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.

Interobserver variability in cytopathology: How much do we agree?

Interobserver variability remains a major challenge for cytopathologists despite the development of standardized reporting and classification systems. Indeed, whereas moderate-to-good interobserver agreement is generally achievable when the differential diagnosis between benign and malignant entities is straightforward, high levels of variability make the diagnostic interpretation of atypical and suspicious samples not consistent. This review explores the landscape of interobserver agreement in cytopathology across different anatomical sites.

Molecular testing in salivary gland cytopathology: A practical overview in conjunction with the Milan system.

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.

Delphi expert consensus for whole slide imaging in thyroid cytopathology.

OBJECTIVE: Despite an increase in thyroid fine needle aspiration (FNA) and advances in whole slide imaging (WSI) adoption, digital pathology is still considered inadequate for primary diagnosis of these cases. Herein, we aim to validate the utility of WSI in thyroid FNAs employing the Delphi method strategy. METHODS: A panel of experts from seven reference cytology centres was recruited. The study consisted of two consecutive rounds: (1) an open-ended, free-response questionnaire generating a list of survey items; and (2) a consensus analysis of 80 selected shared WSIs from 80 cases by six investigators answering six morphological questions utilising a 1 to 5 Likert scale. RESULTS: High consensus was achieved for all parameters, with an overall average score of 4.27. The broad majority of items (84%) were ranked either 4 or 5 by each physician. Two badly scanned cases were responsible for more than half of the low-ranked (≤2) values (57%). Good to excellent (≥3) diagnostic confidence was reached in more than 95.2% of cases. For most cases (78%) WSI assessment was not limited by technical issues linked to the image acquisition process. CONCLUSION: This systematic Delphi study indicates broad consensus among participating physicians on the application of DP to thyroid cytopathology, supporting expert opinion that WSI is reliable and safe for primary diagnostic purposes.

Evaluating local thyroid cytopathology practices by molecular quality metrics: A multi-institutional study on 4651 FNAs with a focus on the role of the interventional cytopathologist.

BACKGROUND: The diagnostic accuracy of thyroid fine-needle aspiration (FNA) can be highly influenced by the technical skills of the operator performing the procedure and by interobserver variability in microscopic interpretation. This is particularly true for the indeterminate categories. Recently, molecular testing has been proposed as an ancillary tool for monitoring the performance of different thyroid cytopathology practices. The objective of this multicenter study was to evaluate the quality of different local cytopathology practices by assessing the impact of interventional cytopathologists on FNA adequacy for molecular testing and the variations in mutation rates across different health care centers operating in the Campania region. METHODS: The study included 4651 thyroid FNA samples diagnosed in different Southern Italian clinical laboratories belonging to the TIRNET (the Tiroide Network). FNA samples were collected by different proceduralists and were classified by local cytopathologists according to The Bethesda System for Reporting Thyroid Cytopathology. FNAs classified as atypia of undetermined significance, follicular neoplasm, suspicious for malignancy, and malignant were centralized for a real-time polymerase chain reaction-based, seven-gene test at the authors' institution. RESULTS: Centers that employed interventional cytopathologists obtained fewer unsatisfactory FNA samples for molecular testing (11.3%) than centers that employed noncytopathologists (16.7%; p < .05). Furthermore, a significant variation in the mutation rate was observed in FNAs diagnosed by different local cytopathologists; indeterminate categories had the highest percentage of mutation rate variability among centers. CONCLUSIONS: Interventional cytopathologists obtained higher yields of diagnostic material for molecular testing. Finally, the current results suggest that the variability in mutation rates among different centers may highlight the low reproducibility of microscopic criteria among cytopathologists, particularly for indeterminate cases.

Spatially Resolved Molecular Approaches for the Characterisation of Non-Invasive Follicular Tumours with Papillary-like Features (NIFTPs).

Noninvasive follicular thyroid neoplasms with papillary-like nuclear features (NIFTP) are low-risk thyroid lesions most often characterised by RAS-type mutations. The histological diagnosis may be challenging, and even immunohistochemistry and molecular approaches have not yet provided conclusive solutions. This study characterises a set of NIFTPs by Matrix-Assisted Laser Desorption/Ionisation (MALDI)-Mass Spectrometry Imaging (MSI) to highlight the proteomic signatures capable of overcoming histological challenges. Archived formalin-fixed paraffin-embedded samples from 10 NIFTPs (n = 6 RAS-mutated and n = 4 RAS-wild type) were trypsin-digested and analysed by MALDI-MSI, comparing their profiles to normal tissue and synchronous benign nodules. This allowed the definition of a four-peptide signature able to distinguish RAS-mutant from wild-type cases, the latter showing proteomic similarities to hyperplastic nodules. Moreover, among the differentially expressed signals, Peptidylprolyl Isomerase A (PPIA, 1505.8 m/z), which has already demonstrated a role in the development of cancer, was found overexpressed in NIFTP RAS-mutated nodules compared to wild-type lesions. These results underlined that high-throughput proteomic approaches may add a further level of biological comprehension for NIFTPs. In the future, thanks to the powerful single-cell detail achieved by new instruments, the complementary NGS-MALDI imaging sequence might be the correct methodological approach to confirm that the current NIFTP definition encompasses heterogeneous lesions that must be further characterised.

Type 2 Deiodinase Thr92Ala Polymorphism and Aging Are Associated with a Decreased Pituitary Sensitivity to Thyroid Hormone.

Background: The DIO2 Thr92Ala polymorphism (rs225014), which occurs in about 15-30% of Caucasian people, determines a less efficient type 2 deiodinase (D2) enzyme. The aim of this study was to determine the impact of DIO2 Thr92Ala polymorphism on the serum thyrotropin (TSH) levels in thyroidectomized patients with hypothyroidism and to evaluate whether TSH levels and aging could be related, at pituitary level, to D2 activity. Methods: This prospective study was performed on 145 thyroid cancer patients, treated with total thyroidectomy, and undergoing radioiodine treatment after 3 weeks of levothyroxine (LT4) withdrawal. A mouse model has been used to determine D2 protein and mRNA levels in pituitary during aging. Results: Genetic analysis identified DIO2 Thr92Ala polymorphism in 56% of participants: 64/145 (44%) patients were homozygous wild type (WT) (Thr/Thr), 64 (44%) heterozygous (Thr/Ala), and 17 (12%) homozygous mutant (Ala/Ala). A significant negative relationship was observed between aging and the rise in serum TSH levels during LT4 withdrawal. However, this negative correlation found in WT was reduced in heterozygous and lost in mutant homozygous patients (Thr/Thr r = -0.45, p = 0.0002, 95% confidence interval [CI] -0.63 to -0.23; Ala/Thr r = -0.39, p = 0.0012, CI -0.60 to -0.67; and Ala/Ala r = -0.30, p = 0.2347; CI -0.70 to 0.20). Accordingly, when we compared the TSH measured in each patient to its theoretical value predicted from age, the TSH did not reach its putative target in 47% of WT patients, in 70% of Ala/Thr, and 76% of Ala/Ala carrying patients (p = 0.0036). This difference was lost in individuals older than 60 years, suggesting a decline of D2 associated with aging. The hypothesis that the pituitary D2 decreases with age was confirmed by the evidence that D2 mRNA and protein levels were lower in pituitary from old versus young mice. Conclusion: An age-related decline in TSH production in response to hypothyroidism was correlated with decreased D2 levels in pituitary. The presence of DIO2 homozygous Ala/Ala polymorphism was associated with a reduced level of TSH secretion in response to hypothyroidism, indicating a decreased pituitary sensitivity to serum thyroxine variation (Institutional Research Ethics board approval number no. 433/21).

Multiple predictive biomarker testing in melanoma: Another challenge in identifying the optimal approach on cytological samples.

BACKGROUND: The management of cutaneous melanoma has changed dramatically in recent years thanks to the development of tyrosine kinase and immune-checkpoint inhibitors (ICIs). Thus, multiple biomarker testing is becoming ever more important for the identification of patients who are potentially eligible for these treatments. One reliable approach to the molecular evaluation of metastatic melanoma is fine needle cytology (FNC). To examine the utility of this approach for assessing PD-L1 expression levels, we evaluated the cellular adequacy of residual cell block (CB) material from metastatic melanomas that were previously tested for BRAF and NRAS mutations. METHODS: We retrieved from our internal archives a series of FNC samples of metastatic melanoma that had been subjected to molecular testing on residual CB material or a dedicated needle rinse between January 2016 and July 2022. Real-time polymerase chain reaction was used to assess BRAF and NRAS status, and an SP263 assay was employed to ascertain PD-L1 expression levels. RESULTS: Overall, n = 19 cases were selected. Of these, 11 (57.9%) cases revealed a BRAF exon 15 p.V600E mutation, one case (5.3%) revealed NRAS mutation, and seven cases (36.8%) showed no mutations. Regarding PD-L1 assessment, 16/19 (84.2%) cases were deemed adequate, meaning they contained at least 100 viable cells. CONCLUSIONS: We highlighted the feasibility of assessing PD-L1 expression levels in residual CB material from metastatic melanomas previously tested for BRAF and NRAS mutations. Moreover, we pointed out that FNC needle rinses may be an alternative source of nucleic acids for molecular testing, preserving CB material for immunocytochemistry evaluation.

Reference standards for gene fusion molecular assays on cytological samples: an international validation study.

AIMS: Gene fusions assays are key for personalised treatments of advanced human cancers. Their implementation on cytological material requires a preliminary validation that may make use of cell line slides mimicking cytological samples. In this international multi-institutional study, gene fusion reference standards were developed and validated. METHODS: Cell lines harbouring EML4(13)-ALK(20) and SLC34A2(4)-ROS1(32) gene fusions were adopted to prepare reference standards. Eight laboratories (five adopting amplicon-based and three hybridisation-based platforms) received, at different dilution points two sets of slides (slide A 50.0%, slide B 25.0%, slide C 12.5% and slide D wild type) stained by Papanicolaou (Pap) and May Grunwald Giemsa (MGG). Analysis was carried out on a total of 64 slides. RESULTS: Four (50.0%) out of eight laboratories reported results on all slides and dilution points. While 12 (37.5%) out of 32 MGG slides were inadequate, 27 (84.4%) out of 32 Pap slides produced libraries adequate for variant calling. The laboratories using hybridisation-based platforms showed the highest rate of inadequate results (13/24 slides, 54.2%). Conversely, only 10.0% (4/40 slides) of inadequate results were reported by laboratories adopting amplicon-based platforms. CONCLUSIONS: Reference standards in cytological format yield better results when Pap staining and processed by amplicon-based assays. Further investigation is required to optimise these standards for MGG stained cells and for hybridisation-based approaches.

Relevance of the College of American Pathologists guideline for validating whole slide imaging for diagnostic purposes to cytopathology.

Whole slide imaging (WSI) allows pathologists to view virtual versions of slides on computer monitors. With increasing adoption of digital pathology, laboratories have begun to validate their WSI systems for diagnostic purposes according to reference guidelines. Among these the College of American Pathologists (CAP) guideline includes three strong recommendations (SRs) and nine good practice statements (GPSs). To date, the application of WSI to cytopathology has been beyond the scope of the CAP guideline due to limited evidence. Herein we systematically reviewed the published literature on WSI validation studies in cytology. A systematic search was carried out in PubMed-MEDLINE and Embase databases up to November 2021 to identify all publications regarding validation of WSI in cytology. Each article was reviewed to determine if SRs and/or GPSs recommended by the CAP guideline were adequately satisfied. Of 3963 retrieved articles, 25 were included. Only 4/25 studies (16%) satisfied all three SRs, with only one publication (1/25, 4%) fulfilling all three SRs and nine GPSs. Lack of a suitable validation dataset was the main missing SR (16/25, 64%) and less than a third of the studies reported intra-observer variability data (7/25, 28%). Whilst the CAP guideline for WSI validation in clinical practice helped the widespread adoption of digital pathology, more evidence is required to routinely employ WSI for diagnostic purposes in cytopathology practice. More dedicated validation studies satisfying all SRs and/or GPSs recommended by the CAP are needed to help expedite the use of WSI for primary diagnosis in cytopathology.

Microsatellite instability evaluation of patients with solid tumour: routine practice insight from a large series of Italian referral centre.

DNA mismatch repair complex is involved in the maintenance of DNA stability. In the recent years, a plethora of technical approaches for microsatellite instability (MSI) analysis emerged. Here, we review the results of our MSI status evaluation by adopting a customised workflow on microfluidic system obtained in 4 years of diagnostic routine practice. Data from MSI status were retrieved from our institutional archive covering the period from January 2017 to December 2021. Microfluidic analysis was carried out on microfluidic platform. Results were inspected with a proprietary software. Overall, microsatellite stability (MSS) and MSI-high (MSI-H) profile was detected in n=423/458 (92.36%) and n=35/458 (7.64%) patients with metastatic CRC (mCRC), respectively. In addition, n=78/86 (90.70%) and n=8/86 (9.30%) patients without CRC showed an MSS and MSI-H profile. This review highlights the suitability of microfluidic approach in patients with cancer for MSI testing.

Thyroid Cancer and Fibroblasts.

Thyroid cancer is the most common type of endocrine cancer, and its prevalence continue to rise. Non-metastatic thyroid cancer patients are successfully treated. However, looking for new therapeutic strategies is of great importance for metastatic thyroid cancers that still lead to death. With respect to this, the tumor microenvironment (TME), which plays a key role in tumor progression, should be considered as a new promising therapeutic target to hamper thyroid cancer progression. Indeed, thyroid tumors consist of cancer cells and a heterogeneous and ever-changing niche, represented by the TME, which contributes to establishing most of the features of cancer cells. The TME consists of extracellular matrix (ECM) molecules, soluble factors, metabolites, blood and lymphatic tumor vessels and several stromal cell types that, by interacting with each other and with tumor cells, affect TME remodeling, cancer growth and progression. Among the thyroid TME components, cancer-associated fibroblasts (CAFs) have gained more attention in the last years. Indeed, recent important evidence showed that thyroid CAFs strongly sustain thyroid cancer growth and progression by producing soluble factors and ECM proteins, which, in turn, deeply affect thyroid cancer cell behavior and aggressiveness. Hence, in this article, we describe the thyroid TME, focusing on the desmoplastic stromal reaction, which is a powerful indicator of thyroid cancer progression and an invasive growth pattern. In addition, we discuss the origins and features of the thyroid CAFs, their influence on thyroid cancer growth and progression, their role in remodeling the ECM and their immune-modulating functions. We finally debate therapeutic perspectives targeting CAFs.

A roadmap for a comprehensive diagnostic approach to fine needle cytology of lymph node metastases.

OBJECTIVE: Fine needle cytology (FNC) is widely used as a first-line procedure in the diagnostic algorithm of lymphadenopathies. In a metastatic setting, a first-line diagnostic approach identifies non-haematopoietic malignancy; however, cytopathologists could also provide a second diagnostic level, identifying the origin of the primary tumour. This paper outlines a comprehensive and practical approach to the cytological diagnosis of lymph node metastases. METHODS: Cytological diagnoses of lymph node metastases performed over a 10-year period were selected and divided into two groups. The first group, labelled "oncological," comprised patients with a previous history of malignancy; the second group, labelled "naïve," included patients with no relevant history. Pathology records were retrieved to record microscopic findings, namely, background appearance, group architecture, and specific cell features; data from cell block (CB) preparations were also collected. RESULTS: Overall, 982 cases were selected: 497 cases (50.61%) in the naïve group, and 485 (49.39%) in the oncological group. Overall, a second diagnostic level was achieved in 834/982 cases (84.92%); cases diagnosed as carcinoma not otherwise specified were more frequent in the naïve group than in the oncological group (17.51% vs. 8.04%, P < 0.01). Notably, although CB material was available in only 44.87% of the naïve cases, we were able to achieve a second diagnostic level thanks to the integration of clinical and cytomorphological findings, plus lymph node topography, in 82.49% of the cases. CONCLUSION: Our results confirmed that in a metastatic setting, FNC can reliably lead to the identification of the origin of the primary tumour.

Evaluation of the Molecular Landscape in PD-L1 Positive Metastatic NSCLC: Data from Campania, Italy.

BACKGROUND: Immune-checkpoint inhibitors (ICIs) have increased and improved the treatment options for patients with non-oncogene-addicted advanced stage non-small cell lung cancer (NSCLC). However, the role of ICIs in oncogene-addicted advanced stage NSCLC patients is still debated. In this study, in an attempt to fill in the informational gap on the effect of ICIs on other driver mutations, we set out to provide a molecular landscape of clinically relevant oncogenic drivers in programmed death-ligand 1 (PD-L1) positive NSCLC patients. METHODS: We retrospectively reviewed data on 167 advanced stage NSCLC PD-L1 positive patients (≥1%) who were referred to our clinic for molecular evaluation of five driver oncogenes, namely, EGFR, KRAS, BRAF, ALK and ROS1. RESULTS: Interestingly, n = 93 (55.7%) patients showed at least one genomic alteration within the tested genes. Furthermore, analyzing a subset of patients with PD-L1 tumor proportion score (TPS) ≥ 50% and concomitant gene alterations (n = 8), we found that n = 3 (37.5%) of these patients feature clinical benefit with ICIs administration, despite the presence of a concomitant KRAS gene alteration. CONCLUSIONS: In this study, we provide a molecular landscape of clinically relevant biomarkers in NSCLC PD-L1 positive patients, along with data evidencing the clinical benefit of ICIs in patient NSCLC PD-L1 positive alterations.

RNA-based next-generation sequencing in non-small-cell lung cancer in a routine setting: an experience from an Italian referral center.

Aim: ALK, ROS1, NTRK and RET gene fusions and MET exon 14 skipping alterations represent novel predictive biomarkers for advanced non-small-cell lung cancer (NSCLC). Therefore, testing patients for these genetic variants is crucial for choosing the best selective treatment. Over the last couple of decades, next-generation sequencing (NGS) platforms have emerged as an extremely useful tool for detecting these variants. Materials & methods: In the present study, we report our NGS molecular records produced during a year of diagnostic activity. Results: Overall, our in-house developed NGS workflow successfully analyzed n = 116/131 (88.5%) NSCLC samples. Of these, eight (6.8%) and five (4.3%) out of 116 patients harbored ALK and RET gene rearrangements, respectively: one case harbored ROS1 gene fusion (0.7%). Conclusion: Our results highlight that an RNA-based NGS analysis can reliably detect gene fusion alterations, thereby playing a pivotal role in the management of NSCLC patients.

Liquid Biopsy in Prostate Cancer Management-Current Challenges and Future Perspectives.

Although appreciable attempts in screening and diagnostic approaches have been achieved, prostate cancer (PCa) remains a widespread malignancy, representing the second leading cause of cancer-related death in men. Drugs currently used in PCa therapy initially show a potent anti-tumor effect, but frequently induce resistance and PCa progresses toward metastatic castration-resistant forms (mCRPC), virtually incurable. Liquid biopsy has emerged as an attractive and promising strategy complementary to invasive tissue biopsy to guide PCa diagnosis and treatment. Liquid biopsy shows the ability to represent the tumor microenvironment, allow comprehensive information and follow-up the progression of the tumor, enabling the development of different treatment strategies as well as permitting the monitoring of therapy response. Liquid biopsy, indeed, is endowed with a significant potential to modify PCa management. Several blood biomarkers could be analyzed for diagnostic, prognostic and predictive purposes, including circulating tumor cells (CTCs), extracellular vesicles (EVs), circulating tumor DNA (ctDNA) and RNA (ctRNA). In addition, several other body fluids may be adopted (i.e., urine, sperm, etc.) beyond blood. This review dissects recent advancements and future perspectives of liquid biopsies, highlighting their strength and weaknesses in PCa management.

MicroRNA profiling predicts positive nodal status in papillary thyroid carcinoma in the preoperative setting.

BACKGROUND: The molecular characterization of thyroid nodules in cytological samples has so far been focused on discriminating between benign and malignant forms in a purely diagnostic setting. The evidence on the impact of molecular biomarkers to determine the risk of aggressiveness in cytologically "neoplastic" lesions is limited to genomic alterations (such as BRAF and TERT mutations). The aim of our study was to assess the preoperative role of microRNAs (miRNAs) in predicting the nodal status of patients with papillary thyroid cancer. METHODS: A pilot series of histological samples of papillary thyroid carcinoma with (6 cases) or without (6 cases) lymph node metastases, matched for other major clinical and pathological features, was analyzed for global miRNA expression in a screening phase. A set of miRNAs was then validated in a series of 63 consecutive cytological samples of papillary carcinomas: 48 pN-negative and 15 pN-positive at histology. RESULTS: Unsupervised cluster analysis segregated surgical pN-negative and pN-positive samples, except for 1 case. The 45 differentially expressed miRNAs in pN-positive versus pN-negative cases were predicted to regulate a wide range of cellular pathways, enriched for Wnt, gonadotropin-releasing hormone receptor, and cerulein/cholecystokinin receptor signaling. In agreement with their profiles in surgical samples, 4 miRNAs of the 10 selected for validation (miR-154-3p, miR-299-5p, miR-376a-3p, and miR-302E) had a significant differential expression in cytological samples of papillary carcinoma with lymph node metastases and predicted the positive nodal status with a relatively good performance. CONCLUSIONS: MiRNA profiling is a potential promising strategy to define papillary carcinoma aggressiveness in the preoperative setting.