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Over the past decade, molecular cytopathology has emerged as a relevant area of modern pathology. Notably, in patients with advanced-stage cancer, cytological samples could be the only material available for diagnosis and molecular biomarker testing to identify patients suitable for targeted therapies. As a result, the contemporary cytopathologist’s role extends beyond morphological assessments to include critical skills such as evaluating the adequacy of the cytological samples and managing these specimens for molecular testing. This case collection can be a valuable source of insight, especially for young pathologists, who should learn to combine the opportunities offered by molecular biology with the basis of morphological evaluation.

Over the past decade, molecular cytopathology has emerged as a relevant area of modern pathology. Notably, in patients with advanced-stage cancer, cytological samples could be the only material available for diagnosis and molecular biomarker testing to identify patients suitable for targeted therapies. As a result, the contemporary cytopathologist's role extends beyond morphological assessments to include critical skills such as evaluating the adequacy of the cytological samples and managing these specimens for molecular testing. This case collection can be a valuable source of insight, especially for young pathologists, who should learn to combine the opportunities offered by molecular biology with the basis of morphological evaluation. [ABSTRACT FROM AUTHOR]

Recently, significant advances in the molecular characterization of salivary gland neoplasms have facilitated the classification and diagnosis of specific diagnostic entities. In the highly challenging diagnostic scenario of salivary malignancies, molecular testing is increasingly being adopted in routine practice to refine the cytological diagnosis of salivary lesions. Here, we reviewed the most recent evidence in the field of salivary glands molecular cytopathology. [ABSTRACT FROM AUTHOR]

The scope and extent of molecular cytopathology in the era of precision medicine has been expanding in recent years. The versatility of cytology specimen preparations has provided ample opportunity for the cytopathology community to evolve, innovate and 'do more with less' using limited amounts of tissue. More recently, cytology-derived supernatant liquid biopsy samples have been identified as a substantial source of high-quality genomic material that can be interrogated for genotyping for therapeutic decision-making, as well as other roles in cancer screening for early-stage disease, longitudinal monitoring for therapeutic response and disease prognostication. These novel substrates, including supernatants from body fluids such as urine, pleural effusion, ascitic fluid, cerebrospinal fluid, as well as fine-needle aspiration (FNA) specimens, serve as a bridge between tissue-based testing and conventional liquid biopsy testing from the patient's plasma. Cytologically derived liquid biopsy samples can only be used in situations where the tissue sample is inadequate for genotyping, or when plasma-based liquid biopsy fails to identify an oncogenic driver alteration, but they can be used as a stand-alone complementary specimen source that can provide reliable genomic information for therapeutic decisions. This review aims to highlight some of the advances in the field and the clinical applications of the cytology-derived supernatant specimen., (© 2025 John Wiley & Sons Ltd.)

Generally, predictive biomarker tests are clinically validated on histological formalin-fixed, paraffin-embedded (FFPE) samples. In addition to FFPE samples, cytological samples have also emerged as a useful approach to detect predictive biomarkers. However, as of today, despite the promising results reported in the recent literature, their full implementation in routine clinical practice is still lagging owing to a lack of standardized preparatory protocols, challenging assessments of cyto-histological correlation, and variable inter-observer agreement. The aim of this report was to explore the possibility of implementing a large-scale validation of predictive biomarker testing on cytological material. To this aim, we evaluated the technical feasibility of PD-L1 assessment on a cell block (CB)-derived tissue microarray (cbTMA). Consecutive and unselected CBs prepared from metastatic lymph node fine-needle cytology (FNC) samples were retrospectively collected and used for TMA construction. PD-L1 immunohistochemistry (IHC) was carried out on cbTMA sections with the companion diagnostic kit SP263 assay. TMA contained 33 CB-derived cores. A total of 20 sections were hematoxylin and eosin (H&E) stained. Overall, 29 (88%) samples were visible at least in one H&E-stained slide. Four cases out of five sections stained with the SP263 assay (4/29, 13.8%) showed PD-L1 positivity in neoplastic and/or immune cells; remarkably, no unspecific background was observed. Although our study was based on a limited and non-selected series, our findings do provide proof of concept for the use of cbTMA in predictive biomarker testing on cytological material in large-scale post-clinical trial validation studies, multicenter studies, and quality control programs.

Breast cancer is the most common cause of cancer-related deaths in the female population worldwide. To the best of our knowledge, breast cancer (BRCA)1/2 gene mutations have not been described yet on breast cancer cytological specimens. Here we describe the case of a 38-year old woman with a family and personal history for breast cancer, who underwent a fine needle aspiration (FNA) procedure for a novel 30 mm lesion located in the external quadrants of the contralateral (left) breast. Cytological findings and ancillary immunostaining confirmed the diagnosis of a triple negative NST carcinoma. BRCA1/2 molecular assessment was carried out on DNA extracted from cytological (November 2020), biopsy (December 2014) and surgical resection (July 2015) specimens, as well as on the resection of a benign fibroadenoma, by using a next generation sequencing approach. Molecular analysis showed a pathogenic BRCA1 insertion (c.5266dupC; p.Q1756PfsTer74) in the cytological specimen (allelic fraction 92.0%), biopsy (allelic fraction 84.2%), surgical resection (allelic fraction 87.8%) and fibroadenoma (58.9%), demonstrating a germinal BRCA mutated status.

Several advances in recent decades in digital imaging, artificial intelligence, and multiplex modalities have improved our ability to automatically analyze and interpret imaging data. Imaging technologies such as optical coherence tomography, optical projection tomography, and quantitative phase microscopy allow analysis of tissues and cells in 3-dimensions and with subcellular granularity. Improvements in computer vision and machine learning have made algorithms more successful in automatically identifying important features to diagnose disease. Many new automated multiplex modalities such as antibody barcoding with cleavable DNA (ABCD), single cell analysis for tumor phenotyping (SCANT), fast analytical screening technique fine needle aspiration (FAST-FNA), and portable fluorescence-based image cytometry analyzer (CytoPAN) are under investigation. These have shown great promise in their ability to automatically analyze several biomarkers concurrently with high sensitivity, even in paucicellular samples, lending themselves well as tools in FNA. Not yet widely adopted for clinical use, many have successfully been applied to human samples. Once clinically validated, some of these technologies are poised to change the routine practice of cytopathology.

The application of next generation sequencing (NGS) technology to cytological samples has significantly modified molecular cytopathology practice. Cytological samples represent a valid source of high‐quality DNA for NGS analysis, especially for predicting patients' response to targeted treatments and for refining the risk of malignancy in indeterminate cytological diagnoses. However, several pre‐analytical factors may influence the reliability of NGS clinical analysis. Here, we briefly review the challenges of NGS in cytology practice, focusing on those pre‐analytical factors that may negatively affect NGS success rates and routine diagnostic applications. Finally, we address the future directions of the field. Molecular cytopathology is a rapidly evolving field. Modern molecular cytopathologists play a key role in bridging the gap between conventional microscopy and novel molecular technologies. Cytological samples represent a valid source of high‐quality DNA for next generation sequencing (NGS) analysis, especially for predicting patients' response to targeted treatments and for refining the risk of malignancy in indeterminate cytological diagnosis. [ABSTRACT FROM AUTHOR]

With a growing number of predictive biomarkers needed to manage patients with non-small cell lung cancer (NSCLC), there has been a paradigm shift in care and handling of diagnostic samples. Among the various testing methods, immunohistochemistry (IHC) is the most cost- effective and widely available. Furthermore, over the past decade immunotherapy has emerged as one of the most promising cancer treatments. In this scenario IHC is the most used testing method available for PDL-1/PD1 immunotherapy. Several monoclonal antibodies targeting programmed death 1 (PD-1)/programmed death ligand-1 (PD-L1) pathways have been integrated into standard-of-care treatments of a wide range of cancer types, once provided evidence of PD-L1 expression in tumor cells by immunohistochemistry (IHC). Since currently available PD-L1 assays have been developed on formalin-fixed paraffin embedded (FFPE) histological specimens, a growing body of research is being dedicated to confirm the feasibility of applying PDL-1 assays also to cytological samples. Albeit promising results have been reported, several important issues still need to be addressed. Among these are the type of cytological samples, pre-analytical issues, cyto-histological correlation, and inter-observer agreement. This review briefly summarizes the knowledge of the role of cytopathology in the analysis of PD-L1 by immunocytochemistry (ICC) and future directions of cytopathology in the immunotherapy setting.

Molecular cytopathology is a rapidly evolving field embracing both conventional microscopy and molecular pathology. Its growing popularity stems from the fact that in many types of advanced cancers, including non small cell lung cancer (NSCLC), cytological samples often constitute the only available specimens for morphomolecular analysis. Indeed, non formalin fixed and paraffin embedded (FFPE) cytological samples feature a higher quality of extracted nucleic acids than histological specimens. However, because of the growing complexity of molecular testing, several efforts should be made to validate the analytical performance of the wide array of currently available molecular technologies, including next generation sequencing (NGS). This technology has the terrific advantage of allowing simultaneous detection of scores of predictive biomarkers even in low-input DNA/RNA specimens. Here, we briefly review the role of the modern cytopathologist in the morphomolecular diagnosing of advanced stage NSCLC and the adoption of NGS in conventional cytopreparations (cell blocks, direct smears, and liquid-based cytology) and supernatants.

In this review, we describe molecular pathology testing to predict response to targeted treatment of solid tumors, focusing on Italian routine clinical practice. The combination of the universal health care system organized at national, regional, and local levels has led a decentralized model, with a large number of local laboratories performing in‐house molecular testing following guidelines issued and external quality assessment organized by the Italian Society of Pathology and Cytopathology–Italian Division of the International Academy of Pathology. In this framework, in the early days of predictive testing, sponsored informatics platforms support to set up national programs that aimed to integrate the activity of oncologists and pathologists to test cancer patients for druggable alterations. More recently, reimbursement for molecular testing is being covered completely by the Italian National Health Service. In the near future, considering the development of complex technologies, we expect that outsourcing samples to next‐generation sequencing referral laboratories will take place. In this review, we describe molecular pathology testing to predict response to targeted treatment of solid tumors, focusing on Italian routine clinical practice. [ABSTRACT FROM AUTHOR]

In recent years, cytopathology has established itself as an independent diagnostic modality to guide clinical management in many different settings. The application of molecular techniques to cytological samples to identify prognostic and predictive biomarkers has played a crucial role in achieving this goal. While earlier studies have demonstrated that single biomarker testing is feasible on cytological samples, currently, this provides only limited and increasingly insufficient information in an era where an increasing number of biomarkers are required to guide patient care. More recently, multigene mutational assays, such as next-generation sequencing (NGS), have gained popularity because of their ability to provide genomic information on multiple genes. The cytopathologist plays a key role in ensuring success of NGS in cytological samples by influencing the pre-analytical steps, optimizing preparation types and adequacy requirement in terms of cellularity and tumor fraction, and ensuring optimal nucleic acid extraction for DNA input requirements. General principles of the role and potential of NGS in molecular cytopathology in the universal healthcare (UHC) European environment and examples of principal clinical applications were discussed in the workshop that took place at the 30th European Congress of Pathology in Bilbao, European Society of Pathology, whose content is here comprehensively described. [ABSTRACT FROM AUTHOR]

Background: Artificial genomic reference standards in a cytocentrifuge/cytospin format with well‐annotated genomic data are useful for validating next‐generation sequencing (NGS) on routine cytopreparations. Here, reference standards were optimized to be stained by different laboratories before DNA extraction and to contain a lower number of cells (2 × 105). This was done to better reflect the clinical challenge of working with insufficient cytological material. Methods: A total of 17 worldwide laboratories analyzed customized reference standard slides (slides A‐D). Each laboratory applied its standard workflow. The sample slides were engineered to harbor epidermal growth factor receptor (EGFR) c.2235_2249del15 p.E746_A750delELREA, EGFR c.2369C>T p.T790M, Kirsten rat sarcoma viral oncogene homolog (KRAS) c.38G>A p.G13D, and B‐Raf proto‐oncogene, serine/threonine kinase (BRAF) c.1798_1799GT>AA p.V600K mutations at various allele frequencies (AFs). Results: EGFR and KRAS mutation detection showed excellent interlaboratory reproducibility, especially on slides A and B (10% and 5% AFs). On slide C (1% AF), either the EGFR mutation or the KRAS mutation was undetected by 10 of the 17 laboratories (58.82%). A reassessment of the raw data in a second‐look analysis highlighted the mutations (n = 10) that had been missed in the first‐look analysis. BRAF c.1798_1799GT>AA p.V600K showed a lower concordance rate for mutation detection and AF quantification. Conclusions: The data show that the detection of low‐abundance mutations is still clinically challenging and may require a visual inspection of sequencing reads to detect. Genomic reference standards in a cytocentrifuge/cytospin format are a valid tool for regular quality assessment of laboratories performing molecular studies on cytology with low‐AF mutations. Cytological genomic reference standards are a valid educational and validation tool and show highly reproducible results. The detection of low‐abundance mutations is challenging and may require a visual inspection of sequencing reads. [ABSTRACT FROM AUTHOR]

Introduction: Thyroid malignancy is one of the most common types of cancer in developed nations. Currently, fine-needle aspiration cytology (FNAC) is the most practical screening test for thyroid nodules. However, cytologically indeterminate samples comprise approximately 15%-30% of cases. These include cases classified as atypia of undetermined significance (AUS), follicular neoplasm (FN), and suspicious for malignancy (SFM). Indeterminate cases can be sent for molecular testing for more definitive classification to help guide management and prevent overtreatment of benign thyroid nodules. We conducted a retrospective review on molecular testing of indeterminate thyroid FNAC and reviewed subsequent histologic diagnoses in resection specimens to assess how molecular testing supported a diagnosis and its effect on clinical management of patients at our institution., Methods: A retrospective chart review was performed on all thyroid FNAC specimens, corresponding molecular testing, and subsequent surgical resection specimens over a 6-year period., Results: A total of 10,253 thyroid FNAC were performed in our hospital system during our study period, of which 10% (n = 1102/10,253) had indeterminate FNAC results. Molecular testing was performed in 16% (n = 178/1102) of indeterminate cytology cases. Genetic alterations were identified in 39% (n = 69/178) of the cases sent for molecular testing. The majority of cytologically indeterminate cases sent for molecular testing were follicular-patterned lesions and their corresponding resection specimens revealed mostly low grade follicular derived neoplasms (i.e., follicular adenoma, non-invasive follicular thyroid neoplasm with papillary-like nuclear features, and follicular variant of papillary thyroid carcinoma). Of the cases with identified genetic alterations, 75% (n = 52/69) were treated surgically. In cases with no genetic alterations identified, only 18% (n = 20/109) were treated surgically., Discussion/conclusion: Molecular testing on cytologically indeterminate thyroid nodules can help provide a more accurate risk of malignancy assessment in patients with lesions that are difficult to diagnosis based solely on FNAC morphology. The types of genetic alterations identified in the resected thyroid lesions were consistent with what has been previously described in the literature. Additionally, we found that in the patients with indeterminate thyroid FNAC with adjunct molecular testing, more than half did not undergo surgical resection. This finding emphasizes the value of adding molecular testing in patients, particularly when attempting to reduce unnecessary surgical intervention., (© 2023 The Authors. Diagnostic Cytopathology published by Wiley Periodicals LLC.)

Minimally invasive procedures such as endobronchial ultrasound‐guided transbronchial needle aspiration (EBUS‐TBNA) must yield not only good quality and quantity of material for morphological assessment, but also an adequate sample for analysis of molecular markers to guide patients to appropriate targeted therapies. In this context, cytopathologists worldwide should be familiar with minimum requirements for refereeing cytological samples for testing. The present manuscript is a review with comprehensive description of the content of the workshop entitled Cytological preparations for molecular analysis: pre‐analytical issues for EBUS TBNA, presented at the 40th European Congress of Cytopathology in Liverpool, UK. The present review emphasises the advantages and limitations of different types of cytology substrates used for molecular analysis such as archival smears, liquid‐based preparations, archival cytospin preparations and FTA (Flinders Technology Associates) cards, as well as their technical requirements/features. These various types of cytological specimens can be successfully used for an extensive array of molecular studies, but the quality and quantity of extracted nucleic acids rely directly on adequate pre‐analytical assessment of those samples. In this setting, cytopathologists must not only be familiar with the different types of specimens and associated technical procedures, but also correctly handle the material provided by minimally invasive procedures, ensuring that there is sufficient amount of material for a precise diagnosis and correct management of the patient through personalised care. [ABSTRACT FROM AUTHOR]

Training in molecular cytopathology testing is essential in developing and maintaining skills in modern molecular technologies as they are introduced to a universal health care system such as extant in the UK and elsewhere. We review the system in place in Northern Ireland (NI) for molecular testing of solid tumours, as an example to train staff of all grades, including pathologists, clinical scientists, biomedical scientists and equivalent technical grades. We describe training of pathologists as part of the NI Deanery medical curriculum, the NI training programme for scientists and laboratory rotation for Biomedical Scientists. Collectively, the aims of our training are two‐fold: to provide a means by which individuals may extend their experience and skills; and to provide and maintain a skilled workforce for service delivery. Through training and competency, we introduce new technologies and tests in response to personalised medicine therapies with a competent workforce. We advocate modifying programmes to suit individual needs for skill development, with formalised courses in pre‐analytical, analytical and postanalytical demands of modern molecular pathology. This is of particular relevance for cytopathology in small samples such those from formalin‐fixed paraffin‐embedded cell blocks. We finally introduce how university courses can augment training and develop a skilled workforce to benefit the delivery of services to our patients. [ABSTRACT FROM AUTHOR]

Background: Molecular testing of cytological lung cancer specimens includes, beyond epidermal growth factor receptor (EGFR), emerging predictive/prognostic genomic biomarkers such as Kirsten rat sarcoma viral oncogene homolog (KRAS), neuroblastoma RAS viral [v-ras] oncogene homolog (NRAS), B-Raf proto-oncogene, serine/threonine kinase (BRAF), and phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit α (PIK3CA). Next-generation sequencing (NGS) and other multigene mutational assays are suitable for cytological specimens, including smears. However, the current literature reflects single-institution studies rather than multicenter experiences.Methods: Quantitative cytological molecular reference slides were produced with cell lines designed to harbor concurrent mutations in the EGFR, KRAS, NRAS, BRAF, and PIK3CA genes at various allelic ratios, including low allele frequencies (AFs; 1%). This interlaboratory ring trial study included 14 institutions across the world that performed multigene mutational assays, from tissue extraction to data analysis, on these reference slides, with each laboratory using its own mutation analysis platform and methodology.Results: All laboratories using NGS (n = 11) successfully detected the study's set of mutations with minimal variations in the means and standard errors of variant fractions at dilution points of 10% (P = .171) and 5% (P = .063) despite the use of different sequencing platforms (Illumina, Ion Torrent/Proton, and Roche). However, when mutations at a low AF of 1% were analyzed, the concordance of the NGS results was low, and this reflected the use of different thresholds for variant calling among the institutions. In contrast, laboratories using matrix-assisted laser desorption/ionization-time of flight (n = 2) showed lower concordance in terms of mutation detection and mutant AF quantification.Conclusions: Quantitative molecular reference slides are a useful tool for monitoring the performance of different multigene mutational assays, and this could lead to better standardization of molecular cytopathology procedures. Cancer Cytopathol 2017;125:615-26. © 2017 American Cancer Society. [ABSTRACT FROM AUTHOR]

With increasing requests for the evaluation of prognostic and predictive molecular biomarkers, great attention must be paid to the preanalytical issues regarding sample quality and DNA/RNA yield from all different types of cytological preparations. The objectives of this review were: 1) to provide an update regarding the importance of specimen triage as well as specimen handling and collection; 2) to discuss the different cell preparations that can be used for molecular testing, their advantages and limitations; and 3) to highlight the strategies for biobanking cytology samples. Good-quality DNA/RNA can be harvested from fresh cells in cell suspensions, formalin-fixed paraffin-embedded cell blocks, archival stained smears, archival unstained cytospin preparations, liquid-based cytology slides, FTA cards, and cryopreserved cells. In contrast to formalin-fixed paraffin-embedded tissue specimens (small biopsies and surgical resections), the multitude of types of sample preparations as well as the diversity in sample collection and processing procedures make cytology an ideal specimen for most genomic platforms, with less DNA and RNA degradation and a purer sample, usually with a higher concentration of tumor cells. The broad incorporation of cytological specimens into clinical practice. A should increase the number of samples potentially available for molecular tests and avoid repeat invasive procedures for tissue procurement, thereby increasing patient safety. In this context, it is of utmost importance that cytopathologists become familiar with the variables that can affect test results and embrace the goal of excellence in sample quality. Cancer Cytopathol 2017;125(6 suppl):455-64. © 2017 American Cancer Society. [ABSTRACT FROM AUTHOR]

The results from molecular assays can be affected significantly by the preanalytic condition of cytologic samples. The authors review current knowledge on the use of cytologic samples for epidermal growth factor receptor (EGFR) mutation testing in non-small cell lung cancer with a focus on preanalytic parameters. A systematic electronic search of the MEDLINE database was performed to identify original articles that reported the use of cytologic samples for EGFR molecular analysis and included a minimum of 100 samples. The information collected included author(s), journal, and year of publication; number of patients and samples; sampling method; type of preparation; type of fixative; staining techniques; mutation analysis techniques; tumor cellularity; the percentage of tumor cells; data on DNA quantity, quality, and concentration; failed assays; and the mutation rate. EGFR mutation analysis was conducted on 4999 cytologic samples from 22 studies that fulfilled the inclusion criteria. Fine-needle aspirates and pleural effusions were the most common types of specimens used. DNA was mainly extracted from cell blocks and smears, and the most commonly reported fixatives included formalin, ethanol, and CytoLyt. Cellularity assessments and DNA yields were available from 5 studies each. The average success rate for the assays that used cytologic specimens was 95.87% (range, 85.2%-100%). The mutation rate ranged from 6% to 50.46%, and a higher mutation detection rate and lower numbers of insufficient cases were reported for pleural effusions and lymph node samples from endobronchial ultrasound-guided transbronchial needle aspiration compared with histologic specimens. Low cellularity and a low percentage of tumor cells were associated with higher test failure rates. Future guidelines should consider the current data for specific recommendations regarding cytologic samples. [ABSTRACT FROM AUTHOR]

Molecular cytopathology has gene sequencing as its core technology. Until recently, cytological samples were only tested by sequential single-gene mutational tests. Today, with the better understanding of the molecular events involved in malignancy and the mechanisms of pharmacotherapy, larger gene panels are more informative than a single biomarker. Next-generation sequencing ( NGS), matched with the multiplex capture of targeted gene regions and analysed by sophisticated bioinformatics tools, enables the simultaneous detection of multiple mutations in multiple genes. With the development of miniaturised technology and benchtop sequencers, it is not unlikely that NGS will soon be adopted for routine molecular diagnostics, including cytological samples. This review addresses (1) the most relevant methodological and technical aspects of the NGS analysis workflow and the diverse platforms available; (2) the issues related to daily practice implementation, namely, the cytological sample requirement and the validation procedures; and (3) the opportunities that NGS offers in different fields of cytopathology, to increase mutation detection sensitivity in paucicellular smears and to extend the analysis to a larger number of gene regions. Cytopathologists involvement and coordination in this rapidly evolving field is crucial for the effective implementation of NGS in the present and future cytological practice. [ABSTRACT FROM AUTHOR]

Objective Detection of Epstein-Barr virus ( EBV) status might help in the diagnosis of EBV-related neoplasms. The rate of successful assays for the detection of EBV-infected cells in cytological preparations has not been fully explored. Our aims were to examine the rate of successful in situ hybridization ( ISH) assays for EBV-encoded RNA ( EBER) in cytological specimens and to explore reasons for failure. Methods An electronic search selected cases with ISH- EBER assays performed on cytological preparations during a 10-year period. Data regarding patient age, gender and immune status, sample type and site, type of preparation, ISH- EBER results, immunophenotyping and immunohistochemistry results, final diagnosis and correspondent histopathological samples were retrieved. Results Sixty specimens from 58 patients with diagnoses of lymphoproliferative disorder ( n = 35), carcinoma ( n = 24) and sarcoma ( n = 1) were identified. ISH- EBER assays were performed on 50 cell block sections and on 10 cytospin preparations, with 22 positive and 32 negative results. Six tests (four cytospins and two cell block sections) failed owing to loss of material during the assay and background staining, with an overall failure rate of 10% and 4% if cytospins were excluded. Assays were performed on 13 cytology and surgical specimens from the same site, with only one discrepant result. Conclusions Cell block sections had more successful ISH- EBER assays when compared with cytospins. Reasons for failure were loss of material on the slide and background staining. A high concordance rate with surgical specimens emphasizes the usefulness of cytological samples for determining EBV status in patients with exhausted or no histological material available. [ABSTRACT FROM AUTHOR]

Here we report the combined cytological and molecular diagnosis of a lung mass. The cytology and extensive immunohistochemistry on an endobronchial ultrasound-guided fine needle aspiration biopsy were inconclusive. By genomic profiling of the cell block material, we identified a MET exon 14 skipping mutation that indicated a lung origin and made the patient eligible for the tyrosine kinase inhibitor, crizotinib. This case is a prime example of complementing adequate aspiration and cell block processing techniques with molecular testing. Such an approach would augment the usability of fine needle aspiration biopsy, both as a diagnostic modality and as the first line to find therapeutic targets in the era of precision medicine., (© 2021 Wiley Periodicals LLC.)

This short article describes the method of digital cytopathology using Z-stack scanning with or without extended focusing. This technology is suitable to observe such thick clusters as adenocarcinoma on cytologic specimens. Artificial intelligence (AI) has been applied to histological images, but its application on cytologic images is still limited. This article describes our attempt to apply AI technology to cytologic digital images. For molecular analysis, cytologic materials, such as smear, LBC, and cell blocks, have been successfully used for targeted single gene detection and multiplex gene analysis with next-generation sequencing. As a future perspective, the system can be connected to full automation by combining digital cytopathology with AI application to detect target cancer cells and to perform molecular analysis. The literature review is updated according to the subjects., (© 2021 The Author(s) Published by S. Karger AG, Basel.)

Background: Thyroid fine-needle aspirates (FNAs) with undetermined morphology can be outsourced to centralized laboratories for comprehensive molecular profiling. When a local, rapid screening rules out easily detectable BRAF and NRAS mutations outsourcing is minimized, leading to cost savings. The fully automated Idylla technology, that does not require trained staff, is an emerging option. However, Idylla platform has only been validated to process formalin fixed paraffin embedded (FFPE) sections. Here we investigate whether also the FNA needle rinse could be genotyped by the same cytopathologist who performs the FNA, a procedure that can be termed rapid on site molecular evaluation (ROME)., Methods: To validate this approach, the Idylla BRAF and NRAS Test was performed on the rinses from 25 simulated (bench-top) FNAs, in a first part of the study. Genotyping data were compared with those obtained on matched histological FFPE blocks. The second part of the study was carried out on 25 prospectively collected routine FNAs to assess the performance of the Idylla BRAF and NRAS assay against a gold standard real time polymerase chain reaction method., Results: Idylla NRAS-BRAF Mutation Test was performed on needle rinse as well as histological FFPE blocks. A sensitivity of 88.9%, a specificity of 100.0% were obtained comparing the Idylla NRAS-BRAF Mutation Test on needle rinse to the reference method., Conclusions: The FNA needle rinse can be directly genotyped. This obviates the need of cell block preparation, making possible a rapid combined morphological and molecular evaluation. Since DNA extraction is no longer necessary, the cytopathologist can perform ROME him/herself., (© 2020 Wiley Periodicals, Inc.)

Aim: The rapid and fully automated Idylla EGFR Mutation Assay has been specifically designed to process formalin-fixed, paraffin-embedded sections without requiring preliminary DNA extraction. This study evaluates whether this approach can also process archival smears from patients with non-small cell lung cancer (NSCLC) by scraping the stained cellular material directly into the cartridge., Methods: The study was divided into two parts. In the first part, we carried out Idylla EGFR Mutation Assay on archival stained smears from 39 patients with NSCLC. Among these, 14 cases harboured a mutation in either exon 19 (n=11) or exon 21 (n=3), previously detected on DNA extracts by fragment length and TaqMan assays. In the second part, we evaluated whether de-staining of the smears could reduce background fluorescence., Results: The Idylla EGFR Mutation Assay confirmed the presence of EGFR mutation in 11 instances (78.6%). However, concordance was higher for exon 19 deletions (10/11) than for exon 21 p.L858R assessments. Raw data showed a high background fluorescence in channel 2, where the EGFR exon 21 p.L858R mutation was detected. This interference, due to dye residues from the original staining, was partially reduced by de-staining the cytological material., Conclusions: Our data, although preliminary, show that the Idylla EGFR Mutation Assay can reliably process most archival smears without requiring preliminary DNA extraction. Results may be further improved by de-staining the cellular material before insertion into the cartridge., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2019. No commercial re-use. See rights and permissions. Published by BMJ.)

Utilization of fine-needle aspiration biopsy (FNAB) cytology for the diagnosis of diseases of the breast has been met with both excitement and uncertainty during the last couple of decades. Presently, FNAB for the diagnosis of primary and metastatic breast lesions is on the rise again. This is probably due to its fast turnaround time, cost efficiency, and minimal invasiveness, characteristics of this sampling modality which are particularly crucial for patients requiring frequent repeat biopsy in the setting of metastatic lesions. In this article, we will briefly review the main modern applications of FNAB of the breast when coupled with contemporary ancillary techniques. Such contemporary ancillary techniques range from classic immunocytochemistry (ICC) to the most modern molecular techniques, particularly next-generation sequencing. Coupled with contemporary ICC and molecular methods, FNAB of the breast can be used for several applications. The applications reviewed in this article include the primary diagnosis of a breast lesion, the identification of the breast as a primary source of a metastatic lesion, the evaluation of breast prognostic/predictive markers, and the tracking of tumor evolution. In our opinion, FNAB of the breast is an ideal sampling method, sharing many of the advantages of truly liquid and of tissue biopsies. Ultimately, we aim at demystifying the complexity of many of the challenges traditionally associated with the application of ancillary techniques to FNAB of the breast and provide insights into some of the most cutting-edge and clinically useful application scenarios., (© 2019 S. Karger AG, Basel.)

This review is focused on the challenges in standardising and optimising molecular testing workflow in cytopathology. Although cytological samples yield optimal quality DNA, whose minimal amounts in most cases suffice even for multigene mutational profiling, the success of molecular testing is strongly dependent on standardised preanalytical protocols for maximising DNA yield and quality. Sample cytopreparation influences, even more, the quality of RNA and consequently the potential success of reverse transcription-PCR. Here, the educational and technical involvement of the cytopathologist as a relevant component of a multidisciplinary team, in the issues related to test request, specimen collection, fixation, processing, staining, tumour fraction enrichment, DNA quality/quantity assessment and storage conditions is discussed. In addition, the specific sample requirements related to more recent technological developments are examined, underlining the modern role of the cytopathologist, whose continuous education is crucial to meet the opportunities of molecular medicine., Competing Interests: Competing interests: None declared., (© Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2017. All rights reserved. No commercial use is permitted unless otherwise expressly granted.)