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The 5th edition of the World Health Organization (WHO) classification of endocrine tumors was released in 2022. Several novelties have been introduced concerning the nomenclature and histopathological diagnosis of follicular-derived thyroid neoplasms. Tumor types have been sharply classified according to prognostic risk categories into benign tumors, low-risk neoplasms and malignant neoplasms. A grading system for differentiated thyroid carcinomas has been implemented with the aim of improving the stratification of tumors. Particular attention has been paid to the molecular profile of well-differentiated histotypes. In this review, the main changes introduced by the latest edition of the WHO system are presented. The practical effects on the diagnostic pathology of thyroid tumors, along with the clinical implications expected with the new classification scheme, are critically discussed.

Abstract Altered circulating hormone and metabolite levels have been reported during and post-COVID-19. Yet, studies of gene expression at the tissue level capable of identifying the causes of endocrine dysfunctions are lacking. Transcript levels of endocrine-specific genes were analyzed in five endocrine organs of lethal COVID-19 cases. Overall, 116 autoptic specimens from 77 individuals (50 COVID-19 cases and 27 uninfected controls) were included. Samples were tested for the SARS-CoV-2 genome. The adrenals, pancreas, ovary, thyroid, and white adipose tissue (WAT) were investigated. Transcript levels of 42 endocrine-specific and 3 interferon-stimulated genes (ISGs) were measured and compared between COVID-19 cases (virus-positive and virus-negative in each tissue) and uninfected controls. ISG transcript levels were enhanced in SARS-CoV-2-positive tissues. Endocrine-specific genes (e.g., HSD3B2, INS, IAPP, TSHR, FOXE1, LEP, and CRYGD) were deregulated in COVID-19 cases in an organ-specific manner. Transcription of organ-specific genes was suppressed in virus-positive specimens of the ovary, pancreas, and thyroid but enhanced in the adrenals. In WAT of COVID-19 cases, transcription of ISGs and leptin was enhanced independently of virus detection in tissue. Though vaccination and prior infection have a protective role against acute and long-term effects of COVID-19, clinicians must be aware that endocrine manifestations can derive from virus-induced and/or stress-induced transcriptional changes of individual endocrine genes. Key messages • SARS-CoV-2 can infect adipose tissue, adrenals, ovary, pancreas and thyroid. • Infection of endocrine organs induces interferon response. • Interferon response is observed in adipose tissue independently of virus presence. • Endocrine-specific genes are deregulated in an organ-specific manner in COVID-19. • Transcription of crucial genes such as INS, TSHR and LEP is altered in COVID-19.

The tall-cell variant of papillary thyroid carcinoma (TCPTC) is the most common aggressive variant of papillary thyroid carcinoma (PTC) and typically occurs in older patients. In this study, we analyzed retrospectively the largest mono-institutional series of PTCs with tall-cell features (989 patients) over a 17-year period, re-evaluating tumors based on age at presentation and outcomes in different age groups. We divided patients into three age groups following different criteria (the criterion from the American Joint Committee on Cancer Tumor Node Metastasis (AJCC TNM) guidelines, criterion for the statistical division into tertiles and adolescent/post-adolescent criterion) to analyze the clinicopathological characteristics in different age groups, especially in terms of recurrence-free survival (RFS) and distant recurrence-free survival (DRFS). We obtained three main results: 1. the population is distributed among the different age groups, and therefore, this type of cancer is not exclusively found among those of an older age; 2. in the RFS analysis, we can see a higher probability of local recurrence in the younger and older groups and, unexpectedly, a lower probability of local recurrence in the “median age” group; and 3. in the DRFS analysis, we can observe a higher probability of distant recurrence in older patients. From a molecular perspective, no significant differences in the mutational status of BRAF were detected according to different age groups, while mutations in the TERT promoter were exclusively present in older patients of all age groups, highlighting the potential prognostic implications of TERT promoter mutations in PTCs. In conclusion, the results of this series confirm that TC morphology alone in PTCs does not have the same negative prognostic significance in the younger population as in the older population. The reason for these different outcomes remains unclear and needs further studies.

Context Involvement of the pituitary gland in SARS-CoV-2 infection has been clinically suggested by pituitary hormone deficiency in severe COVID-19 cases, by altered serum adrenocorticotropic hormone (ACTH) levels in hospitalized patients, and by cases of pituitary apoplexy. However, the direct viral infection of the gland has not been investigated. Objective To evaluate whether the SARS-CoV-2 genome and antigens could be present in pituitary glands of lethal cases of COVID-19, and to assess possible changes in the expression of immune-related and pituitary-specific genes. Methods SARS-CoV-2 genome and antigens were searched in the pituitary gland of 23 patients who died from COVID-19 and, as controls, in 12 subjects who died from trauma or sudden cardiac death. Real-time reverse transcription polymerase chain reaction (PCR), in situ hybridization, immunohistochemistry, and transmission electron microscopy were utilized. Levels of mRNA transcripts of immune-related and pituitary-specific genes were measured by the nCounter assay. Results The SARS-CoV-2 genome and antigens were detected in 14/23 (61%) pituitary glands of the COVID-19 group, not in controls. In SARS-CoV-2–positive pituitaries, the viral genome was consistently detected by PCR in the adeno- and the neurohypophysis. Immunohistochemistry, in situ hybridization, and transmission electron microscopy confirmed the presence of SARS-CoV-2 in the pituitary. Activation of type I interferon signaling and enhanced levels of neutrophil and cytotoxic cell scores were found in virus-positive glands. mRNA transcripts of pituitary hormones and pituitary developmental/regulatory genes were suppressed in all COVID-19 cases irrespective of virus positivity. Conclusion Our study supports the tropism of SARS-CoV-2 for human pituitary and encourages exploration of pituitary dysfunction after COVID-19.

Introduction: In recent years, there has been a growing development of molecularly targeted therapies for various types of solid tumors—in particular, in non-small-cell lung cancer (NSCLC). This has required the need for greater quantities of tissue that is able to support ancillary studies, alongside cyto-histological diagnoses for the assessment of molecular targets. Conventional TBNA (cTBNA) and EBUS-guided TBNA (EBUS-TBNA) have shown a high diagnostic yield for malignant mediastinal and/or hilar lymph node enlargement and peribronchial masses, however, few studies have compared these two procedures. We retrospectively compared TBNA patients (EBUS-TBNA and cTBNA) in order to determine the diagnostic yield and material adequacy for subsequent ancillary analyses. Materials and Methods: We retrospectively evaluated 318 patients with clinical suspicion of lung cancer or with disease recurrence. All of the patients underwent TBNA (either EBUS-TBNA or cTBNA) on enlarged mediastinal and/or hilar lymph nodes and peribronchial masses between January 2017 and June 2021 at the University Hospital of Pisa, Italy. After a definitive diagnosis, molecular analyses and an evaluation of PD-L1 expression were performed in the cases of adenocarcinoma, squamous cell carcinoma, and NSCLC, not otherwise specified (NOS). Results: EBUS-TBNA was performed in 199 patients and cTBNA was performed in 119 patients with 374 and 142 lymph nodes, respectively. The overall diagnostic yield for positive diagnoses was 59% (diagnostic rate of 61% in EBUS-TBNA, and 55% in cTBNA). Adenocarcinoma (ADC) was the most frequent diagnosis in both methods. EBUS-TBNA diagnostic adequacy was 72% for molecular analysis, while it was 55.5% for cTBNA, showing a statistical trend (p = 0.08) towards the significance of EBUS. The average percentage of neoplastic cells was also statistically different between the two methods (p = 0.05), reaching 51.19 ± 22.14 in EBUS-TBNA and 45.25 ± 22.84 in cTBNA. With regard to the PD-L1 protein expression, the percentage of positivity was similar in both procedures (86% in EBUS-TBNA, 85% in cTBNA). Conclusions: Conventional TBNA (cTBNA) and EBUS-guided TBNA (EBUS-TBNA) are minimally invasive diagnostic methods that are associated with a high diagnostic yield. However, EBUS-TBNA has an improved diagnostic adequacy for molecular analysis compared to cTBNA, and is associated with a higher average percentage of neoplastic cells.

Altered blood hormone and metabolite levels during and post-COVID-19 have been extensively reported. Yet, studies of gene expression at the tissue level that can help identify the causes of endocrine dysfunctions are scarce. We analyzed transcript levels of endocrine-specific genes in five endocrine organs of lethal COVID-19 cases. Overall, 116 autoptic specimens from 77 individuals (50 COVID-19 and 27 uninfected controls) were included. All samples were tested for SARS-CoV-2 genome. Investigated organs included adrenals, pancreas, ovary, thyroid and white adipose tissue (WAT). Transcript levels of 42 endocrine-specific and 3 IFN-stimulated genes (ISGs) were measured and compared between COVID-19 cases (virus-positive and virus-negative in tissue) and uninfected controls. ISG transcript levels were enhanced in tissues positive for SARS-CoV-2. Endocrine-specific genes (e.g., HSD3B2, INS, IAPP, TSHR, FOXE1, LEP, CRYGD) were deregulated in COVID-19 cases in an organ-specific manner. Transcription of organ-specific genes was suppressed in virus-positive specimens of ovary, pancreas and thyroid but enhanced in adrenals. In WAT of COVID-19 cases transcription of ISGs and leptin was enhanced independently of the presence of virus. Our findings suggest that, in COVID-19, endocrine dysfunctions may arise especially when SARS-CoV-2 invades endocrine organs and that transcriptional alterations of endocrine-specific genes may contribute to endocrine manifestations.

Patients with advanced thyroid cancer harboring NTRK rearrangements can be treated with highly effective selective inhibitors. Immunohistochemistry (IHC) analysis, to detect Trk protein expression, represents an appealing screening strategy for NTRK rearrangements, but its efficacy has been poorly explored in thyroid cancer. The aim of this study is to investigate the diagnostic utility of Trk IHC in the identification of NTRK rearrangements. A series of 26 follicular-derived thyroid tumors, positive for NTRK rearrangements, and 28 NTRK fusion-negative controls were retrospectively analyzed by IHC using the pan-Trk monoclonal antibody (clone EPR17341) on the Ventana system. Area under the curve (AUC), sensitivity and specificity were calculated by ROC analysis. Trk expression was detected in 25 samples, including 22 out of the 26 NTRK-rearranged (84.6%) and three out of 28 NTRK-negative samples (10.7%). Four out of twenty-six NTRK-rearranged thyroid tumors were negative for Trk expression (15.4%), all carrying the ETV6/NTRK3 fusion. The AUC, sensitivity and specificity were 0.87, 0.85 and 0.89, respectively. A screening based on IHC analysis showed limited sensitivity and specificity in the identification of NTRK-rearranged tumors. Since falsely negative results could preclude the administration of effective targeted drugs, alternative detection strategies should be considered for thyroid cancer.

BackgroundTumor immune cells influence the efficacy of immune-checkpoint inhibitors (ICIs) and many efforts aim at identifying features of tumor immune microenvironment able to predict benefit from ICIs in proficient mismatch repair (pMMR)/microsatellite stable (MSS) metastatic colorectal cancer (mCRC).MethodsWe characterized tumor immune cell infiltrate, by assessing tumor-infiltrating lymphocytes (TILs), Immunoscore, Immunoscore-IC, and programmed death ligand-1 (PD-L1) expression in tumor samples of patients with mCRC enrolled in the AtezoTRIBE study, a phase II randomized trial comparing FOLFOXIRI/bevacizumab/atezolizumab to FOLFOXIRI/bevacizumab, with the aim of evaluating the prognostic and predictive value of these features.ResultsOut of 218 patients enrolled, 181 (83%), 77 (35%), 157 (72%) and 162 (74%) specimens were successfully tested for TILs, Immunoscore, Immunoscore-IC and PD-L1 expression, respectively, and 69 (38%), 45 (58%), 50 (32%) and 21 (13%) tumors were classified as TILs-high, Immunoscore-high, Immunoscore-IC-high and PD-L1-high, respectively. A poor agreement was observed between TILs and Immunoscore or Immunoscore-IC (K of Cohen ConclusionsThe digital evaluation of tumor immune cell infiltrate by means of Immunoscore-IC or Immunoscore identifies the subset of patients with pMMR mCRC achieving more benefit from the addition of the anti-PD-L1 to the upfront treatment. Immunoscore-IC stands as the most promising predictor of benefit from ICIs.

Pleural effusions are among the first clinical manifestations of malignant pleural mesothelioma (MPM) and often constitute the only available material for diagnosis. Although an MPM diagnosis can be reliable on cytology, the reported sensitivity is low (30% to 75%). Particularly, it can be hard to discriminate epithelioid MPM, the most common histotype, from reactive mesothelial hyperplasia (MH). Currently, BRCA1-associated protein 1 (BAP1) and CDKN2A (p16), evaluated by immunohistochemistry and fluorescent in situ hybridization, respectively, are the most valuable markers to discriminate MPM and MH. Both markers have a high specificity, but their sensitivity is not always satisfying, even when used together. We have recently developed a 117-gene expression panel, based on Nanostring technology, able to differentiate epithelioid MPM from MH pleural tissues better than BAP1 and p16. Herein, we evaluated the efficacy of the same panel on an independent retrospective cohort of 23 MPM and 11 MH pleural effusions (cell blocks and smears). The overall sensitivity and specificity of the panel were equal to 0.9565 and 1, respectively. Moreover, the panel performance was compared with BAP1 and p16 on 25 cell blocks. Sensitivity levels of gene panel, BAP1 alone, p16 alone, and BAP1 plus p16 were 1, 0.5882, 0.4706, and 0.7647, respectively. Specificity was always 1. Although further validation is needed, this gene panel could really facilitate patients' management, allowing a definitive MPM diagnosis directly on pleural effusions.

Simple Summary Non-invasive follicular thyroid neoplasms with papillary-like nuclear features (NIFTP) are follicular thyroid neoplasm with an indolent clinical behaviour. In this study, we evaluated a retrospective series of 451 NIFTPs, of which 197 (43.7%) presented in coexistence with collateral thyroid tumours. Unifocal and multifocal NIFTPs did not show peculiar ultrasound, cytological, molecular, and histo-pathological characteristics. Considering the high rate of coexisting carcinomas, NIFTP patients might benefit from monitoring of the contralateral lobe. Abstract Background: Non-invasive follicular thyroid neoplasms with papillary-like nuclear features (NIFTPs) were introduced in thyroid pathology in 2016. NIFTPs are a group of follicular neoplasm with an indolent behaviour. In this study, we gathered a large retrospective cohort of NIFTPs and compared those presenting as solitary lesions and NIFTPs found in multifocal setting. Methods: A retrospective search of NIFTPs was performed, and the clinico-pathological features were recorded. For a subgroup of patients, pre-surgical ultrasound (US) evaluation, cytological diagnosis, and molecular analysis were available. Results: We collected 451 NIFTPs; 254 (56.3%) were truly solitary tumours, while 197 coexisted with one or more NIFTP/cancer. Contrasting unifocal and multifocal settings, NIFTPs size was the only significantly different parameter. Preoperatively, NIFTP nodules mostly showed low-risk US characteristics, indeterminate cytology and a RAS-like molecular profile. Conclusion: NIFTPs often coexist with collateral thyroid tumours. However, no clinical-pathological differences can be observed between solitary and “multifocal” NIFTPs. Despite the well-established clinical indolence of NIFTP, a careful monitoring of the contralateral lobe should not be excluded.

Purpose The value of molecular markers in refining preoperative risk assessment of indeterminate thyroid nodules is being widely investigated. MicroRNAs (miRNA) are emerging as promising biomarkers for diagnostic and prognostic purposes. The aim of this study is to identify miRNAs specifically deregulated in mutation-negative indeterminate thyroid nodules. Methods Ninety-eight nodules preoperatively diagnosed as TIR 3A or TIR 3B with available histological diagnosis of follicular adenoma (FA), noninvasive follicular neoplasm with papillary-like nuclear features (NIFTP), and follicular variant papillary thyroid carcinoma (FV-PTC) have been retrospectively selected. Mutations in BRAF and RAS genes have been tested in all samples by real-time PCR; miRNAs were purified from cytology slides of 60 samples; expression analysis of 798 miRNAs was measured by the nCounter system. Results Point mutations in BRAF and RAS genes were detected in 32 out of 98 nodules (32.7%), the majority of which in FV-PTCs. Differential expression of miRNA in wild-type nodules highlighted that two miRNAs, namely miR-7-5p and miR-548ar-5p, were downregulated in FV-PTCs compared to FAs. The combined expression of these miRNAs, tested by ROC analysis, showed an area under the curve of 0.79. Sensitivity and negative predictive value were high both in wild-type (93% and 92%, respectively) and in mutated nodules (94% and 85%, respectively). Conclusion The analysis of miR-7-5p and miR-548ar-5p expression in indeterminate thyroid nodules demonstrated a promising value in ruling out malignancy.

Objective: Since many jobs imply driving, a relevant part of all road traffic crashes (RTC) is related to work. Statistics considering all crashes suggest that they are significantly associated with consumption of substances, but the root causes are not yet clear. The objective of the present paper was to systematically review the scientific literature concerning substances consumption and work-related RTC. We queried the PubMed and Scopus electronic databases according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. Articles were included if they reported all necessary data and survived a quality assessment. We selected a final sample of 30 articles from an initial pool of 7113. As hypothesized, taking any of the considered substances was found to increase the risk of work-related RTC. Descriptive statistics on work-related RTC showed a higher average positivity rate for medicines (14.8%) than for alcohol (3.02%) and drugs (0.84%). Interestingly, the impact of some medications found an unconvincing explanation in the mere occurrence of side effects, and it suggests that psychosocial and/or medical conditions could be better predictors of RTC. We therefore propose an intervention and prevention model that also considers biopsychosocial factors, for which further studies are needed in future research.

Pembrolizumab has been approved as first-line treatment for advanced Non-small cell lung cancer (NSCLC) patients with tumors expressing PD-L1 and in the absence of other targetable alterations. However, not all patients that meet these criteria have a durable benefit. In this monocentric study, we aimed at refining the selection of patients based on the expression of immune genes. Forty-six consecutive advanced NSCLC patients treated with pembrolizumab in first-line setting were enrolled. The expression levels of 770 genes involved in the regulation of the immune system was analysed by the nanoString system. PD-L1 expression was evaluated by immunohistochemistry. Patients with durable clinical benefit had a greater infiltration of cytotoxic cells, exhausted CD8, B-cells, CD45, T-cells, CD8 T-cells and NK cells. Immune cell scores such as CD8 T-cell and NK cell were good predictors of durable response with an AUC of 0.82. Among the immune cell markers, XCL1/2 showed the better performance in predicting durable benefit to pembrolizumab, with an AUC of 0.85. Additionally, CD8A, CD8B and EOMES showed a high specificity (&gt, 0.86) in identifying patients with a good response to treatment. In the same series, PD-L1 expression levels had an AUC of 0.61. The characterization of tumor microenvironment, even with the use of single markers, can improve patients’ selection for pembrolizumab treatment.

The alteration of miRNA processing is a driver event in several tumors including thyroid cancer. In particular, somatic DICER1 mutations, reported in follicular-patterned lesions, are shared by benign as well as malignant tumors. In the present study, we investigated the effects of alterations in the miRNA processing genes on the miRNA profile. The study included 19 follicular adenomas (FAs) and 22 follicular variant of papillary thyroid carcinomas (FVPTCs). The mutational status in the hot spot regions of DICER1, DROSHA, TARBP2, DGCR8 and the most commonly affected genes in thyroid tumors was investigated on both tumor and paired normal tissues. The miRNA profile and the mRNA expression levels of DICER1, DROSHA, TARBP2, DGCR8 and XPO5 were also evaluated. Two DICER1 RNase IIIb domain mutations were found in FAs. These lesions presented a considerable loss of 5p miRNAs. Fifteen miRNAs were specifically deregulated in DICER1-mutant lesions compared to FAs and FVPTCs. These miRNAs regulate crucial pathways in cancer such as Hippo, p53 and TGF-beta signalling. DICER1 somatic mutations in the RNase IIIb domain are not specific for malignancy, but the miRNA imbalance that they cause is remarkable, especially with regard to the loss of 5p miRNAs. DICER1-mutant lesions have a characteristic miRNA deregulation, which is different from that of FVPTCs; nevertheless, this impairment is consistent with malignant transformation. Further studies providing the real risk of malignancy associated with DICER1 mutations and the evolution of DICER1-mutant lesions are needed to make them useful in the clinical practice.

Papillary thyroid carcinoma (PTC) presents distinct clinico-pathological and molecular differences in children compared with adult patients. Whether the presence of rearrangements or point mutations is associated with aggressive PTC clinical presentation is still controversial. In this study, PTCs diagnosed in patients aged less than 18 years were retrospectively searched from the institutional archive and tumor tissue was tested for point mutations in BRAF and RAS genes and for rearrangements in RET, NTRK1, NTRK3, ALK, PPARG, BRAF and THADA. A total of 163 PTCs were analyzed. Point mutations were found in 83 (51%) and gene fusions in 48 cases (30%). The most frequent alteration was the BRAFV600E mutation (36.8%), followed by NTRK3 fusion (11%), NRAS mutation (10.4%) and RET fusion (10.4%). Fusion-driven PTCs showed more frequently infiltrative growth, larger tumors, extrathyroidal extension and N1b disease. PTCs showing solid growth pattern were significantly enriched in gene fusions. This is one of the largest cohorts of pediatric PTCs. Fusion-driven tumors most frequently show aggressive pathological features, the search for rearrangements, especially in tumors with solid areas, could improve the characterization of pediatric PTCs and offer possible therapeutic options.

Background: Hashimoto’s thyroiditis and Graves’ disease are autoimmune thyroid disorders (AITD) of unknown origin. Enterovirus (EV) infection of thyroid cells has been implicated as a possible initiator of cell damage and of organ-specific autoimmunity. We asked whether persistent infection of human epithelial cells with EV strains obtained from thyroid tissue of AITD patients could be associated with transcriptional changes capable of fostering immunopathology. Methods: EV isolates obtained from thyroid tissue of AITD cases were used to infect the AV3 epithelial cell line. AV3 cells incubated with a virus-free medium from thyroid tissue of subjects without evidence of thyroid autoimmunity were used as uninfected controls. Transcripts of immune-related genes were compared in infected vs. uninfected cells. Results: The EV genome and antigens were detected only in the cells exposed to AITD-derived virus isolates, not in control cells. Persistent EV infection, while suppressing transcription of several type I IFN and cytokine determinants, was associated with enhanced transcription of NFKB1/RELA, IFNAR1, JAK1/STAT1, i.e., the determinants that play key immunologic roles. Infection also led to upregulation of the CCL2 chemokine and the IL-18 pro-inflammatory interleukin. Conclusion: As in the case of EV strains obtained from autoimmune diabetes, results show that the EV strains that are present in the thyroid of AITD cases do repress IFN and cytokine pathways. JAK1/STAT1 upregulation supports activation of TLR pathways and aberrant T cell signaling. In the early phases of AITD, our results highlight the potential benefit of interventions aimed at blocking the viral infection and easing the inflammatory response.

Context Recent diagnostic criteria updates of the tall cell variant of papillary thyroid carcinoma (TCPTC) by the World Health Organization (WHO) have determined the inclusion of tumors with 30% to 49% of tall cells. However, the impact of tall cell percentage on papillary thyroid carcinoma (PTC) patients’ prognosis is still debated. Objective We aimed to evaluate whether tall cell percentage affects patient outcome in the absence of aggressive features. Methods Rates of aggressive features, recurrence-free survival (RFS), and distant RFS (5-year median follow-up) were compared among tumors with less than 30%, 30% to 49% and at least 50% tall cells. We also evaluated the impact of the new tall cell cutoff on patient management. Results Overall, 3092 tumors (15.7% of all PTCs) were collected: A total of 792 PTCs had less than 30%, 503 had 30% to 49%, and 1797 had 50% or more tall cell areas. With the new WHO definition, the number of TCPTCs increased by 28%. There were no differences in recurrence rates according to tall cell percentage. The coexistence of BRAF and TERT promoter mutations predicted a worse RFS. Considering the new definition of TCPTC, the level of risk according to the American Thyroid Association increased from low to intermediate in 4.2% of cases. However, the recurrence rate within this subgroup was comparable to low risk. Conclusion TCPTC and PTC with tall cell areas can be considered as a unique group with similar recurrence risk. However, whenever aggressive features are absent, tumors have a low risk of recurrence independently of tall cell percentage.

Purpose The SARS-CoV-2 genome has been detected in a variety of human samples including blood, urine, semen, and faeces. However, evidence of virus presence in tissues other than lung are limited. Methods We investigated whether SARS-CoV-2 could be detected in 50 autoptic specimens of endocrine organs from 29 patients who died of COVID-19. Results The virus was detected in 25 specimens including ten abdominal subcutaneous adipose tissue samples (62%), six testes (67%), and nine thyroid (36%) samples. The analysis of multiple endocrine organ samples obtained from the same patients showed that, in virus-positive cases, the viral genome was consistently detected in all but two matched specimens. Conclusion Our findings show that the virus spread into endocrine organs is a common event in severe cases. Further studies should assess the rate of the phenomenon in clinically mild cases. The potential long-term effects of COVID-19 on endocrine functions should be taken into consideration.

Delta-like protein 3 (DLL3) is a protein of the Notch pathway, and it is a potential therapeutic target for high-grade lung neuroendocrine tumors (NETs), i.e., small cell lung carcinoma (SCLC) and large cell neuroendocrine carcinoma (LCNEC). However, DLL3 prevalence in lung NETs and its association with clinicopathological characteristics and prognosis remained unclear. We analyzed the immunohistochemical expression of DLL3 and its prognostic role in a consecutive series of 155 surgically resected lung NETs, including typical carcinoid (TC), atypical carcinoid (AC), LCNEC, and SCLC patients. The DLL3 expression was categorized as high (>50% positive tumor cells) or low (vs.

The disruption of the Hippo pathway occurs in many cancer types and is associated with cancer progression. Herein, we investigated the impact of 32 Hippo genes on overall survival (OS) of cancer patients, by both analysing data from The Cancer Genome Atlas (TCGA) and reviewing the related literature. mRNA and protein expression data of all solid tumors except pure sarcomas were downloaded from TCGA database. Thirty-two Hippo genes were considered; for each gene, patients were dichotomized based on median expression value. Survival analyses were performed to identify independent predictors, taking into account the main clinical-pathological features affecting OS. Finally, independent predictors were correlated with YAP1 oncoprotein expression. At least one of the Hippo genes is an independent prognostic factor in 12 out of 13 considered tumor datasets. mRNA levels of the independent predictors coherently correlate with YAP1 in glioma, kidney renal clear cell, head and neck, and bladder cancer. Moreover, literature data revealed the association between YAP1 levels and OS in gastric, colorectal, hepatocellular, pancreatic, and lung cancer. Herein, we identified cancers in which Hippo pathway affects OS; these cancers should be candidates for YAP1 inhibitors development and testing.

The demonstration of tissue invasion by histology is an essential criterion for the differential diagnosis of benign and malignant mesothelial proliferations. When tissue invasion cannot be identified, the use of ancillary tests is sometimes necessary. Among investigated markers, the loss of BRCA1 associated protein 1 (BAP1) protein expression and the homozygous deletion of p16 have shown 100% specificity in separating benign and malignant mesothelial lesions. However, beyond the excellent specificity of these two markers, their low sensitivity limits their clinical utility. In this context, a previous study developed and tested a novel tool for use in the differential diagnosis of malignant pleural mesothelioma (MPM) using the NanoString System and a classification algorithm. In the current study, the performance of gene classifiers were compared using BAP1 and p16 testing. p16 FISH and BAP1 immunohistochemistry were performed on the same series of 34 epithelioid MPM and 20 benign pleural lesions, which were previously analyzed by the system. The diagnostic performance of p16, BAP1 and our classification models were compared using ROC analysis. It was observed that BAP1 loss and p16 deletion were highly specific for MPM, since they were not detected in benign lesions. However, their AUC values were not completely satisfying (BAP1: 0.8235; p16: 0.7647) particularly due to their low sensitivities. As expected, combining BAP1 and p16 tests increased the diagnostic sensitivity, thus improving the AUC (0.8824). In the same series of cases, our MPM tool outperformed BAP1 and p16 tests using the 22 and 40-gene classification models (AUC 22-gene model: 0.9996; AUC 40-gene model: 0.9990). In conclusion, the present gene-expression-based classification exhibited great potential and further validation is required to support these findings in a prospective fashion, in order to provide a solid alternative for pleural proliferation diagnosis.

Patients with indeterminate thyroid nodules (Bethesda III and IV) are often treated with diagnostic lobectomy, which in most cases represents an overtreatment. A reliable rule-out molecular test could spare patients unnecessary surgery. Stained smears of 88 indeterminate thyroid nodules with histologic diagnosis of follicular-patterned tumors were selected: 34 follicular adenomas (FAs), 34 follicular variant papillary thyroid carcinomas (FVPTCs), and 20 noninvasive follicular neoplasms with papillary-like nuclear features (NIFTPs). The expression level of 126 genes was measured by digital counting. Mutation testing was performed for the main gene mutations and fusions. Performance of gene expression and mutation tests was calculated by receiver operating characteristic analysis. The gene expression model showed an area under the curve (AUC) of 88%, with 91% negative predictive value in FAs and FVPTCs only. Part of NIFTPs was labeled as benign, and part was labeled as malignant; thus, the classifier performance worsened. Two FAs (5.9%), eight NIFTPs (40%), and 22 FVPTCs (64.7%) were mutation positive. Mutation testing AUC was 79% in FAs and FVPTCs, and decreased by including NIFTPs. This gene expression–based test was feasible in thyroid-stained smears, showed higher AUC than mutation test, and had a high negative predictive value—making it a good candidate as a rule-out test for indeterminate thyroid cytology. NIFTPs have a heterogeneous phenotype, and their preoperative diagnosis requires further investigation.

Pulmonary sarcomatoid carcinomas (PSC) are a rare group of lung cancer with a median overall survival of 9–12 months. PSC are divided into five histotypes, challenging to diagnose and treat. The identification of PSC biomarkers is warranted, but PSC molecular profile remains to be defined. Herein, a targeted whole transcriptome analysis was performed on 14 PSC samples, evaluated also for the presence of the main oncogene mutations and rearrangements. PSC expression data were compared with transcriptome data of lung adenocarcinomas (LUAD) and squamous cell carcinomas (LUSC) from The Cancer Genome Atlas. Deregulated genes were used for pathway enrichment analysis; the most representative genes were tested by immunohistochemistry (IHC) in an independent cohort (30 PSC, 31 LUAD, 31 LUSC). All PSC cases were investigated for PD-L1 expression. Thirty-eight genes deregulated in PSC were identified, among these IGJ and SLMAP were confirmed by IHC. Moreover, Forkhead box signaling and Fanconi anemia pathways were specifically enriched in PSC. Finally, some PSC harboured alterations in genes targetable by tyrosine kinase inhibitors, as EGFR and MET. We provide a deep molecular characterization of PSC; the identification of specific molecular profiles, besides increasing our knowledge on PSC biology, might suggest new strategies to improve patients management.

Follicular Variant of Papillary Thyroid Carcinoma (FVPTC) is usually associated with a good outcome. Nevertheless, in rare cases, it develops distant metastases (1–9%). Our goal was to investigate whether mRNA and miRNA expression profiles may help distinguish between metastatic versus non-metastatic FVPTCs. Twenty-four primary FVPTCs, 12 metastatic and 12 non-metastatic, with similar clinicopathological features were selected and analyzed by nanoString nCounter technology using two distinct panels for expression analysis of 740 mRNA and 798 miRNAs. Data analysis was performed using the nanoString nSolver 3.0 software. Forty-seven mRNA and 35 miRNAs were differentially expressed between the two groups. Using these mRNA and miRNAs, metastatic and non-metastatic FVPTCs were clearly divided into two distinct clusters. Our results indicate that FVPTCs with metastatic abilities have different expression profiles compared to the non-metastatic. A prospective validation is needed to evaluate the usefulness of this molecular approach in the early identification of high-risk FVPTCs.

Simple Summary Thyroid tumors that derive from follicular cells are not a homogeneous entity, showing variable morphological appearance and different degrees of differentiation. Molecular markers are useful for both diagnostic purposes and prognostic stratification of patients. In presurgical setting, molecular testing of indeterminate thyroid nodules on aspirates provides useful diagnostic information; the molecular analysis on tumor tissues can also reveal the presence of genetic alterations related to patients’ prognosis. In recent years, the molecular characterization of these tumors has acquired even more importance thanks to the introduction of targeted drugs. This review summarizes the current literature on the molecular landscape of follicular-derived thyroid tumors. Abstract Thyroid cancer is the most common type of endocrine-related malignancy, whose incidence rates have increased dramatically in the last few decades. Neoplasms of follicular origin generally have excellent prognosis, with the exception of less differentiated tumors. Follicular-derived thyroid cancer can manifest as a variety of morphologically distinct entities, characterized by various degrees of differentiation and invasiveness. Histological evaluation is thus crucial for the definition of patients’ prognosis. However, within each histological subtype, tumor behavior can be highly variable, and, in this respect, molecular characterization can provide insightful information to refine the risk stratification of tumors. In addition to the importance of its prognostic role, molecular testing can be used to support the differential diagnosis of thyroid nodules in the absence of marked cyto-morphological aberrations. Finally, with the advent of targeted drugs, the presence of molecular alterations will guide the therapeutic strategies for patients with advanced tumors who do not respond to standard treatment. This review aims to describe the genetic landscape of follicular-derived thyroid tumors also highlighting differences across histological subtypes.

The distinction between follicular thyroid carcinomas (FTCs) and follicular-patterned benign lesions is almost impossible on fine-needle aspiration cytology. Furthermore, minimally invasive FTCs (MI-FTCs) with less than 4 vascular invasion foci generally have an excellent prognosis, but there are exceptions and, so far, no molecular marker appears able to identify them reliably. We aimed to distinguish benign lesions from low- and high-risk FTCs by a small-scale combination of genes. The expression analysis of 75 selected genes was performed on 18 follicular adenomas (FAs), 14 MI-FTCs and 6 widely invasive FTC (WI-FTCs). The mutational status of the RAS genes, TERT promoter and PAX8-PPARG rearrangements was also investigated. Seven samples were mutated, namely 3 MI-FTCs and 4 WI-FTCs. Twenty-five genes were differentially expressed (FDR ECM1, RXRG, SDPR, SLC26A4, TIFF3, TIMP1) were also differently expressed among MI-FTCs and FAs or WI-FTCs and were considered to build a classification model, which was tested to classify samples according to their histological class. Hence, 31 out of 38 were correctly classified, and accuracy remained high after cross-validation (27/38). The 2 MI-FTCs incorrectly classified as WI-FTCs harbored both RAS and TERT promoter mutations. The capability of these six genes to stratify benign, low- and high-risk lesions appears to be promising in supporting the diagnosis of indeterminate thyroid nodules.

Noninvasive encapsulated follicular variants of papillary thyroid carcinomas have been recently reclassified as noninvasive follicular thyroid neoplasms with papillary-like nuclear features (NIFTPs). NIFTPs exhibit a behavior that is very close to that of follicular adenomas but different from the infiltrative and invasive follicular variants of papillary thyroid carcinomas (FVPTCs). The importance of miRNAs to carcinogenesis has been reported in recent years. miRNAs seem to be promising diagnostic and prognostic molecular markers for thyroid cancer, and the combination of miRNA expression and mutational status might improve cytological diagnosis. The aim of the present study was to evaluate the miRNA expression profile in wild-type, RAS- or BRAF-mutated NIFTPs, infiltrative and invasive FVPTCs, and follicular adenomas using the nCounter miRNA Expression assay (NanoString Technologies). To identify the significant Kyoto Encyclopedia of Genes and Genomes (KEGG) molecular pathways associated with deregulated miRNAs, we used the union of pathways option in DNA Intelligent Analysis (DIANA) miRPath software. We have shown that the miRNA expression profiles of wild-type and mutated NIFTPs could be different. The expression profile of wild-type NIFTPs seems comparable to that of follicular adenomas, whereas mutated NIFTPs have an expression profile similar to that of infiltrative and invasive FVPTCs. The upregulation of 4 miRNAs (miR-221-5p, miR-221-3p, miR-222-3p, miR-146b-5p) and the downregulation of 8 miRNAs (miR-181a-3p, miR-28-5p, miR-363-3p, miR-342-3p, miR-1285-5p, miR-152-3p, miR-25-3p, miR-30e-3) in mutated NIFTPs compared to wild-type ones suggest a potential invasive-like phenotype by deregulating the specific pathways involved in cell adhesion and cell migration (Hippo signaling pathway, ECM-receptor interaction, adherens junction, regulation of actin cytoskeleton, fatty acid biosynthesis and metabolism).

The follicular variant (FV) of papillary thyroid cancer (PTC) is one of the most common variants of PTC. Clinically, non-infiltrative FVPTC is considered a low-risk variant of PTC, and the non-invasive encapsulated forms of FVPTC represent a group of thyroid tumors with a particularly good prognosis. Consequently, these neoplasms have been very recently reclassified as non-invasive follicular neoplasms with papillary-like nuclear features (NIFTP). From a molecular standpoint, NIFTP appears to be similar to follicular neoplasms. However, only limited data are currently available regarding their gene expression profile.The aim of this study was to identify specific molecular signatures of 26 NIFTPs compared to those of 19 follicular adenomas (FAs) and 18 infiltrative FVPTCs (IFVPTCs). A nanoString custom assay was used to perform mRNA expression analysis. All cases were also genotyped for BRAF, N-, H-, and K-RAS mutations. Samples were grouped on the basis of gene expression profiles by Pearson's correlation and non-negative matrix factorization clustering analysis. Finally, the uncorrelated shrunken centroid machine-learning algorithm was used to classify the samples.The results revealed distinct expression profiles of FAs and IFVPTCs. NIFTP samples can exhibit different expression profiles, more similar to FAs (FA-like) or to IFVPTCs (IFVPTC-like), and these different expression profiles largely depend on the presence of different mutations (RAS or BRAF).In conclusion, although further validation of the model is required by using a larger group of prospective cases, these data reinforce the hypothesis that IFVPTC-like NIFTPs might represent precursors of IFVPTC.

Follicular variants of papillary thyroid carcinoma include encapsulated (with or without capsular/vascular invasion) and infiltrative forms, which have different clinical behaviors. The encapsulated forms that lack capsular invasion have an indolent clinical behavior that is similar to benign lesions; therefore, they were recently reclassified as 'noninvasive follicular thyroid neoplasms with papillary-like nuclear features' (NIFTPs). Because NIFTPs have nuclear features of papillary carcinomas, distinguishing between NIFTPs and infiltrative follicular variant of papillary thyroid carcinoma is almost impossible with cytological examination. The aim of this study is to determine whether miRNA expression profiles may help distinguish between NIFTPs versus follicular adenomas and infiltrative follicular variant of papillary thyroid carcinomas. The expression profiling of 798 miRNAs was tested in 54 thyroid tumors, including 18 follicular adenomas, 19 NIFTPs and 17 infiltrative follicular variant of papillary thyroid carcinomas, using nCounter Nanostring. We found that miR-146-5p, miR-221-5p, miR-222-3p, miR-30e-3p, and miR-152-3p could discriminate between benign and malignant lesions with a very high level of significance (P-value

Fine-needle aspiration (FNA) cytology is the gold standard for the evaluation of thyroid nodules, but about 30% are cytologically indeterminate. The inability to resolve preoperative thyroid nodules as benign or malignant is therefore a challenge. Molecular tests in FNA are useful to assist the diagnosis and the management of patients. However, if a thyroid nodule is indeterminate and genetic tests are inconclusive, surgical excision may be considered for definitive diagnosis. To overcome these limitations, novel diagnostic procedures and molecular tests are required. Among these, miRNAs have been reported as promising molecular markers in thyroid cancer. The aim of this study was to evaluate the potential diagnostic utility of miRNA expression in preoperative diagnosis of thyroid nodules with indeterminate cytology and wild type in RAS family genes and BRAF gene. The miRNA expression profile was evaluated in a small cohort of 12 wild-type Follicular Adenomas (FAs) and 4 wild-type Infiltrative Follicular Variant of Papillary Thyroid Carcinomas (IFVPTCs) with pre-surgical indeterminate cytology or in patients with cytological diagnosis not available. The two groups have been selected with similar characteristics in terms of age of patients, nodules size and with a ratio Male-Female 1:3. The expression of 798 miRNAs has been evaluated by using nCounter Nanostring technology, a sensitive and reproducible method. The differential miRNAs expression between FAs and IFVPTCs was investigated by applying the non-parametric Mann-Whitney U-test. In addition, an unsupervised hierarchical clustering analysis by Pearson correlation was performed. We found a set of 13 miRNAs differentially expressed between malignant and benign lesions. miR-222-3p, miR-30d-5p, miR-100-5p, miR-1972, miR-125b-5p, miR-155-5p were significantly up-regulated, whereas miR-574-5p, let-7f-5p, miR-148a-3p, miR-598-3p, miR-195-5p, miR-301a-3p, miR-5196-3p+hsa-miR-6732-3p were significantly down-regulated in IFVPTCs compared to FAs (p-value Although this is a preliminary study, the deregulation of this set of miRNAs might help identify malignant nodules that are negative for well-known gene alterations in preoperative FNAs. Citation Format: Maria Denaro, Vincenzo Condello, Clara Ugolini, Elisabetta Macerola, Anello Marcello Poma, Gabriele Materazzi, Paolo Vitti, Fulvio Basolo. A panel of miRNAs for diagnosis of wild-type thyroid nodules with pre-surgical indeterminate cytology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5402.