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.

Enhancing PET Degrading Enzymes: A Combinatory Approach.

Plastic waste has become a substantial environmental issue. A potential strategy to mitigate this problem is to use enzymatic hydrolysis of plastics to depolymerize post-consumer waste and allow it to be reused. Over the last few decades, the use of enzymatic PET-degrading enzymes has shown promise as a great solution for creating a circular plastic waste economy. PsPETase from Piscinibacter sakaiensis has been identified as an enzyme with tremendous potential for such applications. But to improve its efficiency, enzyme engineering has been applied aiming at enhancing its thermal stability, enzymatic activity, and ease of production. Here, we combine different strategies such as structure-based rational design, ancestral sequence reconstruction and machine learning to engineer a more highly active Combi-PETase variant with a melting temperature of 70 °C and optimal performance at 60 °C. Furthermore, this study demonstrates that these approaches, commonly used in other works of enzyme engineering, are most effective when utilized in combination, enabling the improvement of enzymes for industrial applications.

rs822336 binding to C/EBPß and NFIC modulates induction of PD-L1 expression and predicts anti-PD-1/PD-L1 therapy in advanced NSCLC.

Efficient predictive biomarkers are needed for immune checkpoint inhibitor (ICI)-based immunotherapy in non-small cell lung cancer (NSCLC). Testing the predictive value of single nucleotide polymorphisms (SNPs) in programmed cell death 1 (PD-1) or its ligand 1 (PD-L1) has shown contrasting results. Here, we aim to validate the predictive value of PD-L1 SNPs in advanced NSCLC patients treated with ICIs as well as to define the molecular mechanisms underlying the role of the identified SNP candidate. rs822336 efficiently predicted response to anti-PD-1/PD-L1 immunotherapy in advanced non-oncogene addicted NSCLC patients as compared to rs2282055 and rs4143815. rs822336 mapped to the promoter/enhancer region of PD-L1, differentially affecting the induction of PD-L1 expression in human NSCLC cell lines as well as their susceptibility to HLA class I antigen matched PBMCs incubated with anti-PD-1 monoclonal antibody nivolumab. The induction of PD-L1 expression by rs822336 was mediated by a competitive allele-specificity binding of two identified transcription factors: C/EBPß and NFIC. As a result, silencing of C/EBPß and NFIC differentially regulated the induction of PD-L1 expression in human NSCLC cell lines carrying different rs822336 genotypes. Analysis by binding microarray further validated the competitive allele-specificity binding of C/EBPß and NFIC to PD-L1 promoter/enhancer region based on rs822336 genotype in human NSCLC cell lines. These findings have high clinical relevance since identify rs822336 and induction of PD-L1 expression as novel biomarkers for predicting anti-PD-1/PD-L1-based immunotherapy in advanced NSCLC patients.

Benchmarking digital displays (monitors) for histological diagnoses: the nephropathology use case.

AIM: The digital transformation of the pathology laboratory is being continuously sustained by the introduction of innovative technologies promoting whole slide image (WSI)-based primary diagnosis. Here, we proposed a real-life benchmark of a pathology-dedicated medical monitor for the primary diagnosis of renal biopsies, evaluating the concordance between the 'traditional' microscope and commercial monitors using WSI from different scanners. METHODS: The College of American Pathologists WSI validation guidelines were used on 60 consecutive renal biopsies from three scanners (Aperio, 3DHISTECH and Hamamatsu) using pathology-dedicated medical grade (MG), professional grade (PG) and consumer-off-the-shelf (COTS) monitors, comparing results with the microscope diagnosis after a 2-week washout period. RESULTS: MG monitor was faster (1090 vs 1159 vs 1181 min, delta of 6-8%, p<0.01), with slightly better performances on the detection of concurrent diseases compared with COTS (κ=1 vs 0.96, 95% CI=0.87 to 1), but equal concordance to the commercial monitors on main diagnosis (κ=1). Minor discrepancies were noted on specific scores/classifications, with MG and PG monitors closer to the reference report (r=0.98, 95% CI=0.83 to 1 vs 0.98, 95% CI=0.83 to 1 vs 0.91, 95% CI=0.76 to 1, κ=0.93, 95% CI=077 to 1 vs 0.93, 95% CI=0.77 to 1 vs 0.86, 95% CI=0.64 to 1, κ=1 vs 0.50, 95% CI=0 to 1 vs 0.50, 95% CI=0 to 1, for IgA, antineutrophilic cytoplasmic antibody and lupus nephritis, respectively). Streamlined Pipeline for Amyloid detection through congo red fluorescence Digital Analysis detected amyloidosis on both monitors (4 of 30, 13% cases), allowing detection of minimal interstitial deposits with slight overestimation of the Amyloid Score (average 6 vs 7). CONCLUSIONS: The digital transformation needs careful assessment of the hardware component to support a smart and safe diagnostic process. Choosing the display for WSI is critical in the process and requires adequate planning.

Advances in radiology and pathology of prostate cancer: a review for the pathologist.

Multiparametric magnetic resonance imaging (mpMRI) has improved systematic prostate biopsy procedures in the diagnosis of clinically significant prostate cancer (csPCa) by reducing the number of unnecessary biopsies; numerous level one evidence studies have confirmed the accuracy of MRI-targeted biopsy, but, still today, systematic prostate biopsy is recommended to reduce the 15-20% false negative rate of mpMRI. New advanced imaging has been proposed to detect suspicious lesions and perform targeted biopsies especially when mpMRI cannot be performed. Transrectal ultrasound (TRUS) modalities are emerging as methods with greater sensitivity and specificity for the detection of PCa compared to the traditional TRUS; these techniques include elastography and contrast-enhanced ultrasound, as well as improved B-mode and Doppler techniques. These modalities can be combined to define a novel ultrasound approach: multiparametric ultrasound (mpUS). More recently, micro-ultrasound (MicroUS) and prostate-specific membrane antigen (PSMA) positron emission tomography/computed tomography (PET/CT) have demonstrated to be sensitive for the detection of primary prostatic lesions resulting highly correlated with the aggressiveness of the primary prostatic tumor. In parallel, artificial intelligence is advancing and is set out to deeply change both radiology and pathology. In this study we address the role, advantages and shortcomings of novel imaging techniques for Pca, and discuss future directions including the applications of artificial intelligence-based techniques to imaging as well as histology. The significance of these findings for the practicing pathologist is discussed.

Digital odyssey: lessons learnt from a reverse transition from a digital to a manual pathology workflow.

In the fully digital Caltagirone pathology laboratory, a reverse shift from a digital to a manual workflow occurred due to a server outage in September 2023. Here, insights gained from this unplanned transition are explored. Surveying the affected pathologists and technicians revealed unanimous preferences for the time-saving and error-reducing capabilities of the digital methodology. Conversely, the return to manual methods highlighted increased dissatisfaction and reduced efficiency, emphasising the superiority of digital workflows. This case study underscores that transition challenges are not inherent to digital workflows but to transitioning itself, advocating for the adoption of digital technologies in all pathology practices.

Resident training in interventional pathology: Ultrasound-guided fine-needle aspiration and rapid on site evaluation-5 years of teaching experience in a single university hospital.

INTRODUCTION: Despite the established role of the interventional pathologist, their diagnostic performance is difficult to establish. At least in Spain training of pathology residents in ultrasound-guided interventional procedures for specimen collection is limited or absent in most institutions. We present our teaching experience in the instruction of ultrasound-guided fine-needle aspiration (FNA) to pathology residents in a tertiary-level hospital. MATERIALS AND METHODS: The training of pathology residents who rotated through the interventional unit of the pathology department and the application of ultrasound-guided FNA and rapid on-site evaluation (U-ROSE) was documented over 5 years. The training period was broken down into learning phases and included the number of ultrasound-guided FNA performed, anatomical location, and their diagnostic performance, among other aspects. RESULTS: Nineteen (19) pathology residents were trained in U-ROSE, and performed a total of 4003 procedures, with a mean of 211 per resident. In 53% of cases only one pass was required for an adequated sample. The specimen was diagnostic in more than 97% of cases. The most frequently sampled anatomical sites were the thyroid gland (n = 2347), followed by lymph node (n = 667), soft tissues (n = 663) and salivary glands (n = 322). CONCLUSION: The results support the training programme followed by pathology residents in learning U-ROSE, which is essential to lay the foundations for the future interventional pathologist.

Combined fine needle aspiration cytology and core needle biopsy in the same setting: A two-years' experience.

INTRODUCTION: Fine needle aspiration cytology (FNAC) combined with rapid on-site evaluation (ROSE) and ancillary techniques is an accurate diagnostic tool for many pathologies. However, in some cases, it may not be sufficient for actionable diagnoses or molecular testing, especially for cases that require large immunohistochemical panels or cases in which histological features are mandatory for the diagnosis. Core needle biopsy (CNB), on the contrary, provides samples that are suitable for histological features and sufficient for all ancillary studies. However, CNB is often performed by radiologists or clinicians without the direct participation of cytopathologists, which can lead to missed or delayed diagnoses. This study reports on the experience of combining FNAC and CNB performed in one setting by cytopathologists. The aim was to evaluate the impact of CNB on FNAC and the diagnostic efficiency of the combined procedures. MATERIALS AND METHODS: One hundred forty-two FNAC and CNB procedures performed in the same setting over a period of 2 years were analysed. The FNAC diagnoses were compared and integrated with the subsequent CNB diagnoses. The impact of CNB was categorized as follows: non-contributory, in cases of inadequate samples; confirmed, when the CNB and FNAC diagnoses were the same; improved, when the CNB diagnosis was consistent with the FNAC diagnosis and further specified the corresponding entity; allowed, when CNB produced a diagnosis that could not be reached by FNAC; changed, when the CNB changed the previous FNAC diagnosis. RESULTS: CNB confirmed the FNAC diagnosis in 40.1% of cases (n = 57/142). CNB improved the FNAC diagnosis in 47.2% of cases (n = 67/142). CNB allowed a diagnosis that could not be performed on FNAC in 2.1% of cases (n = 3/142). CNB changed a previous FNAC diagnosis in 2.1% of cases (n = 3/142). CNB was non-contributory in 8.4% of cases (n = 12/142). CNB produced a positive impact on the whole diagnostic procedure in 51.4% of total cases (n = 73/142). The combined FNAC and CNB resulted in actionable diagnoses in 91.5% of all cases (n = 130/142). A complete molecular assessment was successfully performed in 14.7% of cases (n = 21/142) utilizing either FNAC or CNB material. CONCLUSIONS: The combined use of FNAC and CNB in one setting improves the diagnostic accuracy of both procedures. This approach exploits the advantages of each procedure, enhancing the accuracy of the final diagnosis.

Changes in mucosal IgG4+- and IL-10+-cell frequencies in adults with eosinophilic esophagitis on a two-food elimination diet.

Eosinophilic esophagitis (EoE) is increasingly diagnosed in patients with dysphagia. Type-2 immunity can induce EoE histopathology via non-IgE-dependent mechanisms, possibly involving IgG4 and IL-10. To elucidate the contribution of this response to EoE pathogenesis, we examined its association with clinical and histologic endpoints in adult EoE patients given a two-food elimination diet. IgG4- and IL-10-expressing cells were counted in esophageal biopsies and serum food-specific IgG4 measured at baseline and follow-up. Variables were correlated with histologic measures of disease activity. Patients exhibited significant reduction in esophageal eosinophilia and overall histology. A significant decrease in IL-10+-cell frequencies correlated with histologic changes. In contrast, a decline in serum and esophageal IgG4, while substantial, did not correlate with IL-10+-cell frequencies or histologic parameters. These results suggest a critical role of IL-10 in EoE pathogenesis. Conversely, IgG4 expression, while reflecting exposure to food antigens, is not obviously related to EoE histopathology or IL-10 expression.

A Retrospective Multicenter Italian Analysis of Epidemiological, Clinical and Histopathological Features in a Sample of Patients with Acinic Cell Carcinoma of the Parotid Gland.

BACKGROUND: The acinic cell carcinoma (AciCC) of the parotid gland is a rare tumor with an indolent behavior; however, a subgroup of this tumor presents an aggressive behavior with a tendency to recur. The aim of this multicenter study was to identify and stratify those patients with AciCC at high risk of tumor recurrence. METHODS: A retrospective study was carried out involving 77 patients treated with surgery between January 2000 and September 2022, in different Italian referral centers. Data about tumor characteristics and its recurrence were collected. The histological specimens and slides were independently reviewed by a senior pathologist coordinator (L.C.) and the institution's local head and neck pathologist. RESULTS: The patients' age average was 53.6 years, with a female prevalence in the group. The mean follow-up was 67.4 months (1-258, SD 59.39). The five-year overall survival (OS) was 83.2%. The 5-year disease-free survival (DFS) was 60% (95% CI 58.2-61.7). A high incidence of necrosis, extraglandular spread, lymphovascular invasion (LVI), atypical mitosis, and cellular pleomorphism was observed in the high-risk tumors compared to the low-risk ones. CONCLUSION: AciCC generally had an indolent behavior, optimal OS, DFS with few cervical node metastases, and rare distant relapses. This multicenter retrospective case series provides evidence of the need for clinical-epidemiological-histological stratification for patients at risk of poor outcomes. Our results suggest that the correct definition of high-risk AciCC should include tumor size, the presence of necrosis, extraglandular spread, LVI, atypical mitosis, and cellular pleomorphism.

A quinolin-8-ol sub-millimolar inhibitor of UGGT, the ER glycoprotein folding quality control checkpoint.

Misfolded glycoprotein recognition and endoplasmic reticulum (ER) retention are mediated by the ER glycoprotein folding quality control (ERQC) checkpoint enzyme, UDP-glucose glycoprotein glucosyltransferase (UGGT). UGGT modulation is a promising strategy for broad-spectrum antivirals, rescue-of-secretion therapy in rare disease caused by responsive mutations in glycoprotein genes, and many cancers, but to date no selective UGGT inhibitors are known. The small molecule 5-[(morpholin-4-yl)methyl]quinolin-8-ol (5M-8OH-Q) binds a CtUGGTGT24 "WY" conserved surface motif conserved across UGGTs but not present in other GT24 family glycosyltransferases. 5M-8OH-Q has a 47 µM binding affinity for CtUGGTGT24in vitro as measured by ligand-enhanced fluorescence. In cellula, 5M-8OH-Q inhibits both human UGGT isoforms at concentrations higher than 750 µM. 5M-8OH-Q binding to CtUGGTGT24 appears to be mutually exclusive to M5-9 glycan binding in an in vitro competition experiment. A medicinal program based on 5M-8OH-Q will yield the next generation of UGGT inhibitors.

Upper Esophageal Sphincter Metrics across Eosinophilic Esophagitis, Gastroesophageal Reflux Disease and Functional Dysphagia: A Pilot Study.

BACKGROUND: Recent studies have evaluated the upper esophageal sphincter (UES) with high-resolution manometry (HRM) in some esophageal diseases, but not eosinophilic esophagitis (EoE). The aim of our study was to evaluate the function of the UES across EoE, gastroesophageal reflux disease (GERD), functional dysphagia (FD), and the relationship with esophageal symptoms, esophageal body contraction, and esophagogastric junction (EGJ) metrics. METHODS: HRM was performed on 30 EoE, 18 GERD, and 29 FD patients according to the Chicago Classification 3.0. The study data were exported to the online analysis platform Swallow Gateway. The UES was assessed in terms of UES Resting Pressure (UES-RP), UES Basal Pressure (UES-BP), UES Integrated Relaxation Pressure (UES-IRP), UES Relaxation Time (UES-RT), Basal UES Contractile Integral (Basal UES-CI), Post-Deglutitive UES Contractile Integral (Post-Deglutitive UES-CI), and Proximal Contractile Integral (PCI). RESULTS: ANOVA analysis showed significantly higher values of Post-Deglutitive UES-CI in EoE patients compared with FD patients (p = 0.001). Basal UES-CI and UES-RP showed significantly higher values in EoE (p = 0.002, p = 0.038) and GERD (p < 0.001, p = 0.001) patients compared with FD patients. Correlations between LES-CI and Post-Deglutitive UES-CI, Basal UES-CI, and UES-RP (p ≤ 0.001, p = 0.027, p = 0.017, respectively), and between LES-BP and Post-Deglutitive UES-CI (p = 0.019), independent of diagnosis, were shown. No correlations have been demonstrated between the UES, EGJ metrics, and esophageal symptoms. CONCLUSIONS: Some differences in UES metrics in the three different diseases were found. Further studies are needed to confirm the results of our pilot study and possible applications in clinical practice.

Digital pathology world tour.

Objective: Digital pathology (DP) is currently in the spotlight and is rapidly gaining ground, even though the history of this field spans decades. Despite great technological progress, the adoption of DP for routine clinical diagnostic use remains limited. Methods: A systematic search was conducted in the electronic databases Pubmed-MEDLINE and Embase. Inclusion criteria were all published studies that encompassed any application of DP. Results: Of 4888 articles retrieved, 4041 were included. Relevant articles were categorized as "diagnostic" (147/4041, 4%) where DP was utilized for routine diagnostic workflow and "non-diagnostic" (3894/4041, 96%) for all other applications. The "non-diagnostic" articles were further categorized according to DP application including "artificial intelligence" (33%), "education" (5%), "narrative" (17%) for reviews and editorials, and "technical" (45%) for pure research publications. Conclusion: This manuscript provided temporal and geographical insight into the global adoption of DP by analyzing the published scientific literature.

Cytopathological differential diagnosis of malignant tumor cells in the cerebrospinal fluid: A retrospective analysis.

Leptomeningeal metastasis is the spread of cancer to the leptomeninges and subarachnoid space and represents a dreadful complication of cancer. The most commonly responsible neoplasms are high-grade lymphomas, leukemias, and some solid tumors, chiefly breast and lung cancer as well as melanoma. Herein we report our ten-year retrospective experience on 715 cases of cerebrospinal fluid cytology, 21 (2.9%) of which were positive for leptomeningeal metastasis. Sample collection and processing, clinical history, interdisciplinary dialog, and ancillary techniques such as immunocytochemistry and flow cytometry are all fundamental in reaching the correct diagnosis and thus optimally caring for patients with leptomeningeal metastasis.