[Molecular diagnostics enable targeted therapies for anaplastic and poorly differentiated thyroid cancer]. / Molekylär diagnostik ger bättre behandling av tyreoideacancer.

Anaplastic and poorly differentiated thyroid cancer (ATC, PDTC) are rare and highly aggressive tumors that historically have been associated with a short life expectancy and low chance of cure. Molecular pathology and the introduction of highly effective targeted drugs have revolutionized the possibilities of management of patients with ATC and PDTC, with BRAF and MEK inhibitors as the most prominent example. Here we provide updated recommendations regarding diagnostics and management, including primary surgical management and targeted therapies based on specific molecular pathological findings.

Regional variations in the management of primary hyperparathyroidism in Sweden: population-based case-control study.

BACKGROUND: Substantial disparities in the utilization of parathyroidectomy for primary hyperparathyroidism have been reported. This study aimed to analyse regional variations in parathyroidectomy incidence with respect to the patient's disease burden and socioeconomic status. METHODS: A population-based case-control study included all patients with primary hyperparathyroidism who underwent parathyroidectomy in Sweden between 2008 and 2017 and 10 matched controls. Data on demographic and socioeconomic variables, co-morbidities and drug prescriptions were collected from relevant national registers. Conditional logistic regression was used to analyse predictors of parathyroidectomy. RESULTS: A total of 8626 patients with primary hyperparathyroidism (77% women) underwent parathyroidectomy during the study interval. The annual incidence of parathyroidectomy was 9.0 per 100 000 persons. The annual age-adjusted regional incidences of parathyroidectomy varied between 3.3 and 16.9 operations per 100 000 inhabitants. Except for a small underrepresentation of patients with lower education, no effect of socioeconomic variables was observed. Compared with matched controls, the parathyroidectomy group had increased odds ratios of having developed classical symptoms of primary hyperparathyroidism and being prescribed medication against cardiovascular disorders and psychiatric illness at the time of parathyroidectomy. Increased risks of kidney stones and osteoporosis were observed 5 years before parathyroidectomy. Patients with primary hyperparathyroidism selected for parathyroidectomy from regions with a low incidence of operations had a higher prevalence of kidney stones, osteoporosis and hypertension, as well as larger adenomas and higher calcium levels at the time of parathyroidectomy compared with patients in high-incidence regions. CONCLUSION: The considerable variation in parathyroidectomy seems more likely associated with different clinical thresholds for detection of primary hyperparathyroidism and referral to surgery than socioeconomic disparities.

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.

Spatial Transcriptomics in a Case of Follicular Thyroid Carcinoma Reveals Clone-Specific Dysregulation of Genes Regulating Extracellular Matrix in the Invading Front.

Follicular thyroid carcinoma (FTC) is recognized by its ability to invade the tumor capsule and blood vessels, although the exact molecular signals orchestrating this phenotype remain elusive. In this study, the spatial transcriptional landscape of an FTC is detailed with comparisons between the invasive front and histologically indolent central core tumor areas. The Visium spatial gene expression platform allowed us to interrogate and visualize the whole transcriptome in 2D across formalin-fixated paraffin-embedded (FFPE) tissue sections. Four different 6 × 6 mm areas of an FTC were scrutinized, including regions with capsular and vascular invasion, capsule-near area without invasion, and a central core area of the tumor. Following successful capturing and sequencing, several expressional clusters were identified with regional variation. Most notably, invasive tumor cell clusters were significantly over-expressing genes associated with pathways interacting with the extracellular matrix (ECM) remodeling and epithelial-to-mesenchymal transition (EMT). Subsets of these genes (POSTN and DPYSL3) were additionally validated using immunohistochemistry in an independent cohort of follicular thyroid tumors showing a clear gradient pattern from the core to the periphery of the tumor. Moreover, the reconstruction of the evolutionary tree identified the invasive clones as late events in follicular thyroid tumorigenesis. To our knowledge, this is one of the first 2D global transcriptional mappings of FTC using this platform to date. Invasive FTC clones develop in a stepwise fashion and display significant dysregulation of genes associated with the ECM and EMT - thus highlighting important molecular crosstalk for further investigations.

Adrenal and periadrenal schwannoma: histological, molecular and clinical characterization of an institutional case series.

PURPOSE: Adrenal schwannoma (AS) and periadrenal schwannoma (PAS) are exceedingly rare Schwann cell tumors that develop from the adrenal medulla and periadrenal peripheral nerves respectively. The underlying genetic events are elusive. METHODS: We searched our institutional database for AS/PAS cases and reviewed the histology and clinical outcome. Comprehensive molecular work-up was performed. RESULTS: We found reports of 4 AS/PAS cases diagnosed between 1992 and 2022 among the 1248 adrenal lesions submitted for histopathology during the same time period (0.32%). Two patients were male, two were female, and the age span was 59-80 years. Median size was 70 mm (range 50-100 mm), and from a radiology perspective, the lesions were initially suspected of malignant lesions originating from either adrenals or kidneys. Hormonal analyses were normal in all cases. Histologically, three cases were annotated as cellular AS or PAS, and one case was annotated as microcystic AS. Molecular characterization using focused next-generation sequencing did not identify SMARCB1 or NF2 mutations, alterations previously associated to schwannoma at other anatomical sites. The postoperative period was without complications for all patients, and follow-up did not show any signs of relapse or metastatic disease. CONCLUSION: AS/PAS are rare neoplasms that are most often benign, and the molecular etiology is most likely not related to mutations in established schwannoma-related genes. Since these tumors may be misinterpreted as malignant, knowledge of this entity is essential for radiologists, endocrinologists, surgeons and pathologists.

5hmC Immunohistochemistry: A Predictor of TERT Promoter Mutational Status in Follicular Thyroid Carcinoma?

Telomerase reverse transcriptase (TERT) gene aberrancies correlate to adverse prognosis in follicular thyroid carcinoma (FTC). As loss of 5-hydroxymethylcytosine (5hmC) has been associated with TERT promoter mutations in papillary thyroid carcinoma, this study sought to analyze the levels of 5hmC in a cohort of follicular thyroid tumors with available TERT data. A total of 29 tumors (26 FTCs, 2 follicular thyroid tumors of uncertain malignant potential, and 1 oncocytic thyroid carcinoma) with known TERT promoter mutational status and TERT gene expression were assessed for 5hmC immunoreactivity using two antibodies (clones RM236 and 4D9.) Slides were analyzed using a semiquantitative scoring system. Of the 10 tumor cases with aberrant TERT, only 1 scored negative with both antibodies (1/10; 10%), whereas the remaining 9 cases (9/10; 90%) exhibited some positivity for at least one antibody. Of the 19 TERT wild-type tumors, no case was scored negative using RM236, and 2 cases (2/19; 11%) using 4D9. The differences between TERT promoter mutated and wild-type groups were non-significant. The sensitivity and specificity for 5hmC immunohistochemistry (IHC) to detect mutated cases were 10% and 100% (RM236) and 20% and 89% (4D9). Therefore, 5hmC IHC is not a sensitive marker for detecting TERT promoter mutations in follicular thyroid tumors.

[Many adrenal incidentalomas are not adequately evaluated - updated guidelines to improve follow-up are presented]. / Många binjureincidentalom får ingen adekvat utredning.

The use of cross-sectional imaging in Sweden has increased more than twofold in the last 20 years. Inadvertently discovered adrenal lesions, adrenal incidentalomas, are reported in about one per cent of abdominal investigations. The first Swedish guidelines for the management of adrenal incidentalomas were published in 1996 and have since then been regularly revised. Still, data indicate that less than half of patients receive adequate follow-up. Here we comment on the newly updated guidelines and briefly review the recommended clinical and radiological work-up.

[Adrenohepatic fusion - an unknown but not uncommon phenomenon of clinical importance]. / Adrenohepatisk fusion ­ ett okänt fenomen med klinisk betydelse.

Adrenohepatic fusion (AHF) is a union of the right adrenal gland and the liver with intermingling of parenchymal adrenal and liver cells. The phenomenon can be of clinical importance when evaluating patients with adrenal tumors. Using conventional imaging techniques such as computed tomography, a benign adrenal adenoma developing in an adrenohepatic fusion may mimic an invasive hepatocellular carcinoma or adrenal cortical carcinoma. This study presents a comprehensive review of the literature and shows a prevalence of 5.6 percent in autopsy studies. Moreover, 19 patients with adrenal masses in AHF are presented together with their clinical data.

Digital droplet PCR TERT promoter mutational screening in fine needle aspiration cytology of thyroid lesions: A highly specific technique for pre-operative identification of high-risk cases.

BACKGROUND: Despite the advent of comprehensive molecular testing in surgical pathology, most centers still rely on the morphological assessment of fine-needle aspiration cytology (FNAC) to triage patients with thyroid nodules for surgery. Subsets of patients could benefit from the inclusion of molecular testing to increase the diagnostic and/or prognostic properties of the cytology analysis, including the assessment of TERT promoter mutations, an event coupled with thyroid malignancy, and poor prognosis. METHODS: In this prospective study, preoperative FNAC material from 65 cases was assessed for TERT promoter hotspot mutations C228T and C250T using the digital droplet PCR (ddPCR) technique on frozen pellets and re-evaluated postoperatively. RESULTS: Our cohort consisted of 15 B-III (23%), 26 B-IV (40%), 1 B-V (2%), and 23 (35%) B-VI lesions according to the Bethesda System for Reporting Thyroid Cytopathology. TERT promoter mutations were detected in 7 cases; 4 papillary thyroid carcinomas (all with preoperative B-VI status), two follicular thyroid carcinomas (one B-IV and one B-V status), and one poorly differentiated thyroid carcinoma (with B-VI status). All mutated cases were verified by mutational analysis of tumor tissue derived from postoperative formalin-fixed paraffin-embedded tissue, while all cases identified as wild-type on FNAC remained wild-type postoperatively. Moreover, the occurrence of a TERT promoter mutation was significantly associated with malignant disease and higher Ki-67 proliferation indices. CONCLUSION: In the present cohort, we found that ddPCR is a highly specific method for detecting high-risk TERT promoter mutations on thyroid FNAC material that could guide different surgical approaches in subsets of indeterminate lesions if reproduced in larger materials.

Thyroglobulin expression, Ki-67 index, and lymph node ratio in the prognostic assessment of papillary thyroid cancer.

The clinical significance of thyroglobulin (Tg) expression in papillary thyroid cancer (PTC) has not been systematically explored in relation to the Ki-67 index, lymph node ratio (LNR), or other conventional prognostic predictors. In this retrospective study of 327 patients with PTC, we investigated the immunohistochemical expression of Tg in both primary tumors and their matching lymph node metastases in relation to the Ki-67 index, LNR, and clinical data. Tumoral Tg immunoreactivity was inversely correlated to the Ki-67 index and tumor recurrence. The Ki-67 index was higher in lymph node metastases (mean 4%) than in the primary tumors (mean 3%). Reduced Tg expression, estimated as 0-25% Tg positive tumor cells, was more common in lymph node metastases compared to primary tumors. In addition to advanced metastatic burden (defined as N1b stage and LNR ≥ 21%), low Tg expression (0-25% positive tumor cells) in lymph node metastases had a significant prognostic impact with shorter recurrence-free survival. These findings support the potential value of histopathological assessment of Tg expression and Ki-67 index in lymph node metastases as complementary predictors to anticipate the prognosis of PTC patients better.

Altered expression of the IGF2­H19 locus and mitochondrial respiratory complexes in adrenocortical carcinoma.

Abnormalities of the insulin­like growth factor 2 (IGF2)­H19 locus with the overexpression of IGF2 are frequent findings in adrenocortical carcinoma (ACC). The present study assessed the expression of RNAs and microRNAs (miRNAs/miRs) from the IGF2­H19 locus using PCR­based methods in ACC and adrenocortical adenoma (ACA). The results were associated with proteomics data. IGF2 was overexpressed in ACC, and its expression correlated with that of miR­483­3p and miR­483­5p hosted by IGF2. The downregulated expression of H19 in ACC compared to ACA correlated with miR­675 expression hosted by H19. Several proteins exhibited an inverse correlation in expression and were predicted as targets of miR­483­3p, miR­483­5p or miR­675. Subsets of these proteins were differentially expressed between ACC and ACA. These included several proteins involved in mitochondrial metabolism. Among the mitochondrial respiratory complexes, complex I and IV were significantly decreased in ACC compared to ACA. The protein expression of NADH:ubiquinone oxidoreductase subunit C1 (NDUFC1), a subunit of mitochondrial respiratory complex I, was further validated as being lower in ACC compared to ACA and normal adrenals. The silencing of miR­483­5p increased NDUFC1 protein expression and reduced both oxygen consumption and glycolysis rates. On the whole, the findings of the present study reveal the dysregulation of the IGF2­H19 locus and mitochondrial respiration in ACC. These findings may provide a basis for the further understanding of the pathogenesis of ACC and may have potential values for diagnostics and treatment.

Metastatic Neuroendocrine Neoplasms of Unknown Primary: Clues from Pathology Workup.

Neuroendocrine neoplasms (NENs) are diverse tumors arising in various anatomical locations and may therefore cause a variety of symptoms leading to their discovery. However, there are instances in which a NEN first presents clinically as a metastatic deposit, while the associated primary tumor is not easily identified using conventional imaging techniques because of small primary tumor sizes. In this setting (which is referred to as a "NEN of unknown primary"; NEN-UP), a tissue biopsy is often procured to allow the surgical pathologist to diagnose the metastatic lesion. If indeed a metastatic NEN-UP is found, several clues can be obtained from morphological assessment and immunohistochemical staining patterns that individually or in concert may help identify the primary tumor site. Herein, histological and auxiliary analyses of value in this context are discussed in order to aid the pathologist when encountering these lesions in clinical practice.

Prognostic Utility of the Ki-67 Labeling Index in Follicular Thyroid Tumors: a 20-Year Experience from a Tertiary Thyroid Center.

Follicular thyroid tumors pose a diagnostic challenge on the preoperative level, as the discrimination between follicular thyroid carcinoma (FTC) and adenoma (FTA) demands careful histopathological investigation. Moreover, prognostication of FTCs is mostly based on tumor size and extent of invasive properties, while immunohistochemical markers pinpointing high-risk cases are lacking. We have routinely established a Ki-67 labeling index for follicular thyroid tumors since 1999. To assess the potential value of Ki-67 as an adjunct tool to (1) correctly separate FTCs from FTAs and (2) help identify poor-prognosis FTCs, we collected histopathological and clinical data from 818 follicular thyroid tumors with a histological Ki-67 labeling index established in clinical routine practice (516 FTAs, 252 FTCs, and 50 follicular thyroid tumors of uncertain malignant potential (FT-UMPs)). The Ki-67 labeling index was higher in FTCs (mean 5.8%) than in FTAs (mean 2.6%) (P < 0.001), and a receiver operating characteristic curve analysis revealed a cut-off value of 4% to separate FTC from FTA with a sensitivity and specificity of 65% and 83%, respectively. Similarly, a Ki-67 labeling index above 4% was found to identify FTCs that later metastasized from clinically indolent FTCs with a sensitivity and specificity of 80% and 48%, respectively. Ki-67 constituted an independent predictor of future FTC metastases/recurrence and death of disease, and a value > 4% was a reliable prognostic marker within individual pT staging groups. We conclude that Ki-67 is a potentially valuable marker for the prognostication of FTCs, and future implementation in the histopathological assessments of follicular thyroid tumors could be beneficial if reproduced in international series.

Synergistic effects of telomerase reverse transcriptase and regulator of telomere elongation helicase 1 on aggressiveness and outcomes in adrenocortical carcinoma.

Adrenocortical carcinoma (ACC) is one of the deadliest endocrine malignancies and telomere maintenance by activated telomerase is critically required for ACC development and progression. Because telomerase reverse transcriptase (TERT) and regulator of telomere elongation helicase 1 (RTEL1) play key roles in telomere homeostasis, we determined their effect on ACC pathogenesis and outcomes. Analyses of TCGA and GEO datasets showed significantly higher expression of RTEL1 but not TERT in ACC tumors, compared to their benign or normal counterparts. Furthermore, gains/amplifications of both TERT and RTEL1 genes were widespread in ACC tumors and their expression correlated with their gene copy numbers. Higher expression of either TERT or RTEL1 was associated with shorter overall and progression-free survival (OS and PFS) in the TCGA ACC patient cohort, and higher levels of both TERT and RTEL1 mRNA predicted the shortest patient OS and PFS. However, multivariate analyses showed that only RTEL1 independently predicted patient OS and PFS. Gene set enrichment analysis further showed enrichments of wnt/ß-catenin, MYC, glycolysis, MTOR, and DNA repair signaling pathways in ACC tumors expressing high TERT and RTEL1 mRNA levels. Taken together, TERT and RTEL1 promote ACC aggressiveness synergistically and may serve as prognostic factors and therapeutic targets for ACC.

Synchronous lateral lymph node metastases from papillary and follicular thyroid carcinoma: case report and review of the literature.

BACKGROUND: Follicular thyroid carcinomas (FTCs) rarely metastasize to regional lymph nodes, and descriptions of synchronous lateral lymph node metastases of FTC and papillary thyroid carcinoma (PTC) are lacking. CASE PRESENTATION: We describe a 43-year-old female with a preoperative cytology indicating a right-sided PTC with lateral lymph node metastases. She underwent a total thyroidectomy and central and lateral lymph node dissection, and histopathology confirmed a multifocal tall cell variant PTC together with a 12 mm minimally invasive FTC in the ipsilateral lobe. While the central compartment demonstrated metastatic PTC, the lateral compartment contained PTC metastases alongside a 15 mm large follicular-patterned mass in a separate lymph node. As the cells lacked PTC associated nuclear changes, the possibility of a lateral lymph node metastasis of FTC was considered, with the possibility of ectopic thyroid tissue as a differential diagnosis. By utilizing next-generation sequencing, a Q61R NRAS mutation was pinpointed, thus proving the tissue as tumorous. The patient underwent radioiodine treatment and is currently monitored following a negative whole-body scan. CONCLUSIONS: This is probably the first case report of a patient with co-existing lateral lymph node PTC and FTC metastases. Consulting previous publications, there is currently a gap of knowledge in terms of how patients with regional FTC metastases should be followed-up and treated, especially when co-occurring with spread high-risk PTC subtypes. Moreover, what guides a seemingly indolent FTC to spread via the lymphatic system remains to be defined from a molecular standpoint.

Cyto-morphological features of parathyroid lesions: Fine-needle aspiration cytology series from an endocrine tumor referral center.

BACKGROUND: Descriptions of parathyroid cell cyto-morphology are limited. Fine-needle aspiration cytology (FNAC) with immunocytochemistry (ICC) or biochemical PTH measurements may help verify the parathyroid origin in extraordinary cases, although these methods are nowadays largely replaced by imaging techniques. METHODS: We collected all available FNAC reports of parathyroid lesions from our department spanning 20 years, to characterize the clinical use of parathyroid FNAC, and to assess morphological correlates between cytology and subsequent histopathology. RESULTS: Twenty-eight cases with assessable cytological smears were found, of which 21 cases were surgically resected and 20 available for histological review (15 adenomas, 2 carcinomas, 2 atypical tumors and a single case with secondary hyperplasia). FNAC was predominantly performed to localize the diseased gland in cases with inconclusive imaging, in cases with a suspicion of intrathyroidal localization or in cases with persistent hypercalcemia following unsuccessful surgery. The diagnosis was verified using either PTH ICC and/or PTH measurements for most cases, and the procedure affected the clinical decision-making for the majority of patients in this selected cohort. Cytological differences between parathyroid adenomas and carcinomas were found, as only carcinomas showed pleomorphism with irregular nuclei and prominent nucleoli. Morphologically, no correlations to predominant cell types or growth patterns visualized at histological investigations among adenomas were noted, and biopsy artifacts were evident in 40% of cases. CONCLUSIONS: Parathyroid FNAC could be considered as a complementary analysis for a small group of selected patients, but benefits have to be weighed against the risk of biopsy artifacts in histological preparations.

Pan-Genomic Sequencing Reveals Actionable CDKN2A/2B Deletions and Kataegis in Anaplastic Thyroid Carcinoma.

Anaplastic thyroid carcinoma (ATC) is a lethal malignancy characterized by poor response to conventional therapies. Whole-genome sequencing (WGS) analyses of this tumor type are limited, and we therefore interrogated eight ATCs using WGS and RNA sequencing. Five out of eight cases (63%) displayed cyclin-dependent kinase inhibitor 2A (CDKN2A) abnormalities, either copy number loss (n = 4) or truncating mutations (n = 1). All four cases with loss of the CDKN2A locus (encoding p16 and p14arf) also exhibited loss of the neighboring CDKN2B gene (encoding p15ink4b), and displayed reduced CDKN2A/2B mRNA levels. Mutations in established ATC-related genes were observed, including TP53, BRAF, ARID1A, and RB1, and overrepresentation of mutations were also noted in 13 additional cancer genes. One of the more predominant mutational signatures was intimately coupled to the activity of Apolipoprotein B mRNA-editing enzyme, the catalytic polypeptide-like (APOBEC) family of cytidine deaminases implied in kataegis, a focal hypermutation phenotype, which was observed in 4/8 (50%) cases. We corroborate the roles of CDKN2A/2B in ATC development and identify kataegis as a recurrent phenomenon. Our findings pinpoint clinically relevant alterations, which may indicate response to CDK inhibitors, and focal hypermutational phenotypes that may be coupled to improved responses using immune checkpoint inhibitors.