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.

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.

The spectrum of the cytopathological features of primary effusion lymphoma and human herpes virus 8-related lymphoproliferative disorders.

INTRODUCTION: Human herpes virus 8-related lymphoproliferative disorders are a complex and heterogeneous group of entities and some of them are eminently diagnosed by cytopathology. In a routine laboratory, these lesions account for less than 1% of the effusion fluids samples. However, they represent up to 30% of all the lymphoma diagnosis from effusion cytological samples and their consideration in the diagnostic flow chart is mandatory, especially in human immunodeficiency virus-positive patients. METHODS: A retrospective series of cytological specimens from cavity effusions (n = 605) were analysed. Five human herpes virus 8-related lymphoproliferative processes were recruited. A combination of morphological criteria (enhanced with May-Grünwald Giemsa staining), cell block-based immunocytochemistry and flow cytometry were undertaken for final characterisation. RESULTS: The identification of malignant cells may be difficult. Some specimens are particularly rich, easily leading to suspect a lymphoproliferative process, whereas in other cases, the presence of abundant reactive mesothelial cells, histiocytes, neutrophils, small reactive T and B lymphocytes may obscure the neoplastic process. The biological behaviour may be very heterogeneous and a standardised therapy for these cases is still lacking, although some patients may benefit from antiretroviral therapy in a human immunodeficiency virus setting. CONCLUSIONS: The present case series highlights some characteristic findings of these entities to reaffirm useful cytopathological diagnostic criteria, stressing the crucial role of the appropriate technical processing of effusion fluids to obtain the best performances.

Digital counting of tissue cells for molecular analysis: the QuANTUM pipeline.

The estimation of tumor cellular fraction (TCF) is a crucial step in predictive molecular pathology, representing an entry adequacy criterion also in the next-generation sequencing (NGS) era. However, heterogeneity of quantification practices and inter-pathologist variability hamper the robustness of its evaluation, stressing the need for more reliable results. Here, 121 routine histological samples from non-small cell lung cancer (NSCLC) cases with complete NGS profiling were used to evaluate TCF interobserver variability among three different pathologists (pTCF), developing a computational tool (cTCF) and assessing its reliability vs ground truth (GT) tumor cellularity and potential impact on the final molecular results. Inter-pathologist reproducibility was fair to good, with overall Wk ranging between 0.46 and 0.83 (avg. 0.59). The obtained cTCF was comparable to the GT (p = 0.129, 0.502, and 0.130 for surgical, biopsies, and cell block, respectively) and demonstrated good reliability if elaborated by different pathologists (Wk = 0.9). Overall cTCF was lower as compared to pTCF (30 ± 10 vs 52 ± 19, p < 0.001), with more cases < 20% (17, 14%, p = 0.690), but none containing < 100 cells for the algorithm. Similarities were noted between tumor area estimation and pTCF (36 ± 29, p < 0.001), partly explaining variability in the human assessment of tumor cellularity. Finally, the cTCF allowed a reduction of the copy number variations (CNVs) called (27 vs 29, - 6.9%) with an increase of effective CNVs detection (13 vs 7, + 85.7%), some with potential clinical impact previously undetected with pTCF. An automated computational pipeline (Qupath Analysis of Nuclei from Tumor to Uniform Molecular tests, QuANTUM) has been created and is freely available as a QuPath extension. The computational method used in this study has the potential to improve efficacy and reliability of TCF estimation in NSCLC, with demonstrated impact on the final molecular results.

Economic assessment of NGS testing workflow for NSCLC in a healthcare setting.

Background: The molecular diagnostic and therapeutic pathway of Non-Small Cell Lung Cancer (NSCLC) stands as a successful example of precision medicine. The scarcity of material and the increasing number of biomarkers to be tested have prompted the routine application of next-generation-sequencing (NGS) techniques. Despite its undeniable advantages, NGS involves high costs that may impede its broad adoption in laboratories. This study aims to assess the detailed costs linked to the integration of NGS diagnostics in NSCLC to comprehend their financial impact. Materials and methods: The retrospective analysis encompasses 210 cases of early and advanced stages NSCLC, analyzed with NGS and collected at the IRCCS San Gerardo dei Tintori Foundation (Monza, Italy). Molecular analyses were conducted on FFPE samples, with an hotspot panel capable of detecting DNA and RNA variants in 50 clinically relevant genes. The economic analysis employed a full-cost approach, encompassing direct and indirect costs, overheads, VAT (Value Added Tax). Results: We estimate a comprehensive cost for each sample of €1048.32. This cost represents a crucial investment in terms of NSCLC patients survival, despite constituting only around 1% of the expenses incurred in their molecular diagnostic and therapeutic pathway. Conclusions: The cost comparison between NGS test and the notably higher therapeutic costs highlights that the diagnostic phase is not the limiting economic factor. Developing NGS facilities structured in pathology networks may ensure appropriate technical expertise and efficient workflows.

Artificial intelligence-based algorithms for the diagnosis of prostate cancer: A systematic review.

OBJECTIVES: The high incidence of prostate cancer causes prostatic samples to significantly affect pathology laboratories workflow and turnaround times (TATs). Whole-slide imaging (WSI) and artificial intelligence (AI) have both gained approval for primary diagnosis in prostate pathology, providing physicians with novel tools for their daily routine. METHODS: A systematic review according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines was carried out in electronic databases to gather the available evidence on the application of AI-based algorithms to prostate cancer. RESULTS: Of 6290 articles, 80 were included, mostly (59%) dealing with biopsy specimens. Glass slides were digitized to WSI in most studies (89%), roughly two-thirds of which (66%) exploited convolutional neural networks for computational analysis. The algorithms achieved good to excellent results about cancer detection and grading, along with significantly reduced TATs. Furthermore, several studies showed a relevant correlation between AI-identified histologic features and prognostic predictive variables such as biochemical recurrence, extraprostatic extension, perineural invasion, and disease-free survival. CONCLUSIONS: The published evidence suggests that AI can be reliably used for prostate cancer detection and grading, assisting pathologists in the time-consuming screening of slides. Further technologic improvement would help widening AI's adoption in prostate pathology, as well as expanding its prognostic predictive potential.

Pathology Laboratory Archives: Conservation Quality of Nucleic Acids and Proteins for NSCLC Molecular Testing.

In the molecular era, proper archival conditions within pathology laboratories are crucial, especially for formalin-fixed paraffin-embedded (FFPE) tissue specimens retrieved years after the original diagnosis. Indeed, improper preservation can impact the integrity of nucleic acids and protein antigens. This study evaluates the quality status of stored FFPE blocks using multilevel omics approaches. FFPE blocks from 45 Non-Small Cell Lung Carcinoma (NSCLC) cases were analyzed. The blocks were collected from six different pathology archives across Italy with distinct environmental characteristics. Nucleic acids' quantity and quality, as well as protein antigens, were assessed using various techniques, including MALDI-MSI. RNA was quantitatively higher, but more fragmented, compared to DNA. DNA quantity and quality were suitable for molecular analyses in 94.4% and 62.3% of samples, respectively. RNA quantity was adequate across all samples, but it was optimal only in 22.3% of cases. DNA quality started to deteriorate after 6-8 years, whereas RNA quality diminished only after 10 years of storage. These data might suggest a particular DNA susceptibility to FFPE blocks conservation. Immunohistochemical intensity decreased significantly after 6-8 years of storage, and MALDI-MSI analysis revealed that younger tissue blocks contained more unique proteomic signals than the older ones. This study emphasizes the importance of proper FFPE archiving conditions for molecular analyses. Governance should prioritize attention to pathology archives to ensure quality preservation and optimize predictive testing. By elucidating the nuances of FFPE block storage, this research paves the way for enhanced molecular diagnostics and therapeutic insights regarding oncology and beyond.

Cost analysis of archives in the pathology laboratories: from safety to management.

CONTEXT: Despite the reluctance to invest and the challenging estimation of necessary supporting costs, optimising the archives seems to be one of the hottest topics in the future management of the pathology laboratories. Historically, archives were only partially designed to securely store and organise tissue specimens, and tracking systems were often flawed, posing significant risks to patients' health and legal ramifications for pathologists. OBJECTIVE: The current review explores the available data from the literature on archives' management in pathology, including comprehensive business plans, structure setup, outfit, inventories, ongoing conservation and functional charges. DATA SOURCES: Electronic searches in PubMed-MEDLINE and Embase were made to extract pertinent articles from the literature. Works about the archiving process and storage were included and analysed to extract information. Prepublication servers were ignored. Italian Institutional Regional databases for public competitive bidding processes were queried too. CONCLUSIONS: A new emergent feeling in the pathology laboratory is growing for archives management; the digital pathology era is a great opportunity to apply innovation to tracking systems and samples preservation. The main aim is a critical evaluation of the return of investment in developing automatic and tracked archiving processes for improving not only quality, efficacy and efficiency of the labs but also patients' healthcare.

The Complex Cyto-Molecular Landscape of Thyroid Nodules in Pediatrics.

Thyroid fine-needle aspiration (FNA) is a commonly used diagnostic cytological procedure in pediatric patients for the evaluation of thyroid nodules, triaging them for the detection of thyroid cancer. In recent years, greater attention has been paid to thyroid FNA in this setting, including the use of updated ultrasound score algorithms to improve accuracy and yield, especially considering the theoretically higher risk of malignancy of these lesions compared with the adult population, as well as to minimize patient discomfort. Moreover, molecular genetic testing for thyroid disease is an expanding field of research that could aid in distinguishing benign from cancerous nodules and assist in determining their clinical management. Finally, artificial intelligence tools can help in this task by performing a comprehensive analysis of all the obtained data. These advancements have led to greater reliance on FNA as a first-line diagnostic tool for pediatric thyroid disease. This review article provides an overview of these recent developments and their impact on the diagnosis and management of thyroid nodules in children.

An Italian Multicenter Perspective Harmonization Trial for the Assessment of MET Exon 14 Skipping Mutations in Standard Reference Samples.

Lung cancer remains the leading cause of cancer deaths worldwide. International societies have promoted the molecular analysis of MET proto-oncogene, receptor tyrosine kinase (MET) exon 14 skipping for the clinical stratification of non-small cell lung cancer (NSCLC) patients. Different technical approaches are available to detect MET exon 14 skipping in routine practice. Here, the technical performance and reproducibility of testing strategies for MET exon 14 skipping carried out in various centers were evaluated. In this retrospective study, each institution received a set (n = 10) of a customized artificial formalin-fixed paraffin-embedded (FFPE) cell line (Custom METex14 skipping FFPE block) that harbored the MET exon 14 skipping mutation (Seracare Life Sciences, Milford, MA, USA), which was previously validated by the Predictive Molecular Pathology Laboratory at the University of Naples Federico II. Each participating institution managed the reference slides according to their internal routine workflow. MET exon 14 skipping was successfully detected by all participating institutions. Molecular analysis highlighted a median Cq cut off of 29.3 (ranging from 27.1 to 30.7) and 2514 (ranging from 160 to 7526) read counts for real-time polymerase chain reaction (RT-PCR) and NGS-based analyses, respectively. Artificial reference slides were a valid tool to harmonize technical workflows in the evaluation of MET exon 14 skipping molecular alterations in routine practice.

Lights on HBME-1: the elusive biomarker in thyroid cancer pathology.

Among the different ancillary immunohistochemical tools that pathologists may employ in thyroid nodules, the so-called Hector Battifora's 'MEsothelioma' 1 (HBME-1) staining is one of the most fascinating, since its real identity is currently unknown. In the present review, the different clinical applications of HBME-1 are analysed, with main emphasis on its role in thyroid pathology with overview on less impactful fields, such as haematopathology or mesothelial lesions. Different acceptable or good diagnostic performances were recorded for HBME-1 in thyroid pathology, being used in routine practice as one of the best tools to screen thyroid malignancy both in terms of sensitivity and specificity. From a speculative point of view, after many attempts to hunt the cryptic target antigen of this antibody, its identity still remains elusive. In this setting, the application of high-throughput technologies (mainly in situ proteomics) may be the exact route to improve the knowledge about the pathophysiology of HBME-1 and to finally unveil its true identity.

Estimating Fatty Pancreas-A Preoperative Bedside Assessment by Bioelectric Impedance Analysis: Implications for Pancreatic Surgery.

OBJECTIVE: The aim of the study was to evaluate whether fatty pancreas could be estimated by fat mass measurement by preoperative bioelectric impedance analysis. Preoperative computed tomography scan and pathologic evaluation were used as validation methods. Moreover, the 3 methodologies were tested for their ability in predicting postoperative pancreatic fistula. METHODS: Seventy-five patients who underwent pancreatic resection were analyzed. Preoperative computed tomography attenuation in Hounsfield unit (CT-HU) was used to assess fatty pancreas. Bioelectric impedance analysis was performed the day before surgery and fat mass index (FMI) was calculated. Pancreatic steatosis was assessed by pathologists at the line of surgical transection. The ability of the methods in predicting postoperative pancreatic fistula was evaluated by the area under the receiver operating characteristics curves. RESULTS: There was a strong correlation between CT-HU values and grade of pancreatic steatosis evaluated at histology ( r = -0.852, P < 0.001) and a moderate correlation between FMI and histologic pancreatic steatosis ( r = 0.612, P < 0.001) and between CT-HU value and FMI ( r = -0.659, P < 0.001) values. The area under the curve (95% confidence interval) was 0.942 (0.879-1) for histology, 0.924 (0.844-1) for CT-HU, and 0.884 (0.778-0.990) for FMI. CONCLUSIONS: Bioelectric impedance analysis represents a valid alternative to assess pancreatic steatosis.

An Indeterminate for Malignancy FNA Report Does Not Increase the Surgical Risk of Incidental Thyroid Carcinoma.

Incidental thyroid carcinomas (ITCs) are a fairly frequent finding in daily routine practice, with papillary thyroid microcarcinoma being the most frequent entity. In our work, we isolated incidental cases arising in thyroids removed for other cytologically indeterminate and histologically benign nodules. We retrospectively retrieved cases with available thyroid Fine Needle Aspiration (FNA, 3270 cases), selecting those with an indeterminate cytological diagnosis (Bethesda classes III−IV, 652 cases). Subsequently, we restricted the analysis to surgically treated patients (163 cases) finding an incidental thyroid carcinoma in 22 of them. We found a 13.5% ITC rate, with ITCs representing 46.8% of all cancer histologically diagnosed in this indeterminate setting. Patients received a cytological diagnosis of Bethesda class III and IV in 41% and 59% of cases, respectively. All ITC cases turned out to be papillary thyroid microcarcinomas; 36% of cases were multifocal, with foci bilaterally detected in 50% of cases. We found an overall ITC rate concordant with the literature and with our previous findings. The assignment of an indeterminate category to FNA did not increase the risk of ITCs in our cohort. Rather, a strong statistical significance (p < 0.01) was found comparing the larger size of nodules that underwent FNA and the smaller size of their corresponding ITC nodule.

Epidermal growth factor receptor exon 20 insertion variants in non-small cell lung cancer patients.

Epidermal growth factor receptor (EGFR) exon 20 insertions occur rarely among different cancer types, with the highest frequency reported among non-small-cell lung cancer (NSCLC) patients, particularly adenocarcinomas (ADCs). Exon 20 insertions fall back in the tyrosine kinase domain, and can be clustered into two principal groups represented by in frame insertions and three to 21 bp (corresponding to 1-7 amino acids) duplications within amino acids 762 and 774. The identification of these alterations is key for an adequate management of NSCLC patients due to the possibility to treat these patients with specific targeted therapies. Next generation sequencing (NGS) technology, able to detect several hotspot gene mutations for different patients simultaneously, is the best detection approach due to its higher sensitivity and specificity compared to other techniques. Here we reviewed the principal biological characteristics, the main detection technologies and treatment options for NSCLC patients harbouring EGFR exon 20 insertions.

Diagnostic Performances of the ACR-TIRADS System in Thyroid Nodules Triage: A Prospective Single Center Study.

Ultrasound scores are used to determine whether thyroid nodules should undergo Fine Needle Aspiration (FNA) or simple clinical follow-up. Different scores have been proposed for this task, with the American College of Radiology (ACR) TIRADS system being one of the most widely used. This study evaluates its ability in triaging thyroid nodules deserving FNA on a large prospective monocentric Italian case series of 493 thyroid nodules from 448 subjects. In ACR 1-2, cytology never prompted a surgical indication. In 59% of cases classified as TIR1c-TIR2, the FNA procedure could be ancillary, according to the ACR-TIRADS score. A subset (37.9%) of cases classified as TIR4-5 would not undergo FNA, according to the dimensional thresholds used by the ACR-TIRADS. Applying the ACR score, a total of 46.5% thyroid nodules should be studied with FNA. The ACR system demonstrated a sensitivity and specificity of 58.9% and 59% in the identification of patients with cytology ≥TIR3A, with a particularly high false negative rate for ACR classes ≥3 (44.8%, 43/96), which would dramatically decrease (7.3%, 7/96) if the dimensional criteria were not taken into account. In ACR 3-4-5, a correspondence with the follow-up occurred in 60.3%, 50.2% and 51.9% of cases. The ACR-TIRADS is a useful risk stratification tool for thyroid nodules, although the current dimensional thresholds could lead to an underestimation of malignant lesions. Their update might be considered in future studies to increase the screening performances of the system.

Use of Diagnostic Criteria from ACR and EU-TIRADS Systems to Improve the Performance of Cytology in Thyroid Nodule Triage.

Objective: The American College of Radiology (ACR) and the European Thyroid Association (EU) have proposed two scoring systems for thyroid nodule classification. Here, we compared the ability of the two systems in triaging thyroid nodules for fine-needle aspiration (FNA) and tested the putative role of an approach that combines ultrasound features and cytology for the detection of malignant nodules. Design and Methods: The scores obtained with the ACR and EU Thyroid Imaging Reporting and Data Systems (TIRADS) from a prospective series of 480 thyroid nodules acquired from 435 subjects were compared to assess their performances in FNA triaging on the final cytological diagnosis. The US features that showed the highest contribution in discriminating benign nodules from malignancies were combined with cytology to improve its diagnostic performance. Results: FNA was recommended on 46.5% and 51.9% of the nodules using the ACR and EU-TIRADS scores, respectively. The ACR system demonstrated a higher specificity as compared to the EU-TIRADS (59.0% vs. 52.4%, p = 0.0012) in predicting ≥ TIR3A/III (SIAPEC/Bethesda) nodules. Moreover, specific radiological features (i.e., echogenic foci and margins), combined with the cytological classes improved the specificity (97.5% vs. 91%, p < 0.0001) and positive predictive values (77.5% vs. 50.7%, p < 0.0001) compared to cytology alone, especially in the setting of indeterminate nodules (TIR3A/III and TIR3B/IV), maintaining an excellent sensitivity and negative predictive value. Conclusions: The ACR-TIRADS system showed a higher specificity compared to the EU-TIRADS in triaging thyroid nodules. The use of specific radiological features improved the diagnostic ability of cytology.

Aplastic anemia in a patient with advanced lung adenocarcinoma during first line osimertinib: A case report and literature review.

OBJECTIVES: Osimertinib is a third-generation, irreversible tyrosine kinase inhibitor of the epidermal growth factor receptor (EGFR-TKI) showing longer progression free survival and overall survival than other EGFR-TKI with an improvement in tolerability. MATERIALS AND METHODS: We report about an advanced lung adenocarcinoma patient with severe aplastic anemia during first line osimertinib. RESULTS AND CONCLUSION: Severe hematologic toxicity is extremely rare but possible with osimertinib and clinicians should be careful about changes in blood cell count during the use of it.