air-dried smears or effusion samples, fixative (formaldehyde versus ethanol versus RPMI), variations in preparatory procedures, staining techniques (DQ versus Pap), the type of and exposure to mounting media, and the type of slide used (positively charged versus frosted). Last but not least, these validation protocols require strict compliance with the oversight structure established by federal and state regulatory bodies and by the representative accrediting bodies such as the CAP and COLA.
- National Comprehensive Cancer Network. NCCN Practice Guidelines in Oncology: Non-Small Cell Lung Cancer. Version 3.2023.
- Roy-Chowdhuri S, Mehrotra M, Bolivar AM, et al. Salvaging the supernatant: next generation cytopathology for solid tumor mutation profiling. Mod Pathol. 2018;31(7):1036–1045.
- Janaki N, Harbhajanka A, Michael CW, et al. Comparison of cytocentrifugation supernatant fluid and formalin-fixed paraffin-embedded tissue for targeted next-generation sequencing. Cancer Cytopathol. 2019;127(5):297–305.
- Lindeman NI, Cagle PT, Aisner DL, et al. Updated molecular testing guideline for the selection of lung cancer patients for treatment with targeted tyrosine kinase inhibitors: guideline from the College of American Pathologists, the International Association for the Study of Lung Cancer, and the Association for Molecular Pathology. J Mol Diagn. 2018;20(2):129–159.
- Roy-Chowdhuri S, Dacic S, Ghofrani M, et al. Collection and handling of thoracic small biopsy and cytology specimens for ancillary studies: guideline from the College of American Pathologists in collaboration with the American College of Chest Physicians, Association for Molecular Pathology, American Society of Cytopathology, American Thoracic Society, Pulmonary Pathology Society, Papanicolaou Society of Cytopathology, Society of Interventional Radiology, and Society of Thoracic Radiology. Arch Pathol Lab Med. 2020;144(8):933–958.
Dr. Sharma is associate director of surgical pathology, Department of Pathology, Renaissance School of Medicine, Stony Brook University, New York. Dr. Antic is professor of pathology, University of Chicago Pritzker School of Medicine, and director of cytopathology, University of Chicago Medical Center. Dr. Reynolds is director of cytology, Department of Laboratory Medicine and Pathology, Mayo Clinic, Jacksonville, Fla. All are members of the CAP Cytopathology Committee.