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.

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 remain elusive. METHODS: A total of 440 FTs from 2 institutions were interrogated for DICER1, DGCR8, and RAS family hotspot mutations using Sanger sequencing. Whole-exome sequencing 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 quantitative RT-PCR analysis. The Cancer Genome Atlas (TCGA) database was also probed for DICER1/DGCR8 mutations and miRNA dysregulation. RESULTS: Fourteen (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. Whole-exome sequencing 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 with wild-type lesions. Moreover, DICER1-mutant cases showed a remarkable reduction of 5' arm miRNAs, findings corroborated in the TCGA cohort. CONCLUSION: DICER1 and DGCR8 hotspot mutations are rare in unselected cohorts of FTs, and mutated cases exhibit a specific miRNA profile. Although DGCR8 mutations may coexist 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.

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.

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.

Predictive Impact of RNF43 Mutations in Patients With Proficient Mismatch Repair/Microsatellite Stable BRAFV600E-Mutated Metastatic Colorectal Cancer Treated With Target Therapy or Chemotherapy.

PURPOSE: Target therapy (TT) with encorafenib plus cetuximab is a standard option in patients with BRAFV600E-mutated (mut) pretreated metastatic colorectal cancer (mCRC). Recently, mutations in RNF43, encoding a negative regulator of the WNT pathway, were associated with longer progression-free survival (PFS) and overall survival (OS) in patients with proficient mismatch repair/microsatellite stable (pMMR/MSS) BRAFV600E-mut mCRC treated with TT. Here, we explored the effect of RNF43 mutations on the efficacy of second-line TT versus standard chemotherapy (CT). METHODS: A retrospective cohort of patients with pMMR/MSS BRAFV600E-mut tumors, available RNF43 mutational status, and treated with second-line TT or oxaliplatin- and/or irinotecan-based CT was analyzed. RESULTS: One hundred thirty-two patients with pMMR/MSS BRAFV600E-mut mCRC were included. RNF43 was found mut in 34 (26%) cases. Among RNF43 mutants, TT was associated with longer PFS (7.7 v 3.0 months; P = .002) and higher overall response rate (ORR; 45% v 0%; P = .009) compared with CT. Conversely, among RNF43 wild-type (wt) patients, only a trend for longer PFS (4.5 v 3.7 months; P = .064) favoring TT, with no differences in ORR (P = .14), was observed. After excluding 36 patients receiving TT in third line or beyond, a longer OS (19.4 v 10.1 months; P = .022) and a numerically OS advantage (10.6 v 6.6 months; P = .068) were reported for TT both in the RNF43-mut and in the RNF43 wt groups. However, no interaction effect was reported between RNF43 mutational status and treatment in ORR (Pinteraction = .96), PFS (Pinteraction = .13), and OS (Pinteraction = .44). CONCLUSION: Patients with pMMR/MSS BRAFV600E-mut mCRC achieve benefit from TT versus CT independently of RNF43 mutational status, although a higher magnitude of benefit from TT is observed in RNF43-mut tumors. These findings deserve confirmation in concluded and ongoing randomized trials.

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.

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.

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.

Molecular Genetics of Follicular-Derived Thyroid Cancer.

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.

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.