Identification of a Novel Non-V600E BRAF Mutation in Papillary Thyroid Cancer.

Papillary thyroid cancer (PTC) is a common endocrine malignancy, and its incidence is reported to be constantly increasing. BRAF mutation is detected in approximately 44% of PTCs, and the most common BRAF mutation is thymine (T) to adenine (A) missense mutation in nucleotide 1796 (T1796A, V600E). Although BRAFV600E represents 95% of all BRAF mutations, uncommon BRAF mutations have been reported in thyroid carcinomas and represent an alternative mechanism of BRAF activation with unclear clinical significance. We report a novel non-V600E mutation (c.1799_1812delinsAT, p.V600_W604delinsD), identified preoperatively with next-generation sequencing (NGS) on the material obtained with fine-needle aspiration cytology (FNAC) performed on a thyroid nodule cytologically suspicious for malignancy in a 35-year-old male patient. The presence of this new variant of BRAF mutation was subsequently confirmed in the postoperative phase by direct Sanger sequencing. In conclusion, we report a new non-V600E variant previously undetected in papillary thyroid cancer. In addition, this case report shows that the NGS technique on cytological tissue allows to detect the presence of rare mutations, thus increasing the diagnostic specificity of molecular analysis.

Prevalence, Molecular Landscape and Clinical Impact of DICER1 and DGCR8 Mutated Follicular-Patterned Thyroid Nodules.

BACKGROUND: Mutations in micro-RNA (miRNA) regulators DICER1 and DGCR8 have recently been uncovered, revealing a potential novel mechanism driving thyroid tumor development. However, the true frequency of these hotspot mutations in follicular-patterned thyroid tumors (FTs) and their relation to established driver gene events remains elusive. METHODS: A total of 440 FTs from two institutions were interrogated for DICER1, DGCR8, and RAS family hotspot mutations using Sanger sequencing. Whole-exome sequencing (WES) was also performed to identify additional driver gene aberrations in DICER1/DGCR8-mutant cases. Subsets of cases were further analyzed using miRNA expression profiling, and key dysregulated miRNAs were validated as markers of DICER1 mutations using qRT-PCR analysis. The TCGA database was also probed for DICER1/DGCR8 mutations and miRNA dysregulation. RESULTS: 14 (3.2%) and 4 (1%) FTs harbored DICER1 and DGCR8 hotspot mutations respectively in the combined cohort, and no cases with normal tissue available were found to exhibit a constitutional variant. Two DGCR8-mutant cases also harbored oncogenic RAS mutations. WES analysis did not identify additional driver gene events in DICER1/DGCR8-positive cases. Comprehensive miRNA expression profiling revealed a unique pattern of dysregulated miRNAs in DICER1/DGCR8-mutant cases compared to wild-type lesions. Moreover, DICER1-mutant cases showed a remarkable reduction of 5' arm miRNAs, findings which were corroborated in the TCGA cohort. CONCLUSIONS: DICER1 and DGCR8 hotspot mutations are rare in unselected cohorts of FTs, and mutated cases exhibit a specific miRNA profile. While DGCR8 mutations may co-exist with established RAS gene alterations, FTs with DICER1 variants were devoid of other driver gene events.

SARS-CoV-2 spread to endocrine organs is associated with obesity: an autopsy study of COVID-19 cases.

PURPOSE: SARS-CoV-2 infection may be limited to the respiratory tract or may spread to multiple organs. Besides disease severity, factors associated with virus spread within the host are elusive. Here, we tried to identify features associated with SARS-CoV-2 spread to endocrine organs. METHODS: In a retrospective autoptic cohort of 51 subjects who died because of COVID-19, we analyzed the severity and type of lung pathology, patients' features and the detection of virus in thyroid, testis, adrenal gland, pancreas, anterior pituitary, and the white adipose tissue (WAT). RESULTS: The SARS-CoV-2 genome was detected in endocrine organs of 30/51 cases. The anterior pituitary and WAT were most frequently positive for virus. While pathological features of lung were not associated with the presence of virus in endocrine organs, obesity (BMI > 30) was significantly associated to virus detection in pancreas (p = 0.01) and thyroid (p = 0.04). WAT infection was detected more frequently in males (p = 0.03). CONCLUSION: In subject with obesity dying of COVID-19, the virus frequently spreads to endocrine organs. The findings emphasize the need for optimal treatment of patients with obesity at the very onset of COVID-19. Since post-COVID conditions remain a major issue worldwide, a rigorous follow-up of endocrine function-especially of thyroid and pancreas-is advocated in subjects with obesity.

Prevalence of Differentiated High-Grade Thyroid Carcinoma Among Well-Differentiated Tumors: A Systematic Review and Meta-Analysis.

Background: The current edition of the World Health Organization (WHO) classification of endocrine tumors introduced grading for follicular cell-derived thyroid cancer. Tumors with necrosis and/or high mitotic count but not fulfilling the Turin criteria for poorly differentiated carcinoma will be reclassified as differentiated high-grade thyroid carcinoma (DHGTC). However, the impact of this reclassification has not been evaluated. In this study, we performed a systematic review and meta-analysis to estimate the prevalence of this new entry across thyroid tumor subtypes. Methods: In this systematic review and meta-analysis, studies reporting data on necrosis and/or mitoses in well-differentiated thyroid carcinoma (WDTC) were used to estimate the prevalence of DHGTC. Heterogeneity and potential publication bias were also evaluated. Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines were followed, and quality assessment was performed using a modification of the Newcastle-Ottawa scale. The study has been registered in the International Prospective Register of Systematic Reviews (PROSPERO, ID: CRD42022378716). Results: In clinically unselected patients, the prevalence of DHGTC in WDTC was 0.072 [95% confidence interval, CI, = 0.045-0.113]. The proportion of high-grade tumors greatly varied across growth patterns and subtypes. Overall, the prevalence of DHGTC was higher in follicular thyroid carcinoma (FTC; 0.146 [CI = 0.101-0.205]) than in papillary thyroid carcinoma (PTC; 0.059 [CI = 0.036-0.097]). Diffuse sclerosing, follicular, and classic subtype PTC had the lowest rates of high-grade features (i.e., 0.018 [CI = 0.004-0.084]; 0.036 [CI = 0.010-0.124]; and 0.042 [CI = 0.027-0.066], respectively), while a greater proportion of solid trabecular and histologically aggressive PTC could be reclassified as DHGTC (i.e., 0.154 [CI = 0.067-0.314] and 0.168 [CI = 0.108-0.252], respectively). Similar proportions were obtained for minimally and widely invasive FTC (i.e., 0.136 [CI = 0.058-0.287] and 0.152 [CI = 0.086-0.254], respectively). Finally, in a cohort of patients with poor prognosis (i.e., fatal cases, metastatic and radioiodine resistant tumors, cases with biochemical recurrence), the proportion of DHGTC was 0.287 [CI = 0.155-0.469]. Conclusions: Following the current WHO indications, some tumors will be reclassified as DHGTC. The proportion of tumors with high-grade features is relevant in FTC, solid trabecular, and histologically aggressive PTC subtypes. A remarkable enrichment in DHGTC among patients with poor prognosis confirms the negative impact of high-grade features on outcome.

Indeterminate Thyroid Nodules: From Cytology to Molecular Testing.

Thyroid cancer is the most common malignancy of the endocrine system. Fine-needle aspiration (FNA) biopsy of thyroid nodules has become the gold standard procedure, in terms of cost and efficacy, for guiding clinicians towards appropriate patients' management. One challenge for cytopathologists is to accurately classify cytological specimens as benign or malignant based on cytomorphological features. In fact, with a frequency ranging from 10% to 30%, nodules are diagnosed as indeterminate. In recent years, the mutational landscape of thyroid tumors has been extensively described, and two molecular profiles have been identified: RAS-like (NRAS, HRAS, and KRAS mutations; EIF1AX mutations; BRAF K601E mutation; and PPARG and THADA fusions) and BRAFV600E-like (including BRAFV600E mutation and RET and BRAF fusions). The purpose of this review is to discuss the latest molecular findings in the context of indeterminate thyroid nodules, highlighting the role of molecular tests in patients' management.

Transcriptional changes in multiple endocrine organs from lethal cases of COVID-19.

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.

Outcomes of the Tall-Cell Variant of Papillary Thyroid Carcinoma in Patients with Different Ages: A 17-Year Mono-Institutional Experience.

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.

The 5th edition of WHO classification of tumors of endocrine organs: changes in the diagnosis of follicular-derived thyroid carcinoma.

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.

Autopsy Study of Testicles in COVID-19: Upregulation of Immune-Related Genes and Downregulation of Testis-Specific Genes.

CONTEXT: Infection by SARS-CoV-2 may be associated with testicular dysfunction that could affect male fertility. OBJECTIVE: Testicles of fatal COVID-19 cases were investigated to detect virus in tissue and to evaluate histopathological and transcriptomic changes. METHODS: Three groups were compared: (a) uninfected controls (subjects dying of trauma or sudden cardiac death; n = 10); (b) subjects dying of COVID-19 (virus-negative in testes; n = 15); (c) subjects dying of COVID-19 (virus-positive in testes; n = 9). SARS-CoV-2 genome and nucleocapsid antigen were probed using RT-PCR, in situ hybridization, and immunohistochemistry (IHC). Infiltrating leukocytes were typed by IHC. mRNA transcripts of immune-related and testis-specific genes were quantified using the nCounter method. RESULTS: SARS-CoV-2 was detected in testis tissue of 9/24 (37%) COVID-19 cases accompanied by scattered T-cell and macrophage infiltrates. Size of testicles and counts of spermatogenic cells were not significantly different among groups. Analysis of mRNA transcripts showed that in virus-positive testes immune processes were activated (interferon-alpha and -gamma pathways). By contrast, transcription of 12 testis-specific genes was downregulated, independently of virus positivity in tissue. By IHC, expression of the luteinizing hormone/choriogonadotropin receptor was enhanced in virus-positive compared to virus-negative testicles, while expression of receptors for androgens and the follicle-stimulating hormone were not significantly different among groups. CONCLUSION: In lethal COVID-19 cases, infection of testicular cells is not uncommon. Viral infection associates with activation of interferon pathways and downregulation of testis-specific genes involved in spermatogenesis. Due to the exceedingly high numbers of infected people in the pandemic, the impact of virus on fertility should be further investigated.

The Italian Consensus for the Classification and Reporting of Thyroid Cytology: Cytohistologic and molecular correlations on 37,371 nodules from a single institution.

BACKGROUND: The Italian Consensus for the Classification and Reporting of Thyroid Cytology (ICCRTC) includes six diagnostic categories (TIR 1/1C, TIR 2, TIR 3A, TIR 3B, TIR 4, and TIR 5), each indicating a different risk of malignancy. The objective of this monocentric retrospective study was to evaluate the distribution of the ICCRTC classes at the authors' institution and assess their cytohistologic correlations. METHODS: The authors retrospectively collected 37,371 consecutive cytologic reports of thyroid nodules and described the clinical-pathologic features of the different cytologic categories. The cytologic diagnoses also were compared with histologic outcomes in a subset of patients. RESULTS: The cytologic classes were distributed as follows: nondiagnostic, 15.6%; benign, 66.5%; low-risk indeterminate, 10% (TIR 3A); high-risk indeterminate, 3.5% (TIR 3B); suspicious, 1.7%; and malignant, 2.6%. According to histology, the risk of malignancy was very high in the nondiagnostic category (29.8%), with young male patients more exposed to malignancy, and it was relatively high among benign (7.8%) and indeterminate nodules (32.5% in TIR 3A; 52.1% in TIR 3B), mainly because of the high prevalence of follicular architecture in malignant tumors. On histology, the malignancy rates were 92.4% and 99.3% for the suspicious and malignant categories, respectively; aggressive variants of papillary thyroid carcinoma were mostly diagnosed in these categories. CONCLUSIONS: In this series, nondiagnostic nodules showed high prevalence and, surprisingly, high malignancy rates. Malignant tumors with follicular architecture represented a diagnostic pitfall in benign and indeterminate nodules. The suspicious and malignant categories had high specificity for malignancy. Importantly, the ICCRTC had high reliability for identifying preoperatively aggressive histotypes of thyroid carcinoma.

Limited Accuracy of Pan-Trk Immunohistochemistry Screening for NTRK Rearrangements in Follicular-Derived Thyroid Carcinoma.

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.

Suppression of Pituitary Hormone Genes in Subjects Who Died From COVID-19 Independently of Virus Detection in the Gland.

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.

Predictive Biomarkers in Thyroid Cancer.

In molecular pathology, predictive biomarkers identify which patients are likely to respond to targeted drugs. These therapeutic agents block specific molecules directly involved in cancer growth, dedifferentiation and progression. Until few years ago, the only targeted drugs available for advanced thyroid cancer included multi-tyrosine kinase inhibitors, mainly targeting the MAPK pathway and the angiogenic signaling. The administration of these drugs does not necessarily require a molecular characterization of tumors to assess the presence of predictive alterations. However, the availability of new selective targeted drugs for thyroid cancer patients is changing the diagnostic strategies for the molecular characterization of these tumors. The search for targetable alterations can be performed directly on tumor tissue by using a variety of methodologies, depending also on the number and type of alterations to test (i.e. single nucleotide variation or gene rearrangement). Herein, a comprehensive review of the currently available targeted treatments for thyroid cancer, related predictive markers and testing methodologies is provided.

Down-regulation of miR-7-5p and miR-548ar-5p predicts malignancy in indeterminate thyroid nodules negative for BRAF and RAS mutations.

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.

Clinical-Pathological Features and Treatment Outcome of Patients With Hobnail Variant Papillary Thyroid Carcinoma.

Papillary thyroid carcinoma (PTC) with hobnail areas above 30% is classified as hobnail variant (HVPTC). Although it is widely accepted that HVPTC has a worse outcome than classical PTC, it is unclear whether PTC with hobnail features below 30% is as aggressive as HVPTC. We gathered the largest mono-institutional series of PTC with hobnail areas and HVPTC to evaluate differences in terms of pathological features of aggressiveness, molecular profile, and treatment outcome. A total of 99 PTC with hobnail features above 5% were retrospectively selected; 34 of them met the criteria for HVPTC (0.4% of all PTC diagnosed at our institution). All tumors showed high rates of extra-thyroidal extension (40.4%), lymph node metastasis (68.1% of patients with lymphadenectomy), and vascular emboli (49.5%), with no differences according to the 30% cutoff. On the other hand, distant metastases were present in HVPTC only (9.4%). Also, advanced age, advanced disease stage, and TERT promoter mutation were associated with HVPTC. More than half of the patients with follow-up had structural or biochemical persistence after 1 year from surgery. Structural persistence was significantly more common in patients with HVPTC (37.5% vs. 8.7%), while no differences were observed considering structural and biochemical persistence together. The presence of hobnail features identifies locally aggressive tumors, and, consequently, it should be always acknowledged in the pathological report. However, tumors with more than 30% hobnail areas frequently present TERT promoter mutations, advanced disease stage, and structural persistence after radioiodine ablation.

Clinical-Pathological and Molecular Evaluation of 451 NIFTP Patients from a Single Referral Center.

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.

NanoString in the screening of genetic abnormalities associated with thyroid cancer.

In the setting of cancer pathology, molecular characterization of tumors providing diagnostic and predictive information is acquiring more and more relevance. Moreover, the advent of innovative technologies continuously improves the knowledge of the molecular landscape of tumors and strengthens the links between clinics, tumor pathology and molecular features. In the clinical management of patients with thyroid nodules and thyroid tumors, the aid of molecular testing is encouraged but still not strongly recommended by current guidelines. Also for this reason this field of study is attracting much interest. The nCounter system is a relatively new technology based on a direct hybridization of fluorescent probes to specific nucleic acid targets, followed by digital measurement of signals; the reaction is highly multiplexable and results are robust and reproducible. This review reports and discusses the available data related to the application of this specific technique to thyroid nodules and thyroid tumors samples. The available data indicate that nCounter system represents a solid approach for the research of relevant diagnostic and prognostic biomarkers in thyroid pathology.

Activation of Type I and Type II Interferon Signaling in SARS-CoV-2-Positive Thyroid Tissue of Patients Dying from COVID-19.

Background: Thyroid dysfunctions have been reported after Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection. However, the biological mechanisms behind these conditions remain unexplored. Herein, we report on changes of the immune transcriptome in autoptic thyroid tissues of people who have died from coronavirus disease 2019 (COVID-19). Methods: Twenty-five autoptic thyroid specimens of subjects dying from COVID-19 were investigated. Eleven autoptic thyroid specimens of subjects dying from causes other than infectious conditions served as controls. RNA transcripts of 770 immune-related genes together with RNA genomes of multiple coronavirus types were measured by the nCounter system. Reverse transcription-polymerase chain reaction for two SARS-CoV-2 genes was used to assess virus positivity. Results were validated by immunohistochemistry. Results: The SARS-CoV-2 genome and antigens were detected in 9 of 25 (36%) thyroid specimens from the COVID-19 cohort. Virus-negative thyroid tissues from COVID-19 subject did not show changes of gene transcription nor significant numbers of infiltrating immune cells. Conversely, SARS-CoV-2-positive thyroid specimens showed marked upregulation of immune genes, especially those proper of the type I and type II interferon (IFN) pathways. In infected tissues, infiltrates of innate immune cells (macrophages and polymorphonuclear neutrophils) were prevalent. Conclusions: The thyroid gland can be directly infected by the SARS-CoV-2. Infection strongly activates IFN pathways. The direct viral insult combined with an intense immune response may trigger or worsen thyroid conditions in predisposed individuals.

Molecular Alterations in Relation to Histopathological Characteristics in a Large Series of Pediatric Papillary Thyroid Carcinoma from a Single Institution.

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.

Tall cell percentage alone in PTC without aggressive features should not guide patients' clinical management.

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.