Derek B. Allison, MD
Deepu Alex, MD, PhD
August 2025—Strides in medical imaging techniques and procurement methods have led to the acquisition of small diagnostic samples obtained by minimally invasive techniques. Over the same period, the breadth of molecular information that can be derived from limited tumor material has increased exponentially. In the age of targeted cancer therapy, the clinical utility of this information is substantial and, when coupled with the decreasing costs of molecular analysis, the information transformed the treatment landscape of cancer. These advances have brought cytology back into the spotlight as a potential source of material for biomarker analysis.1 Historically, the low amount of starting material, alcohol-fixation, and the paucity of validated methods made the task of using these samples challenging. However, recent significant scientific advancements have led to cytologic specimens being used as substrates for a variety of tests, including immunocytochemistry, immunohistochemistry, in situ hybridization, polymerase chain reaction, and next-generation sequencing.
Cytology specimen types and fixatives. Most cytology preparations can be used for molecular testing. These include direct smears, liquid-based cytology, cell blocks, and sample supernatants. Fixatives used in cytology include both formalin-based and alcohol-based agents, each with distinct implications for nucleic acid preservation and test validation. Cytology specimens fixed with alcohol are often better preserved and yield higher-quality nucleic acids that are more stable and better extracted than those from formalin-fixed, paraffin-embedded samples. However, most molecular assays are validated for use with formalin-fixed material, necessitating a distinct preanalytical workflow and separate validation process for alcohol-fixed specimens to ensure assay reliability and compliance. Nevertheless, multiple studies demonstrate that cytology material can provide comparable, if not superior, molecular testing performance relative to FFPE samples. This versatility has enabled the integration of a wide range of molecular techniques into cytology-based workflows.
Molecular testing approaches in cytology. Molecular techniques used on cytology specimens span a broad range of complexity. Commonly employed methods include PCR and multiplex sequencing panels, which target specific mutations in a cost-effective and tissue-conserving manner. These assays are typically low complexity and fast, making them attractive for frontline molecular screening. Clinical examples include single-gene PCR-based assays for EGFR mutation detection in lung cancer and gene expression classifiers for thyroid FNA aspirates with indeterminate cytology.
In addition, ultrarapid cartridge-based assays are emerging as an extension of rapid onsite evaluation, enabling same-day molecular testing from cytology smears. For example, a feasibility study demonstrated that cartridge-based assays targeting mutations in EGFR, KRAS, BRAF, and gene fusions or expression imbalances in ALK, ROS1, RET, MET exon 14, and NTRK1/2/3 could be performed directly from cytology smears within two to three hours.2 These assays demonstrated high concordance with orthogonal NGS and PCR-based methods and required minimal tissue input, making them particularly well suited to cytopathology laboratories.
At the high-complexity end of the spectrum, NGS-based approaches—such as targeted gene panels, whole exome sequencing, and transcriptome analysis—enable broad genomic profiling. These assays require more tissue, incur greater costs, and involve longer turnaround times. However, advances in sequencing technology and bioinformatics now allow for successful application of NGS to cytology specimens, even with limited starting material. Emerging platforms now enable combined whole exome (DNA) and whole transcriptome (RNA) sequencing from a single cytology sample, maximizing data output without increasing tissue burden. This dual-modality approach captures somatic mutations, gene fusions, expression profiles, and structural alterations in a single unified workflow, which is particularly advantageous when material is limited, as is often the case with cytology samples.
Role of rapid onsite evaluation. One of the major advantages of cytology-based sampling, particularly in FNA, is the opportunity for real-time ROSE. This allows pathologists to assess adequacy for both diagnostic and molecular purposes at the time of collection. Samples can be triaged into appropriate fixatives based on anticipated downstream testing needs. ROSE also facilitates optimal test selection and minimizes the likelihood of tumor depletion, thereby reducing the need for repeat procedures. Emerging applications now include pairing ROSE with same-day cartridge-based molecular assays, further streamlining the diagnostic-to-treatment timeline.
Diagnostic and therapeutic relevance of molecular testing in cytology. Following is a brief, non-comprehensive overview of how molecular technologies are being applied now across a range of cytology specimen types to enhance diagnostic precision and guide therapeutic decision-making.
Thyroid FNA: Identification of a BRAF V600E mutation in an aspirate can confirm malignancy, even in cases with equivocal cytomorphology. Molecular testing is now routine for indeterminate thyroid nodules and can also detect syndromic germline alterations (e.g. DICER1) in pediatric lesions. Ultrarapid molecular assays targeting BRAF, RET, and NTRK1/2/3 fusions may expedite treatment planning. A variety of specimen types—including smears, cell blocks, and needle rinses—have proved effective for this testing, with expanded panels demonstrating high sensitivity and specificity for follicular and oncocytic lesions.
Lung cancer: Detection of actionable mutations such as EGFR in cytology specimens not only confirms diagnosis but expedites targeted therapy initiation. Several FDA-approved single-gene assays are available for EGFR mutation testing and commonly used in clinical practice on small samples. Same-day testing of EGFR, KRAS, and BRAF has been shown to be feasible and accurate from cytology smears, including endobronchial ultrasound-guided FNAs. Cytology smears play an essential role in the triage and adequacy assessment for comprehensive molecular profiling in advanced non-small cell lung cancer, particularly when cell blocks and biopsies are unavailable.
Sarcomas and salivary gland tumors: Cytology samples—particularly alcohol-fixed specimens—provide high-quality RNA suitable for gene fusion detection in soft tissue and salivary gland neoplasms. Recurrent fusions (e.g. ETV6::NTRK3, MYB::NFIB, EWSR1::FLI1) have diagnostic utility and can be detected via sequencing or in situ hybridization performed on cell blocks or scraped smears.
Pancreatic lesions: Pancreatic cyst fluid obtained by EUS-FNA is increasingly evaluated by targeted NGS to characterize cyst subtype and risk of malignancy. MAPK pathway gene mutations (KRAS, BRAF) and GNAS mutations are specific for mucinous cysts such as IPMN and MCN, while co-occurring alterations in TP53, SMAD4, CTNNB1, and mTOR pathway genes (e.g. PTEN, PIK3CA) are associated with advanced neoplasia. Alterations in VHL, MEN1, and loss of heterozygosity help classify serous cystadenomas and cystic pancreatic neuroendocrine tumors (PanNETs). These genomic profiles improve diagnostic specificity and support more personalized risk stratification and surgical triage when used alongside cytopathologic assessment.3
Technical limitations and evolving solutions. One limitation of cytology specimens is tumor content, both in terms of relative tumor percentage and absolute tumor cellularity. Fluid-based samples and sparsely cellular smears may contain scant neoplastic material amid abundant inflammatory or stromal elements, complicating downstream molecular testing. Additionally, variable fixation and staining protocols across laboratories may impact nucleic acid quality and assay performance, particularly for RNA-based testing. Despite these challenges, modern molecular platforms have demonstrated high sensitivity and robustness across a range of cytology specimen types. Some same-day molecular assays have validated performance thresholds as low as one percent variant allele frequency and require as little as 10 percent tumor cellularity, expanding their utility in low-yield samples. Use of microdissection, stain-free smears, and validated extraction protocols can further improve performance and reduce test failure rates.
Future directions. As our understanding of tumor heterogeneity and the tumor microenvironment deepens, the role of cytology specimens in molecular diagnostics is expected to grow. Improved procurement, fixation, and processing techniques, along with assay optimization for cytology material, will further enhance the diagnostic yield and clinical relevance of these samples. The convergence of technological innovation and cytologic expertise has positioned cytology as a key contributor to molecular pathology in the era of precision medicine. The incorporation of ultrarapid molecular testing into ROSE may soon enable same-day diagnostic and therapeutic decision-making from a single minimally invasive procedure.
- VanderLaan PA, Roy-Chowdhuri S, Griffith CC, Weiss VL, Booth CN. Molecular testing of cytology specimens: overview of assay selection with focus on lung, salivary gland, and thyroid testing. J Am Soc Cytopathol. 2022;11(6):403–414.
- Iorgulescu JB, Yang RK, Roy-Chowdhuri S, Sura GH. Same-day molecular testing for targetable mutations in solid tumor cytopathology—the next frontier of the rapid on-site evaluation. Cancer Cytopathol. 2025;133(1):e22930.
- Paniccia A, Polanco PM, Boone BA, et al. Prospective, multi-institutional, real-time next-generation sequencing of pancreatic cyst fluid reveals diverse genomic alterations that improve the clinical management of pancreatic cysts. Gastroenterology. 2023;164(1):117–133.e7.
Dr. Allison is associate professor of pathology and urology and vice chair for research, University of Kentucky College of Medicine, Lexington. Dr. Alex is a consultant pathologist at BC Cancer Agency in Vancouver, British Columbia, Canada. Both are members of the CAP Cytopathology Committee.