Precision Medicine in Cytopathology.

Over the last decade, cancer diagnostics has undergone a notable transformation with increasing complexity. Minimally invasive diagnostic tests, driven by advanced imaging and early detection protocols, are redefining patient care and reducing the need for more invasive procedures. Modern cytopathologists now safeguard patient samples for vital biomarker and molecular testing. In this article, we explore ancillary testing modalities and the role of biomarkers in organ-specific contexts, underscoring the transformative impact of precision medicine. Finally, the advent of more than 80 Food and Drug Administration-approved predictive biomarkers signals a new era, guiding cancer care toward personalized and targeted strategies.

Pulmonary Cytopathology: Current and Future Impact of Microscopy and Immunohistochemistry.

With the advancement of tissue procurement techniques, in-depth knowledge of morphology is crucial for cytopathologists to diagnose neoplastic and nonneoplastic lung diseases optimally. Cytopathologists must also be well versed in immunohistochemistry/immunocytochemistry markers and their interpretation for an accurate diagnosis.

Surface Acoustic Wave-Enhanced Multi-View Acoustofluidic Rotation Cytometry (MARC) for Pre-Cytopathological Screening.

Cytopathology, crucial in disease diagnosis, commonly uses microscopic slides to scrutinize cellular abnormalities. However, processing high volumes of samples often results in numerous negative diagnoses, consuming significant time and resources in healthcare. To address this challenge, a surface acoustic wave-enhanced multi-view acoustofluidic rotation cytometry (MARC) technique is developed for pre-cytopathological screening. MARC enhances cellular morphology analysis through comprehensive and multi-angle observations and amplifies subtle cell differences, particularly in the nuclear-to-cytoplasmic ratio, across various cell types and between cancerous and normal tissue cells. By prioritizing MARC-screened positive cases, this approach can potentially streamline traditional cytopathology, reducing the workload and resources spent on negative diagnoses. This significant advancement enhances overall diagnostic efficiency, offering a transformative vision for cytopathological screening.

High-risk human papillomavirus infection and cervical cytopathology: relationship with cervical nitric oxide levels.

BACKGROUND: Nitric oxide (NO) may contribute to the persistence of high-risk human papillomavirus (hrHPV) infection, which has been linked to the development of premalignant lesions and cervical cancer. Our study aimed to examine the relationship between cervical NO metabolite (NOx) levels, hrHPV infection, and cytopathological findings. Additionally, we assessed cervical NOx levels as a biomarker for predicting hrHPV infection and epithelial atypia. METHODS: The study involved 74 women who attended the Gynecology and Obstetrics outpatient clinics at Cairo University Hospitals between November 2021 and August 2022. Cervical samples were subjected to Pap testing, assessment of NOx levels by the Griess method, and detection of hrHPV DNA by real-time polymerase chain reaction. RESULTS: High-risk HPV was detected in 37.8% of women. EA was found in 17.1% of cases, with a higher percentage among hrHPV-positive than negative cases (35.7% vs. 4.3%, p = 0.001). The most prevalent hrHPV genotype was HPV 16 (89.3%). The cervical NOx level in hrHPV-positive cases was significantly higher (37.4 µmol/mL, IQR: 34.5-45.8) compared to negative cases (2.3 µmol/mL, IQR: 1.2-9.8) (p = < 0.001). Patients with high-grade atypia showed significantly higher NOx levels (38.0 µmol/mL, IQR: 24.6-94.7) in comparison to NILM and low-grade atypia cases (5.0 µmol/mL, IQR: 1.6-33.3 and 34.5 µmol/mL, IQR: 11.7-61.7, respectively) (p = 0.006). Although the NOx levels among hrHPV-positive cases with low-grade atypia (40.4 µmol/mL, IQR: 33.3‒61.8) were higher than those with NILM (36.2 µmol/mL, IQR: 35.7‒44.0) and high-grade atypia (38.0 µmol/mL, IQR: 24.6‒94.7), the difference was not significant (p = 0.771). ROC curve analysis indicated that the cervical NOx cut-off values of > 23.61 µmol/mL and > 11.35 µmol/mL exhibited good diagnostic accuracy for the prediction of hrHPV infection and EA, respectively. CONCLUSIONS: The high prevalence of hrHPV infection, particularly HPV 16, in our hospital warrants targeted treatment and comprehensive screening. Elevated cervical NOx levels are associated with hrHPV infection and high-grade atypia, suggesting their potential use as biomarkers for predicting the presence of hrHPV and abnormal cytological changes.

Head and Neck Fine Needle Aspiration: Current and Future Impact on Patient Care.

The discovery of multiple novel biomarkers in head and neck tumors has led to an increasing interest in utilizing head and neck cytology material as the primary specimens for testing diagnostic and prognostic biomarkers. Although human papillomavirus and programmed death ligand 1 are the most well-established biomarkers tested in cytology specimens, their utilization in cytology is limited by the absence of standardized protocols for specimen collection and fixation. This has led to a quest for innovative techniques to explore the genomic landscape in head and neck tumors and its application in cytology.

Pulmonary Cytopathology: Current and Future Impact on Patient Care.

Small biopsies of lung are routinely obtained by many methods, including several that result in cytologic specimens. Because lung cancer is often diagnosed at a stage for which primary resection is not an option, it is critical that all diagnostic, predictive, and prognostic information be derived from such small biopsy specimens. As the number of available diagnostic and predictive markers expands, cytopathologists must familiarize themselves with current requirements for specimen acquisition, handling, results reporting, and molecular and other ancillary testing, all of which are reviewed here.

Thyroid Fine-Needle Aspiration: The Current and Future Landscape of Cytopathology.

Thyroid cytology is a rapidly evolving field that has seen significant advances in recent years. Its main goal is to accurately diagnose thyroid nodules, differentiate between benign and malignant lesions, and risk stratify nodules when a definitive diagnosis is not possible. The current landscape of thyroid cytology includes the use of fine-needle aspiration for the diagnosis of thyroid nodules with the use of uniform, tiered reporting systems such as the Bethesda System for Reporting Thyroid Cytopathology. In recent years, molecular testing has emerged as a reliable preoperative diagnostic tool that stratifies patients into different risk categories (low, intermediate, or high) with varying probabilities of malignancy and helps guide patient treatment.

Pancreas Fine Needle Aspiration: Current and Future Impact on Patient Care.

Pancreatic lesions can be solid or cystic and comprise a wide range of benign, premalignant, and malignant entities. Endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) is the current primary sampling method for the preoperative diagnosis of pancreatic lesions. Optimal handling of cytology/small tissue specimens is critical to ensure that the often-scant diagnostic material is appropriately utilized for ancillary and/or molecular studies when appropriate. Ultimately, evaluation of EUS-FNA cytology and small biopsy material can provide accurate and timely diagnoses to guide patient management and triage them to surveillance or surgical intervention.

Minimally Invasive, Maximally Effective: The Power of Precision Cytoanalysis on Effusion Samples-A Comprehensive Exploration from Traditional Methods to Innovative Approaches.

Precision medicine translates through molecular assays and in minimally invasive diagnosis, evident in analyses of effusions that serve therapeutic and diagnostic purposes. This cost-effective and low-risk approach provides advantages, playing a pivotal role in late-stage oncology and frequently standing as the primary resource for cancer diagnosis and treatment pathways. This article outlines the workflow for managing serous fluid and explores how cytology effusion analysis extends beyond immunocytological diagnosis. Combined with current molecular tests it showcases the potential to be a skillful tool in precision cytopathology.

Artificial Intelligence Applications in Cytopathology: Current State of the Art.

The practice of cytopathology has been significantly refined in recent years, largely through the creation of consensus rule sets for the diagnosis of particular specimens (Bethesda, Milan, Paris, and so forth). In general, these diagnostic systems have focused on reducing intraobserver variance, removing nebulous/redundant categories, reducing the use of "atypical" diagnoses, and promoting the use of quantitative scoring systems while providing a uniform language to communicate these results. Computational pathology is a natural offshoot of this process in that it promises 100% reproducible diagnoses rendered by quantitative processes that are free from many of the biases of human practitioners.

Individual Ultrasonographic Characteristics of Thyroid Nodules and Their Cytopathological Correlation to Determine Malignancy Risk.

Background Ultrasonographic evaluation of thyroid nodules is challenging due to their high frequency and low malignancy rate. The risk stratification system developed by the American College of Radiology (ACR) Thyroid Imaging Reporting and Data System (TI-RADS) focuses on addressing the primary contemporary objectives for these lesions, aiming to decrease unnecessary biopsies while maintaining a similar specificity compared with other risk stratification systems. Generally, when indicative of malignancy by ultrasound findings, the next best step in management is an evaluation by fine needle aspiration biopsy (FNAB) and cytological analysis with The Bethesda System for Reporting Thyroid Cytopathology (TBSRTC) results that determine further evaluation requirements, actions that are based on the risk of malignancy (ROM) of the assigned category, which could include surgical intervention. Objectives To validate and analyze the individual impact of each ultrasonographic finding indicative of malignancy in the ACR TI-RADS guidelines based on their respective correlation with results obtained by TBSRTC. Materials and method Reports for 212 thyroid ultrasound-guided FNABs from 2018 to 2020 were assessed. Only 117 had both ACR TI-RADS and TBSRTC reports available and were analyzed. Nodules were divided into two groups: ROM < 5% (Bethesda 1, 2; n = 58), and ROM > 5% (Bethesda 3, 4, 5, 6; n = 59). Statistical analysis was performed using the x2 test and bivariate logistic regression model for each characteristic included in ACR TI-RADS. Results Individual ultrasound characteristics with a more pronounced distribution towards the Bethesda > 5% malignancy group were: solid or almost completely solid composition (n=53, 62.3%), very hypoechoic echogenicity (n=3, 75%), wider-than-tall shape (n=50, 50.5%), lobulated or irregular margin (n=23, 65.7%), punctate echogenic foci (n=18, 72%), and thyroid isthmus location (n=6, 75%). Statistically significant individual ultrasonographic characteristics indicative of malignancy included solid or almost completely solid (p = 0.005), very hypoechoic echogenicity (p = 0.046), margin lobulated or irregular (p = 0.031), and punctate echogenic foci (p = 0.015). No significant association was found in the taller-than-wide shape for differentiating malignant from benign lesions (p = 0.969). Conclusions Specific ultrasound characteristics identified in the ACR TI-RADS system demonstrate a stronger correlation with an increased risk of malignancy when compared with cytologic evaluation results. These characteristics include a solid composition, lobulated or irregular margins, punctate echogenic foci, and very hypoechoic echogenicity. Our findings revealed that the scale points for the taller-than-wide characteristic do not adequately represent its true influence on the risk of malignancy.

Application of the international system for reporting serous fluid cytopathology on pleural effusion cytology with paired pleural biopsy: A new insight and novel approach on risk of malignancy.

INTRODUCTION: The risk of malignancy (ROM) remains an area of interest for further evaluation in reporting systems including in International System for reporting serous fluid cytopathology (TIS), which is a standardized system for reporting effusion cytology. Herein, we report our findings in further investigation of ROM in TIS by studying on paired pleural effusion specimens and corresponding pleural biopsies with emphasis on negative for malignancy, and atypia of undetermined significance categories. MATERIALS AND METHODS: The  Johns Hopkins Hospital pathology database was retrospectively searched for patients with a pleural biopsy (PBX) and a paired pleural effusion (PF) cytology specimens over a 4-year period. We employed the TIS categories. The following statistical parameters were evaluated: sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and ROM. RESULTS: A total of 223 patient cases were included. Effusions TIS reclassification and ROM were as follows: 1.8% non-diagnostic (ROM 75%), 75.8% negative for malignancy (ROM 23%), 4.9% atypical cells of undetermined significance (ROM 45%), 2.2% suspicious for malignancy (ROM 80%), and 15.2% malignant (ROM 100%). Overall accuracy, sensitivity, specificity, PPV and NPV were calculated and were 79.4%, 45%, 97.7%, 91.2% and 77%, respectively. Among, discordant cases diagnosed negative for malignancy on PF and positive for malignancy on PBX, there were significant number of lymphomas, mesotheliomas, and sarcomas. Lung cancer was the most common carcinoma; however, rare types of carcinomas were noted. Cells blocks and immunohistochemistry (IHC) studies were utilized to confirm either malignant conditions or rule out malignancy in both cell blocks and histology biopsies. CONCLUSION: This study demonstrates the high specificity and ROM for 'malignant' and 'suspicious for malignancy' categories in the TIS reporting system and highlights the modest negative predictive value for the 'negative for malignancy' category. Although Tissue biopsies are usually considered as 'gold standard', any definitive diagnosis of malignancy of body fluid should be considered positive for malignancy in further clinical decision-making.

Epstein-Barr virus-positive inflammatory follicular dendritic cell sarcoma: A brief report of a rare neoplasm diagnosed with cytopathology on a splenic biopsy.

Splenic biopsies for cytology remain challenging due to the inherent difficulty in obtaining adequate samples and the paucity of literature on rare entities arising in the spleen. Among these, are tumors arising from blood vessels, lymphomas and rarely, mesenchymal dendritic cell neoplasms. An important but rarely considered entity primarily arising in the spleen is Epstein-Barr virus-positive inflammatory follicular dendritic cell sarcoma (EBV+ IFDCS). EBV+ IFDCS is an indolent neoplasm with useful cytomorphologic and distinct biologic characteristics that can be evaluated on fine-needle aspiration (FNA) cytology and small biopsies. In this report, we present a challenging case with the final diagnosis facilitated by cytomorphology and diagnostic markers in an ambiguous initial presentation.

Viral Cytopathic Changes in Sputum Cytology in a Patient with Known Human Metapneumovirus.

INTRODUCTION: Viral cytopathic changes seen in sputum cytology have been described in association with infection by viruses such as cytomegalovirus (CMV), herpes simplex virus (HSV), adenovirus, and even measles. However, viral cytopathic changes due to human metapneumovirus (hMPV) have not yet been well described in cytology. hMPV is a relatively new entity, discovered in 2001. It is known to cause upper and lower respiratory tract infections in children, the elderly, and immunocompromised patients. CASE PRESENTATION: We describe the viral cytopathic changes seen in sputum in a 63-year-old male patient with known hMPV. These changes include multinucleation, nuclear enlargement, homogenised nuclei, basophilic nuclear inclusions with perinuclear halos, and small eosinophilic cytoplasmic inclusions. CONCLUSION: We aim to raise awareness that hMPV can cause viral cytopathic changes and to describe these cytological features, which have been elucidated in only 1 case report thus far. Distinction from other viruses with similar changes, such as HSV and CMV, is important due to their differing clinical implications.

Impact of serous fluid volume on next-generation sequencing: a significant step forward for optimization of serous fluid sample collection.

Current literature lacks data regarding the influence of serous fluid volume (SFV) on next-generation sequencing (NGS) performed on malignant cases. In this study, we highlight the impact of SFV and other parameters influencing the outcome of NGS analysis. We evaluated 827 samples of serous fluids from 607 patients. Of these, 72 samples underwent NGS analysis. Effusion volume, tumor cellularity, DNA, and RNA quality metrics, as well as clinicopathologic and molecular data were evaluated. Pleural fluid accounted for 56.3% of the fluid samples collected. The most common primary tumor site was gastrointestinal/pancreatobiliary, adenocarcinoma was the most common histologic type. Overall mean volume was 293 mL. The mean Qubit DNA of the 72 effusion samples that underwent NGS analysis was 14.3 ng/µL and mean Qubit RNA was 28.2 ng/µL. The mean Qubit DNA concentration increases in SFV up to 100 mL only. No correlation exists between SFV and mean tumor cellularity. In addition, 74.6% (50 of 67) of sequenced samples showed oncogenic drivers; KRAS was the most common driver followed by EGFR. Three cases displayed ALK fusions, and 1 case displayed NTRK1 fusion. The DNA yield is higher in SFV of 100 mL as a cutoff. Beyond 100 mL, there is no impact of SFV on DNA yield. SFV does not impact RNA yield and mean tumor cellularity. Effusion samples should be submitted for molecular testing despite low tumor cellularity. Our results as a pilot study are important in optimization of SFV for both diagnosis as well as NGS analysis for improving management.

⍺-Synuclein levels in Parkinson's disease - Cell types and forms that contribute to pathogenesis.

Parkinson's disease (PD) has two main pathological hallmarks, the loss of nigral dopamine neurons and the proteinaceous aggregations of ⍺-synuclein (⍺Syn) in neuronal Lewy pathology. These two co-existing features suggest a causative association between ⍺Syn aggregation and the underpinning mechanism of neuronal degeneration in PD. Both increased levels and post-translational modifications of ⍺Syn can contribute to the formation of pathological aggregations of ⍺Syn in neurons. Recent studies have shown that the protein is also expressed by multiple types of non-neuronal cells in the brain and peripheral tissues, suggesting additional roles of the protein and potential diversity in non-neuronal pathogenic triggers. It is important to determine (1) the threshold levels triggering ⍺Syn to convert from a biological to a pathologic form in different brain cells in PD; (2) the dominant form of pathologic ⍺Syn and the associated post-translational modification of the protein in each cell type involved in PD; and (3) the cell type associated biological processes impacted by pathologic ⍺Syn in PD. This review integrates these aspects and speculates on potential pathological mechanisms and their impact on neuronal and non-neuronal ⍺Syn in the brains of patients with PD.

Serum cholesterol levels are inversely associated with the risk of malignancy in subjects with Bethesda category IV thyroid nodules.

INTRODUCTION: Some epidemiological data suggest that there may be an inverse relationship between cholesterol levels and the risk of thyroid cancer in the overall population. The present study was aimed to evaluate the lipid profile specifically in subjects with Bethesda category IV thyroid nodules, and compare whether there were any differences between those with benign and malignant nodules. METHODS: Single-centre, retrospective study on 204 subjects treated by partial or total thyroidectomy for excision of a Bethesda category IV thyroid nodule, who had undergone a blood lipid profile test in the 12 months prior to surgery. In addition to lipid measures, other demographic, clinical, biochemical and ultrasound data were collected. RESULTS: Seventy-five subjects (36.8%) were diagnosed with thyroid carcinoma in the definitive histopathological examination. Patients with thyroid cancer had lower levels of total cholesterol, LDL-cholesterol and non-HDL-cholesterol than subjects with benign thyroid diseases. There were no differences in HDL-cholesterol, triglycerides or total cholesterol/HDL-cholesterol ratio. There were no differences either between groups in other clinical, biochemical and ultrasound variables, including the use of lipid-lowering drugs. In multivariate analysis, only LDL-cholesterol was independently associated with malignancy. Subjects with follicular carcinoma showed the lowest cholesterol levels, while those with papillary carcinoma had intermediate values between the group with follicular carcinoma and the group with benign thyroid diseases. CONCLUSIONS: In subjects with cytologically indeterminate Bethesda category IV thyroid nodules, levels of total cholesterol, non-HDL-cholesterol and, particularly, LDL-cholesterol are lower among those with malignant nodules.

Bronchoscopy with and without needle-based confocal laser endomicroscopy for peripheral lung nodule diagnosis: protocol for a multicentre randomised controlled trial (CLEVER trial).

INTRODUCTION: Despite many technological advances, the diagnostic yield of bronchoscopic peripheral lung nodule analysis remains limited due to frequent mispositioning. Needle-based confocal laser endomicroscopy (nCLE) enables real-time microscopic feedback on needle positioning, potentially improving the sampling location and diagnostic yield. Previous studies have defined and validated nCLE criteria for malignancy, airway and lung parenchyma. Larger studies demonstrating the effect of nCLE on diagnostic yield are lacking. We aim to investigate if nCLE-imaging integrated with conventional bronchoscopy results in a higher diagnostic yield compared with conventional bronchoscopy without nCLE. METHODS AND ANALYSIS: This is a parallel-group randomised controlled trial. Recruitment is performed at pulmonology outpatient clinics in universities and general hospitals in six different European countries and one hospital in the USA. Consecutive patients with a for malignancy suspected peripheral lung nodule (10-30 mm) with an indication for diagnostic bronchoscopy will be screened, and 208 patients will be included. Web-based randomisation (1:1) between the two procedures will be performed. The primary outcome is diagnostic yield. Secondary outcomes include diagnostic sensitivity for malignancy, needle repositionings, procedure and fluoroscopy duration, and complications. Pathologists will be blinded to procedure type; patients and endoscopists will not. ETHICS AND DISSEMINATION: Primary approval by the Ethics Committee of the Amsterdam University Medical Center. Dissemination involves publication in a peer-reviewed journal. SUPPORT: Financial and material support from Mauna Kea Technologies. TRIAL REGISTRATION NUMBER: NCT06079970.

The evolving role of interventional cytopathology from thyroid FNA to NGS: Lessons learned at Federico II University of Naples.

Fine-needle aspiration (FNA) guided by ultrasound (US) has emerged as a highly precise diagnostic method for managing thyroid nodules, significantly diminishing unnecessary surgeries. The effectiveness of US-guided FNA is high when a single specialist performs the FNA procedure and the microscopy. This paradigm has paved the way for the evolution of interventional cytopathology, a specialist with a pivotal role in the preoperative diagnostic process, encompassing patient history review, clinical examination, FNA execution under US guidance, preparation, and microscopic interpretation of cytological samples. As the landscape of precision medicine unfolds, molecular testing assumes greater importance in thyroid cytopathology, particularly in refining the risk of malignancy for indeterminate nodules. The updated Bethesda classification system underscores the clinical significance of molecular tests, emphasizing their role in refining diagnostic accuracy. With this evolving landscape, interventional cytopathologists must adapt by acquiring expertise in molecular technologies and addressing ongoing challenges in workflow harmonization and optimization. This paper delves into our decade-long experience as interventional cytopathologists, focusing on recent endeavours to ensure adequate samples not only for microscopic diagnosis but also for molecular testing. Additionally, here we review the challenges of integrating next-generation sequencing (NGS) technology into clinical practice, highlighting the importance of integrating clinically meaningful molecular data into comprehensive molecular cytology reports.

Deep learning-based cell segmentation for rapid optical cytopathology of thyroid cancer.

Fluorescence polarization (Fpol) imaging of methylene blue (MB) is a promising quantitative approach to thyroid cancer detection. Clinical translation of MB Fpol technology requires reduction of the data analysis time that can be achieved via deep learning-based automated cell segmentation with a 2D U-Net convolutional neural network. The model was trained and tested using images of pathologically diverse human thyroid cells and evaluated by comparing the number of cells selected, segmented areas, and Fpol values obtained using automated (AU) and manual (MA) data processing methods. Overall, the model segmented 15.8% more cells than the human operator. Differences in AU and MA segmented cell areas varied between - 55.2 and + 31.0%, whereas differences in Fpol values varied from - 20.7 and + 10.7%. No statistically significant differences between AU and MA derived Fpol data were observed. The largest differences in Fpol values correlated with greatest discrepancies in AU versus MA segmented cell areas. Time required for auto-processing was reduced to 10 s versus one hour required for MA data processing. Implementation of the automated cell analysis makes quantitative fluorescence polarization-based diagnosis clinically feasible.