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.

A third update of The Bethesda System for Reporting Thyroid Cytopathology has been published in 2023 following the first (2010) and second (2017) versions. The main modifications are the following 1) a new co-Editor, 2) 4 associate editors, 3 of them from Europe, 3) the inclusion of 65 co-authors, 19 of them from Europe, 4) 2 new chapters: one dealing with pediatrics thyroid cytopathology and the other one describing molecular cytopathology profiling, 5) updated risks of malignancy (ROM), 6) a terminology in line with the 2022 WHO classification of thyroid tumors, 7) diagnostic categories now defined by a unique name, 8) 2 subtypes in the \"Atypia of Undetermined Significance\" category with corresponding ROM.

BACKGROUND: 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.
CONCLUSIONS: 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.

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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.

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.

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.

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 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.

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.