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.

Computer-assisted urine cytology: Faster, cheaper, better?

Recent advancements in computer-assisted diagnosis (CAD) have catalysed significant progress in pathology, particularly in the realm of urine cytopathology. This review synthesizes the latest developments and challenges in CAD for diagnosing urothelial carcinomas, addressing the limitations of traditional urinary cytology. Through a literature review, we identify and analyse CAD models and algorithms developed for urine cytopathology, highlighting their methodologies and performance metrics. We discuss the potential of CAD to improve diagnostic accuracy, efficiency and patient outcomes, emphasizing its role in streamlining workflow and reducing errors. Furthermore, CAD tools have shown potential in exploring pathological conditions, uncovering novel biomarkers and prognostic/predictive features previously unknown or unseen. Finally, we examine the practical issues surrounding the integration of CAD into clinical practice, including regulatory approval, validation and training for pathologists. Despite the promising results, challenges remain, necessitating further research and validation efforts. Overall, CAD presents a transformative opportunity to revolutionize diagnostic practices in urine cytopathology, paving the way for enhanced patient care and outcomes.

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.

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.

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.

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.

Juggling the COVID-19 pandemic: A cytopathology point of view.

Since its first identification in China at the end of 2019, severe acute respiratory syndrome coronavirus 2 has rapidly spread all over the world, becoming an international healthcare emergency. In the era of coronavirus disease-2019 (COVID-19), several aspects of normal life, including those related to the medical activities, have been radically changed. Extraordinary measures have been adopted by different nations to cope with the rapid diffusion of COVID-19 all over the world. In hospitals, careful attention has been paid to manage infected patients with a possible detrimental effect for patients affected by other diseases. As with other medical fields, cytopathology laboratories have also drastically modified their activities to cope with the COVID-19 healthcare emergency. Here, the main effects of COVID-19 pandemic on the routine practice of cytopathology are summarised, focusing on the prioritisation policy adopted by cytopathologists worldwide.

Next generation sequencing in cytology.

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.

[Liquid biopsy as indication of tumour progression in lung cancer: a potential intervention in tertiary prevention]. / La biopsia liquida quale segnale di progressione tumorale nel carcinoma polmonare: potenziale di intervento in prevenzione terziaria.

In the era of personalised therapies, liquid biopsy is considered an important diagnostic tool in the clinical management of cancer patients. Tissue specimen represents the gold standard for molecular evaluation of specific gene targets alterations that lead cancer patients to benefit of a "tailed therapy" based on molecular features of the tumour. This innovative source of nucleic acids was introduced in clinical setting only for non-small-cell lung cancer (NSCLC) patients to test epidermal grow factor receptor (EGFR) mutations when tissue is not available for a number of reasons (difficult access to the lesion, the presence of other disabling pathologies, especially in elderly patients, rejection by the patient, etcetera) or to monitor acquired resistance mutation after a first line of treatment. The present study aimed at assessing the diagnostic potential of liquid biopsy in balanced tertiary screening modelling. The cases relating to 5 years of activity regarding to molecular diagnostics performed on liquid biopsy specimens in the Predictive diagnostic laboratory of the University hospital "Federico II" of Naples (Campania Region, Southern Italy) were reviewed. Laboratory data were collected through the software SPSS. Non-parametric analysis was performed in order to test the differences between "wild type" patients or not. A multivariate logistic model was performed in order to assess the effect of mutation, age, and gender on the tumour progression. The results of the revision concern 515 total cases (almost of all plasma or peripheral blood), which allowed to evaluate the liquid biopsies for women and men. The average age of the patients is 66.3 years, and the 25° percentile is 59 years. The cases are: 221 basal and 294 by progression. The cases with mutation, as expected, have an odds ratio of 4,15, compared to the basal, to have a tumour progression (95%IC 2,7-6,3), regardless of gender and age. The detected mutations were 131 from different types of pulmonary carcinomas. Working on case data, specifying the characteristics of the patients with mutations will drive a further estimate in tertiary prevention screening designs.

Osimertinib.

Epidermal growth factor receptor (EGFR)-mutated (exons 18-21) advanced non-small cell lung cancers (NSCLCs) are generally characterized by exquisite sensitivity to treatment with an EGFR-tyrosine kinase inhibitor (-TKI). First-generation or reversible EGFR-TKIs include gefitinib and erlotinib, while, more recently, second-generation or irreversible EGFR-TKIs have been developed, namely afatinib and dacomitinib, with the aim of overcoming/delaying acquired resistance to treatment. Nevertheless, clinical trials have shown that resistance eventually emerges after a median time of slightly less than one year, regardless of whether first- or second-generation EGFR-TKIs are used. In this context, a secondary EGFR mutation in exon 20, namely T790M, has been found to be responsible for approximately 60% of cases of acquired resistance. Alternatively, T790M resistance mutation can be found de novo, in which case it limits the antitumor activity of both first- or second-generation EGFR-TKIs. Osimertinb is an orally bioavailable, third-generation EGFR-TKI that acts by irreversibly binding both EGFR activating mutations and T790M, while sparing wild-type EGFR. On this basis, osimertinib has proven more efficacious than platinum-based chemotherapy in the setting of EGFR T790M-positive NSCLCs pretreated with a first- or second-generation EGFR-TKI. More recently, in another phase 3 trial, osimertinib outperformed gefitinib or erlotinib as first-line treatment of EGFR-mutated (ex19del or L858R) advanced NSCLCs, thus emerging as a new standard of care in this setting. In the present review, we will discuss the preclinical and clinical development of osimertinib, briefly touching upon its activity in special populations and biomarkers of sensitivity to treatment.

Development of a gene panel for next-generation sequencing of clinically relevant mutations in cell-free DNA from cancer patients.

BACKGROUND: When tumour tissue is unavailable, cell-free DNA (cfDNA)can serve as a surrogate for genetic analyses. Because mutated alleles in cfDNA are usually below 1%, next-generation sequencing (NGS)must be narrowed to target only clinically relevant genes. In this proof-of-concept study, we developed a panel to use in ultra-deep sequencing to identify such mutations in cfDNA. METHODS: Our panel ('SiRe') covers 568 mutations in six genes (EGFR, KRAS, NRAS, BRAF, cKIT and PDGFRα)involved in non-small-cell lung cancer (NSCLC), gastrointestinal stromal tumour, colorectal carcinoma and melanoma. We evaluated the panel performance in three steps. First, we analysed its analytical sensitivity on cell line DNA and by using an artificial reference standard with multiple mutations in different genes. Second, we analysed cfDNA from cancer patients at presentation (n=42), treatment response (n=12) and tumour progression (n=11); all patients had paired tumour tissue and cfDNA previously genotyped with a Taqman-derived assay (TDA). Third, we tested blood samples prospectively collected from NSCLC patients (n=79) to assess the performance of SiRe in clinical practice. RESULTS: SiRe had a high analytical performance and a 0.01% lower limit of detection. In the retrospective series, SiRe detected 40 EGFR, 11 KRAS, 1 NRAS and 5 BRAF mutations (96.8% concordance with TDA). In the baseline samples, SiRe had 100% specificity and 79% sensitivity relative to tumour tissue. Finally, in the prospective series, SiRe detected 8.7% (4/46) of EGFR mutations at baseline and 42.9% (9/21) of EGFR p.T790M in patients at tumour progression. CONCLUSIONS: SiRe is a feasible NGS panel for cfDNA analysis in clinical practice.

Current role of cytopathology in the molecular and computational era: The perspective of young pathologists.

Cytopathology represents a well established diagnostic approach because of its limited cost, reliability, and minimal invasiveness with respect to other methodologies. The evolving complexity of the different classifications systems and the implementation of ancillary techniques to refine the diagnosis is progressively helping in the risk of malignancy stratification, and the adoption of next-generation sequencing techniques contributes to enrich this valuable tool with predictive information, which is always more essential in the tailored medicine era. The recent introduction of digital and computational pathology is further boosting the potentialities of cytopathology, aiding in the interpretation of samples to improve the cost effectiveness of large screening programs and the diagnostic efficiency within intermediate/atypical categories. Moreover, the adoption of artificial intelligence tools is promising to complement molecular investigations, representing a stimulating perspective in the cytopathology field. In this work, the authors tried to summarize the multifaceted nature of this complex and evolving field of pathology, synthesizing the most recent advances and providing the young pathologists' perspective on this fascinating world.

The role of liquid biopsy in epithelial ovarian cancer: State of the art.

The clinical implementation of liquid biopsy has dramatically modified the analytical paradigm for several solid tumors. To date, however, only circulating free DNA (cfDNA) has been approved in clinical practice to select targeted treatments for patients with colorectal cancer (CRC), non-small cell lung cancer (NSCLC), and breast cancer (BC). Interestingly, emerging liquid biopsy analytes in peripheral blood, including circulating tumor cells (CTC), miRNA, and extracellular vesicles (EVs), have been shown to play a crucial role in the clinical management of solid tumor patients. Here, we review how these blood-based biomarkers may positively impact early diagnosis, prognosis, and treatment response in ovarian cancer (OC) patients.

PIK3CA testing in hormone receptor-positive/HER2-negative metastatic breast cancer: real-world data from Italian molecular pathology laboratories.

Introduction: PIK3CA gene mutations occur in approximately 40% of hormone receptor-positive/HER2-negative (HR+/HER2-) metastatic breast cancers (MBCs), electing them to targeted therapy. Testing PIK3CA status is complex due to selection of biological specimen and testing method. Materials & methods: This work investigates real-life experience on PIK3CA testing in HR+/HER2- MBC. Clinical, technical and molecular data on PIK3CA testing were collected from two referral laboratories. Additionally, the results of a nationwide PIK3CA survey involving 116 institutions were assessed. Results: Overall, n = 35 MBCs were PIK3CA-mutated, with mutations mostly occurring in exons 9 (n = 19; 51.4%) and 20 (n = 15; 40.5%). The nationwide survey revealed significant variability across laboratories in terms of sampling methodology, technical assessment and clinical report signing healthcare figures for PIK3CA molecular testing in diagnostic routine practice. Conclusion: This study provides insights into the real-world routine of PIK3CA testing in HR+/HER2- MBC and highlights the need for standardization and networking in predictive pathology.

BAP1 Loss, Nuclear Grading, and Nonepithelioid Features in the Diagnosis of Mesothelioma in Italy: Nevermore without the Pathology Report.

The pathologic diagnosis of pleural mesothelioma is generally based on international guidelines, but no compulsory points based on different drugs approvals in different European countries are required to be reported. According to the last (2021) edition of the World Health Organization classification of pleural tumors, the nuclear grade of epithelioid-type mesothelioma should be always inserted in the pathologic report, while the presence of BRCA-associated protein-1 (BAP1) (clone C4) loss and a statement on the presence of the sarcomatoid/nonepithelioid component are fundamental for both a screening of patients with suspected BAP1 tumor predisposition syndrome and the eligibility to perform first-line immunotherapy at least in some countries. Several Italian experts on pleural mesothelioma who are deeply involved in national scientific societies or dedicated working groups supported by patient associations agreed that the pathology report of mesothelioma of the pleura should always include the nuclear grade in the epithelioid histology, which is an overt statement on the presence of sarcomatoid components (at least 1%, in agreement with the last classification of pleural mesothelioma) and the presence of BAP1 loss (BAP1-deficient mesothelioma) or not (BAP1-retained mesothelioma) in order to screen patients possibly harboring BAP1 tumor predisposition syndrome. This review aims to summarize the most recent data on these three important elements to provide evidence regarding the possible precision needs for mesothelioma.