How Next-Generation Sequencing is Powering Precision Cancer Diagnosis from Cytology Specimens
In the intricate world of cancer diagnosis, sometimes the smallest samples are triggering the biggest revolutions.
For decades, pathology conjured images of large tissue specimens—surgical resections and core biopsies that provided abundant material for diagnosis. Yet in our modern era of precision medicine, where treatment decisions increasingly hinge on identifying specific molecular biomarkers hidden within a tumor's DNA, a paradigm shift is underway. Enter next-generation sequencing (NGS) applied to cytology—the science of extracting a wealth of genetic information from often scant samples obtained through minimally invasive procedures.
Cytology, the study of individual cells, has long played a crucial role in cancer diagnosis. Through techniques like fine-needle aspiration (FNA), physicians can extract cells from suspicious masses using needles thinner than those used for blood draws. These procedures are less invasive, reduce patient discomfort, and lower the risk of complications. However, the resulting samples are inherently small—sometimes just a few thousand cells spread across a microscope slide.
The growing need for molecular testing created a significant challenge: how to perform comprehensive genetic analysis when "tissue is the issue"? As one recent review noted, this created a "false impression that cytological specimens are not suitable for molecular testing by nature" 1 .
This perception is rapidly changing. Modern cytological samples contain the same DNA, RNA, and protein molecules as their histological counterparts—they simply arrive without the tissue context 1 . Furthermore, they often provide nucleic acids of higher quality than standard formalin-fixed tissue samples, which can suffer from DNA damage during processing 5 8 .
The evolution of NGS technology has been the game-changer. Unlike older testing methods that could only examine one gene at a time, NGS panels can simultaneously screen dozens to hundreds of genes, even from the limited DNA yields typical of cytology samples 5 7 .
| Tool/Component | Function in the Process | Application in Cytology |
|---|---|---|
| Cytology Samples (Cell Blocks, Smears, Liquid-based Cytology) | The primary source of tumor DNA/RNA for analysis. | Cell blocks (CBs) are the most common preparation (94.2% of samples in one large study) 3 . |
| Supernatant Cell-Free DNA (ScfDNA) | Rescue material from residual cytology fluids; used when cellular tissue is exhausted. | Enables successful sequencing in 71% of cases where cell block material is depleted 3 . |
| Hybridization Capture (Illumina) or Amplicon-Based (Ion Torrent) NGS Platforms | Different technological approaches to "capture" DNA fragments for sequencing. | Ion Torrent may require fewer cells, potentially making it more suitable for scant cytology specimens 1 . |
| Macrodissection/Laser Microdissection | Techniques to enrich tumor content by selectively isolating tumor cells from surrounding material. | Critical for samples with high overall cellularity but low tumor fraction 1 8 . |
| Bioinformatics Pipelines | Specialized software to analyze massive sequencing data, identify variants, and ensure accuracy. | Must be validated for low-input DNA samples and able to filter out background noise 7 8 . |
A recent groundbreaking study published in Nature Communications provides compelling evidence for the reliability of NGS on cytology samples 3 . Researchers at Memorial Sloan Kettering Cancer Center analyzed 4,871 prospectively sequenced clinical cytology samples using their FDA-cleared MSK-IMPACT™ test, offering an unprecedented look at the real-world performance of comprehensive genetic profiling on these limited samples.
The cytology laboratory adopted a modified HistoGel-based cell-block processing method to improve pellet density, preventing the precious sample from being lost during processing 3 .
The molecular laboratory implemented improved bead-based DNA extraction techniques and began using mineral oil for more effective deparaffinization of cell blocks, maximizing DNA yield from these challenging samples 3 .
The team incorporated dual-index sequencing to reduce cross-sample contamination and, importantly, lowered the minimum DNA input requirement from 50 ng to 30 ng for cell blocks, making testing feasible for more limited samples 3 .
When the cellular material from cell blocks was too scant or exhausted, the team turned to a previously underutilized resource: the residual supernatant fluid (ScfDNA) left over after cytological preparations. This fluid contains cell-free DNA and fragments from whole cells that had broken down during sample processing 3 .
The outcomes of this extensive study were striking. Through continuous process optimization, the overall success rate for NGS testing on cytology samples reached 93% for internally processed specimens 3 . Even when using supernatant cell-free DNA as a rescue source for the most challenging cases, the success rate was a remarkable 71% 3 .
81% Success Rate
81% Success Rate
71% Success Rate
92-93% Success Rate
The genetic information gleaned from these cytology samples proved highly reliable. The study found that cytology samples performed similarly to surgical samples in identifying clinically relevant genomic alterations, with 93.8% of successfully sequenced cases harboring at least one somatic alteration 3 .
| Sample Type | Number of Samples | Success Rate | Key Applications |
|---|---|---|---|
| All Cytology Samples | 4,725 | 81% | Comprehensive genomic profiling across diverse tumor types |
| Cell Blocks (CB) | 4,457 | 81% | Primary material for diagnosis and molecular testing |
| Supernatant Cell-Free DNA (ScfDNA) | 268 | 71% | Rescue material when cellular tissue is exhausted |
| Internally Processed CB | Not specified | 92-93% | Demonstrates effect of optimized collection protocols |
One noteworthy finding was the identification of low-level cross-contamination in 4.7% of cell block samples, a pitfall intrinsic to the processing of paraffin blocks where foreign tissue can become embedded. By contrast, ScfDNA samples showed negligible contamination (0.3%), highlighting another advantage of this alternative source 3 .
The implications of successfully performing NGS on cytology samples are profound, particularly for cancers where minimally invasive sampling is the norm.
In non-small cell lung cancer (NSCLC), for instance, many patients are diagnosed at advanced stages where surgery is not an option. For these individuals, cytological samples obtained through procedures like endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) often constitute the only material available for both diagnosis and molecular testing 1 5 .
Current European Society for Medical Oncology guidelines recommend molecular testing for numerous predictive biomarkers in advanced NSCLC, including EGFR, KRAS, BRAF mutations, and ALK, RET, ROS1 fusions, among others 1 . The rapid expansion of clinically significant biomarkers makes large-panel NGS not just convenient but essential—and cytological samples must be suitable for this requirement.
Similarly, in pancreatic ductal adenocarcinoma, endoscopic ultrasound-guided FNA (EUS-FNA) is the primary diagnostic technique. While obtaining adequate material from these notoriously challenging tumors is difficult, comprehensive molecular profiling helps tailor chemotherapy regimens to individual patients, enhancing treatment effectiveness while avoiding unnecessary toxicity 1 .
Beyond traditional cancer diagnostics, NGS on cytology samples is expanding into other areas like cervical cancer screening. A 2025 study demonstrated the feasibility of using NGS on liquid-based cytology samples to detect not just HPV infection but specific HPV genotypes and sublineages with individual risk profiles—information that could significantly improve risk stratification in screening programs .
| Biomarker Type | Specific Examples | Clinical Significance |
|---|---|---|
| Gene Mutations | EGFR, KRAS (G12C), BRAF (V600), MET | Predict response to targeted tyrosine kinase inhibitors |
| Gene Fusions | ALK, RET, ROS1, NTRK | Targeted therapies available for specific fusion proteins |
| Gene Amplifications | MET, HER2 | Indicate potential responsiveness to targeted treatments |
| Emerging Biomarkers | PIK3CA, BRCA1/2, FGFR alterations | Potential targets for future targeted therapies |
Despite the promising advances, implementing NGS in cytology practice isn't without hurdles. The pre-analytical factors that can affect NGS success in cytology are numerous, including type of preparation, fixatives, stains, specimen cellularity, tumor fraction, and DNA yield 1 6 .
Low cellularity remains one of the major obstacles, though strategies like macrodissection (physically scraping tumor-rich areas from slides) or sample complementation (combining multiple samples from the same patient) can help address this challenge 1 .
There's no universally standardized threshold for tumor cellularity in NGS analysis, though some experts recommend that tumor cellularity should exceed two-fold the detection limit of the molecular technique being used 1 5 . A common benchmark aims for a neoplastic nucleus proportion of at least 10%, ideally 20% or more 1 .
| Challenge | Impact on Testing | Potential Solutions |
|---|---|---|
| Low Cellularity | Insufficient DNA yield for sequencing | Macrodissection, sample complementation, increased passes during collection |
| Low Tumor Fraction | Risk of false-negative results due to dilution by normal cells | Laser microdissection, careful pathological review of tumor content |
| Sample Contamination | False positives or sequence quality issues | Dual-index sequencing, stringent processing protocols, use of ScfDNA |
| DNA Quality Issues | Sequencing failures or artifacts | Optimized extraction methods, alternative fixatives to formalin |
| Platform Sensitivity | Ability to detect low-frequency variants | Selection of appropriate NGS technology with high sensitivity |
The role of the cytopathologist has evolved significantly in this molecular era. Today's molecular cytopathologists serve as crucial links between clinicians and molecular biologists, ensuring adequate specimen management and appropriate test selection 5 8 . Their expertise in evaluating sample adequacy—assessing neoplastic cell content while avoiding contaminants like mucus or necrosis—is vital to the success of molecular testing 5 .
The integration of next-generation sequencing with cytology specimens represents nothing short of a revolution in diagnostic pathology. What was once dismissed as inadequate for molecular interrogation has proven not only sufficient but, in some respects, superior—providing high-quality genetic material through less invasive means.
As the MSK-IMPACT study convincingly demonstrated, with appropriate optimization and expertise, cytology samples can achieve success rates up to 93% in comprehensive genetic profiling, identifying actionable biomarkers that direct life-changing targeted therapies 3 . The strategic use of previously discarded resources like supernatant fluids further enhances the value of these precious samples.
In the constantly evolving landscape of precision oncology, the ability to extract maximal information from minimal samples is proving indispensable. The next generation of cancer diagnostics isn't necessarily about bigger samples—it's about smarter approaches to the small ones we already have. The era of "tissue is the issue" is giving way to a new paradigm where sometimes, the most powerful answers come in the smallest packages.