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Background The spectrum of neuroendocrine neoplasia (NEN) of the head and neck region is wide-ranging and diverse, including a variety of diagnoses stretching from benign and low-malignant tumor forms to highly proliferative, poor prognosis neuroendocrine carcinoma (NEC). Moreover, there are several non-neuroendocrine differential diagnoses to keep in mind as well, displaying various degree of morphological and/or immunohistochemical overlap with bona fide neuroendocrine lesions. Methods Review. Results While the growth patterns may vary, well-differentiated NEN usually display a stippled “salt and pepper” chromatin, a granular cytoplasm, and unequivocal expression of neuroendocrine markers such as chromogranin A and synaptophysin. However, these features are often less pronounced in NEC, which may cause diagnostic confusion—not the least since several non-NEC head and neck tumors may exhibit morphological similarities and focal neuroendocrine differentiation. Conclusion As patients with NEC may require specific adjuvant treatment and follow-up, knowledge regarding differential diagnoses and potential pitfalls is therefore clinically relevant. In this review, the top ten morphological and/or immunohistochemical mimics of NEC are detailed in terms of histology, immunohistochemistry, and molecular genetics.

Pheochromocytoma and abdominal paraganglioma (PPGL) are rare neuroendocrine tumors originating from chromaffin cells. Even though only 10–15% of the tumors metastasize, all PPGLs are considered potentially malignant. Topoisomerase 2A (TOP2A) is a protein involved in cell proliferation and has been found to be over-expressed in metastatic PPGL. To provide support whether TOP2A could serve as a prognostic marker, 88 PPGLs (of which 8 metastatic/relapsing) and 10 normal adrenal gland samples were assessed for TOP2A mRNA expression using quantitative real-time PCR (qRT-PCR) and TOP2A immunohistochemistry. Comparisons to clinical parameters connected to metastatic behavior were made, and The Cancer Genome Atlas was used for validation of the results. A significant association between high TOP2A mRNA expression in primary PPGL and subsequent metastatic events (p = 0.008) was found, as well as to specific histological features and clinical parameters connected to metastatic behavior and mutations in SDHB. TOP2A immunoreactivity was calculated as an index of positive nuclei divided by the total amount of nuclei, and this index associated with TOP2A mRNA levels (p = 0.023) as well as the Ki-67 labeling index (p = 0.001). To conclude, TOP2A is a potential prognostic marker as it is frequently elevated in PPGL displaying subsequent metastatic disease, and future studies in larger cohorts are warranted to determine if a TOP2A index as assessed by immunohistochemistry could be a marker of poor outcome. Additionally, elevated levels of TOP2A could indicate a potential actionable event, and future studies with topoisomerase inhibitors would be of interest.

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 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.

Supplemental Table 1. NK cell specific lysis of human cell lines. Effector-to-target ratio = 3:1. Co-culture = 18h. ATC = anaplastic thyroid carcinoma, PTC = papillary thyroid carcinoma, CML = Chronic myelogenous leukemia: SD = standard deviation.

Supplemental Figure 2. (A) Gating strategy for NKG2D expression on NK cells in FNA and PBMCs from an ATC patient. Lymphocytes are gated according to size (forward scatter FSC) and granularity (side scatter SSC), viability (7AAD- cells), NK cell phenotype (CD3-CD56+ cells) and NKG2D expression is plotted as a histogram where solid grey histogram represent isotype control antibody staining and open histogram represent staining with NKG2D-specific antibody. (B) Gating strategy for ULBP2/5/6 expression in FNA from an ATC patient. Tumor cells are first gated according to size (forward scatter FSC), granularity (side scatter SSC) and viability (LIVE/DEAD- cells). Granulocytes that may appear in the same FSC/SSC population are gated out by excluding the CD11c+ cells. ULBP2/5/6 expression is plotted as a histogram where solid grey histogram represents isotype control. antibody staining and open histogram represents staining with ULBP2/5/6-specific antibody. FNA = fine needle aspirate. PBMC = peripheral blood mononuclear cells.

Purpose: Anaplastic thyroid carcinoma (ATC) is one of the most aggressive forms of cancer with no curative therapies available. To date, strategies to target ATC by immunotherapy have not been evaluated. We investigated whether ATC would be a suitable target for natural killer (NK) cell–based immunotherapy.Experimental Design: We first established seven new cell lines from ATC tumors, three from papillary thyroid carcinoma tumors and analyzed them together with eight additional ATC cell lines. Cells were analyzed for sensitivity to lysis by NK cells and their ability to chemoattract and regulate the activity of NK cells. In addition, fresh tumor samples and peripheral blood from six patients with ATC were analyzed for NK cell infiltration and phenotype.Results: We observed that ATC cell lines are sensitive to lysis by ex vivo expanded NK cells and that the lysis was abrogated upon blockade of NKG2D. Sensitivity of thyroid cancer cell lines to NK cell–mediated lysis correlated with surface expression of UL16-binding protein 2 on tumor cells. Moreover, ATC cell lines produced high levels of CXCL10 and stimulated migration of expanded NK cells and ATC tumors were enriched for NK cells expressing the cognate chemokine receptor CXCR3. However, compared with NK cells in peripheral blood, ATC tumor–derived NK cells displayed a suppressed phenotype with a downregulated expression of NKG2D. In vitro, suppression of NK cell–mediated lysis and NKG2D expression by ATC cells was restored upon neutralization of prostaglandin-E2.Conclusions: ATC cell lines are sensitive to NK cell–mediated lysis via ULBP2/5/6 and chemoattract CXCR3-positive NK cells. Patients with ATC may benefit from NK cell–based immunotherapy. Clin Cancer Res; 20(22); 5733–44. ©2014 AACR.

Supplemental Figure 1. (A) Annexin V/7AAD viability staining of ATC cells (U-Hth 104 cell line), non-malignant thyroid cells(non-toxic goiter), immortalized fibroblast cells (BJ cell line) autologous B cells and T cells cultured with or without expanded NK cells at a 1:1 ratio for 4 h. (B) Mean fluorescence intensity (MFI) of NKG2D-ligands on viable tumor cells from ATC FNA. MFI values from NKG2D-ligand staining have been subtracted with isotype control MFI values (C) Specific lysis of the ATC cell line LUTC-17 by autologous NK cells in a 5h cytotoxicity assay. Blocking antibodies targeting ULBP2 or NKG2D was added 30 minutes prior and during the cytotoxicity assay. Effector-to-target ratio = 2:1. The experiment was performed once.

Purpose TERT promoter mutations and TERT gene expression correlate to adverse prognosis in follicular thyroid carcinoma (FTC), identifying cases at risk of poor outcome. As loss of 5-hydroxymethylcytosine (5hmC) immunoreactivity has been associated with TERT promoter mutations in papillary thyroid carcinoma, this study sought to analyze the levels of 5hmC in a well-characterized cohort of follicular thyroid tumors with available TERT data. Methods 29 tumors (26 FTCs, 2 follicular thyroid tumors of uncertain malignant potential (FT-UMPs), and one oncocytic thyroid carcinoma) with known TERT promoter mutational status and TERT gene expression levels were assessed for 5hmC immunoreactivity using two monoclonal antibodies (clones RM236 and 4D9.) Slides were analyzed using a semiquantitative scoring system. Results Of the 10 tumor cases with a TERT promoter mutation and TERT expression, only one was scored as negative with both antibodies (1/10; 10%), while the remaining 9 cases (9/10; 90%) exhibited various degrees of positivity for at least one antibody. Of the 19 TERT wild-type tumors, no case (0/19; 0%) was scored as negative using the RM236 clone, and two cases (2/19; 11%) using the 4D9 clone. The differences between TERT promoter mutated and wildtype groups were non-significant (Fisher’s Exact test P = 0.35 and 0.59 respectively). The sensitivity and specificity for 5hmC IHC to detect mutated cases were 10% and 100% for RM236 and 20% and 89% for 4D9 respectively. Conclusion 5hmC IHC is not a highly sensitive marker for the detection of TERT promoter mutations in follicular thyroid tumors. Further analyses in larger cohorts are warranted.

Background The genomic and transcriptomic landscape of widely invasive follicular thyroid carcinomas (wiFTCs) and Hürthle cell carcinoma (HCC) are poorly characterized, and subsets of these tumors lack information on genetic driver events. Objective The aim of this study was to bridge this gap. Methods We performed whole-genome and RNA sequencing and subsequent bioinformatic analyses of 11 wiFTCs and 2 HCCs with a particularly poor prognosis, and matched normal tissue. Results All wiFTCs exhibited one or several mutations in established thyroid cancer genes, including TERT (n = 4), NRAS (n = 3), HRAS, KRAS, AKT, PTEN, PIK3CA, MUTYH, TSHR, and MEN1 (n = 1 each). MutSig2CV analysis revealed recurrent somatic mutations in FAM72D (n = 3, in 2 wiFTCs and in a single HCC), TP53 (n = 3, in 2 wiFTCs and a single HCC), and EIF1AX (n = 3), with DGCR8 (n = 2) as borderline significant. The DGCR8 mutations were recurrent p.E518K missense alterations, known to cause familial multinodular goiter via disruption of microRNA (miRNA) processing. Expression analyses showed reduced DGCR8 messenger RNA expression in FTCs in general, and the 2 DGCR8 mutants displayed a distinct miRNA profile compared to DGCR8 wild-types. Copy number analyses revealed recurrent gains on chromosomes 4, 6, and 10, and fusion gene analyses revealed 27 high-quality events. Both HCCs displayed hyperploidy, which was fairly unusual in the FTC cohort. Based on the transcriptome data, tumors amassed in 2 principal clusters. Conclusion We describe the genomic and transcriptomic landscape in wiFTCs and HCCs and identify novel recurrent mutations and copy number alterations with possible driver properties and lay the foundation for future studies.

AimsUpregulation of the telomerase reverse transcriptase (TERT) gene is a frequent finding in follicular thyroid carcinomas (FTCs) with metastatic features. The augmented expression is usually caused by TERT promoter mutations. As TERT protein immunohistochemistry might not correlate to TERT mRNA levels in follicular thyroid tumours, we therefore sought to determine if visualisation of TERT mRNA through in situ hybridisation could highlight high-risk cases.MethodsWe collected formalin-fixated paraffin-embedded tissues from 26 follicular thyroid tumours; 7 FTCs, 2 follicular thyroid tumours of uncertain malignant potential (FT-UMPs) and a single Hürthle cell carcinoma with established TERT promoter mutations and gene expression, as well as 16 FTCs with no TERT gene aberrancy or gene expression, and assessed them using RNA Scope in situ hybridisation (ISH) and TERT probes targeting the two main TERT transcripts (TERT1 and TERT2).ResultsTERT 1 and/or 2 mRNA was found by ISH in 8/10 cases with established promoter mutations and mRNA expression, whereas all 16 cases without TERT gene aberrancies or gene expression were negative (Fisher’s exact pTERT mRNA was visualised in the nuclear compartment only, thereby corroborating earlier studies suggesting a non-conventional role for TERT in tumour biology. Moreover, TERT mRNA expression was scattered across the tissue sections and only found in a few percentages of tumour nuclei.ConclusionsTERT mRNA seems to be focally expressed and localised exclusively to the nucleus in TERT promoter mutated follicular thyroid tumours, possibly reflecting a true biological and unorthodox phenomenon worthy of further investigations.

Abdominal paragangliomas and pheochromocytomas (PPGLs) are rare neuroendocrine tumors of the infradiaphragmatic paraganglia and adrenal medulla, respectively. Although few pathologists outside of endocrine tertiary centers will ever diagnose such a lesion, the tumors are well known through the medical community—possible due to a combination of the sheer rarity, their often-spectacular presentation due to excess catecholamine secretion as well as their unrivaled coupling to constitutional susceptibility gene mutations and hereditary syndromes. All PPGLs are thought to harbor malignant potential, and therefore pose several challenges to the practicing pathologist. Specifically, a responsible diagnostician should recognize both the capacity and limitations of histological, immunohistochemical, and molecular algorithms to pinpoint high risk for future metastatic disease. This focused review aims to provide the surgical pathologist with a condensed update regarding the current strategies available in order to deliver an accurate prognostication of these enigmatic lesions.

Contains fulltext : 245825.pdf (Publisher’s version ) (Closed access) BACKGROUND: Phaeochromocytoma or paraganglioma (collectively known as PPGL) in pregnant women can lead to severe complications and death due to associated catecholamine excess. We aimed to identify factors associated with maternal and fetal outcomes in women with PPGL during pregnancy. METHODS: We did a multicentre, retrospective study of patients with PPGL and pregnancy between Jan 1, 1980, and Dec 31, 2019, in the International Pheochromocytoma and Pregnancy Registry and a systematic review of studies published between Jan 1, 2005, and Dec 27, 2019 reporting on at least five cases. The inclusion criteria were pregnancy after 1980 and PPGL before or during pregnancy or within 12 months post partum. Eligible patients from the retrospective study and systematic review were included in the analysis. Outcomes of interest were maternal or fetal death and maternal severe cardiovascular complications of catecholamine excess. Potential variables associated with these outcomes were evaluated by logistic regression. FINDINGS: The systematic review identified seven studies (reporting on 63 pregnancies in 55 patients) that met the eligibility criteria and were of adequate quality. A further 197 pregnancies in 186 patients were identified in the International Pheochromocytoma and Pregnancy Registry. After excluding 11 pregnancies due to potential overlap, the final cohort included 249 pregnancies in 232 patients with PPGL. The diagnosis of PPGL was made before pregnancy in 37 (15%) pregnancies, during pregnancy in 134 (54%), and after delivery in 78 (31%). Of 144 patients evaluated for genetic predisposition for phaeochromocytoma, 95 (66%) were positive. Unrecognised PPGL during pregnancy (odds ratio 27·0; 95% CI 3·5-3473·1), abdominal or pelvic tumour location (11·3; 1·5-1440·5), and catecholamine excess at least ten-times the upper limit of the normal range (4·7; 1·8-13·8) were associated with adverse outcomes. For patients diagnosed during pregnancy, α-adrenergic blockade therapy was associated with fewer adverse outcomes (3·6; 1·1-13·2 for no α-adrenergic blockade vs α-adrenergic blockade), whereas surgery during pregnancy was not associated with better outcomes (0·9; 0·3-3·9 for no surgery vs surgery). INTERPRETATION: Unrecognised and untreated PPGL was associated with a substantially higher risk of either maternal or fetal complications. Appropriate case detection and counselling for premenopausal women at risk for PPGL could prevent adverse pregnancy-related outcomes. FUNDING: US National Institutes of Health.

The majority of parathyroid disease encountered in routine practice is due to single parathyroid adenoma, of which the majority arise as sporadic tumors. This is usually a straightforward diagnosis in endocrine pathology when in the appropriate clinical setting, although subsets of cases will exhibit atypical histological features that may warrant additional immunohistochemical and genetic analyses to estimate the malignant potential. Parathyroid carcinomas on the other hand, are bona fide malignant tumors characterized by their unequivocal invasion demonstrated through routine histology or metastasis. The ultimate endpoint for any molecular marker discovered through laboratory investigations is to be introduced in clinical routine practice and guide the surgical pathologist in terms of diagnostics and prognostication. For parathyroid tumors, the two main diagnostic challenges include the distinction between parathyroid adenoma and parathyroid carcinoma, as well as the pinpointing of hereditable disease for familial screening purposes. While numerous markers on genetic, epigenetic, and protein levels have been proposed as discriminative in these aspects, this review aims to condense the scientific coverage of these enigmatic topics and to propose a focused surgical pathology approach to the subject.

While minimally invasive follicular thyroid cancer (miFTC) generally has low risk of recurrence or death, encapsulated angioinvasive (eaFTC) or widely invasive (wiFTC) histological subtypes display significantly worse prognosis. Drivers of invasion are incompletely understood. Therefore, tissue samples including miFTC, eaFTC, and wiFTC tumors, as well as histologically normal thyroid adjacent to benign follicular adenomas, were selected from a cohort (n = 21) of thyroid tumor patients, and the gene expression of selected transcription factors was characterized with quantitative PCR. Invasion-relevant spatial expression patterns of selected transcription factors were subsequently characterized with immunohistochemistry. E2F1 was over-expressed in all 3 subtypes (pSP1 was differentially expressed in eaFTC and wiFTC compared with normal (p=0.01 and 0.04, respectively). TCF7L2 was significantly upregulated in wiFTC specifically (p

Neuroendocrine neoplasms (NENs) have traditionally been identified via expression of proteins associated to the regulation of secretory vesicles and granules. We report the clinical usage of the “second-generation” proteins ISL LIM homeobox 1 (ISL1), INSM transcriptional repressor 1 (INSM1), and secretagogin (SECG) as immunohistochemical markers of neuroendocrine differentiation since their introduction in clinical routine and compare the results with the established proteins chromogranin A (CGA) and synaptophysin (SYP). In total, 161 tumors, including 139 NENs and 22 “non-NENs” (unrelated tumors with an initial suspicion of NEN), were informatively stained for ISL1, and subsets were also interrogated for INSM1 and/or SECG. Diffuse or focal positive immunoreactivity was noted for ISL1 in 91/139 NENs (65%) and in 6/22 (27%) non-NENs, for INSM1 in 76/85 NENs (89%) and in 2/5 (40%) non-NENs, and for SECG in 49 out of 64 NENs (77%) and in 0/5 non-NENs (0%). Generally, ISL1, INSM1, and SECG exhibited sensitivities in line with or slightly below that of CGA and SYP—largely attributable to tissue-specific patterns regarding tumoral origin. Moreover, for pancreatic and small intestinal NENs, the two largest subgroups, ISL1 staining results were consistent irrespectively of tumor source and WHO grade. We verify previously suggested immunohistochemical schemes of neuroendocrine markers of first- and second-generations to facilitate the diagnostic process for NENs and confirm that the second-generation neuroendocrine markers display tissue-specific patterns. We therefore recommend their implementation in tertiary endocrine pathology centers, not least to aid in the identification of primary tumors when analyzing metastases. Electronic supplementary material The online version of this article (10.1007/s12022-020-09645-y) contains supplementary material, which is available to authorized users.

Benign thyroid lesions such as multinodular goiter and adenomatoid nodules are well-circumscribed lesions displaying a macrofollicular growth pattern and lack of nuclear atypia. The highly unusual macrofollicular variant of follicular thyroid carcinoma (MV-FTC) mirrors these attributes and is thereby misclassified by cytological examination of fine-needle aspiration biopsies. The MV-FTC diagnosis is instead suggested following histological investigation, in which malignant attributes, most commonly capsular invasion, are noted. The bulk of MV-FTCs described in the literature arise in younger female patients and carry an excellent prognosis. A recent coupling to mutations in the DICER1 tumor suppressor gene has been proposed, possibly indicating aberrancies in micro-RNA (miRNA) patterns as responsible of the tumorigenic process. We describe the cytological, histological and molecular phenotype of a 35 mm large MV-FTC arising in the right thyroid lobe of a 33-year-old female with a family history of multinodular goiter. The tumor was encapsulated and strikingly inconspicuous in terms of cellularity and atypia, but nevertheless displayed multiple foci with capsular invasion. A next-generation molecular screening of tumor DNA revealed missense variants in DICER1 (p. D1709N) and MET (p. T1010I), but no established fusion gene events. After sequencing of germline DNA, the DICER1 mutation was confirmed as somatic, while the MET variant was constitutional. The patient is alive and well, currently awaiting radioiodine treatment. This MV-FTC mirrors previous publications, suggesting that these tumors carry a favorable prognosis and predominantly arise in younger females. Moreover, DICER1 mutations should be considered a common driver event in the development of MV-FTCs.

Context Riedel thyroiditis (RT) is a rare inflammatory autoimmune disease that is often a clinically diagnostic dilemma because of its insidious presentation and nonspecific symptoms. Objective The aim of the present systematic review and meta-analysis is to clarify the presentation, management, and outcomes of RT. Study Selection A systematic search of PubMed/MEDLINE and Web of Science was conducted to identify relevant reports published up to September 2019. Data Extraction First author, country, patient sex, ethnicity, presentation, biochemical status, duration of symptoms, histology, treatment, follow-up duration, and short- and long-term outcomes. Data Synthesis Data from 212 RT patients were retrieved. The mean age was 47 years with a predominantly female population (81%). Neck swelling (89%), dyspnea (50%), and neck pain (41%) were the most common presenting symptoms. Inflammatory markers were elevated in 70% to 97% and thyroid antibody positivity was present in less than 50%. Up to 82% underwent surgical intervention, with the most common being total thyroidectomy in 34% of individuals. Glucocorticoids were used in 70% of individuals with median duration 3 months. Prognosis was reasonable with 90% having resolution or improvement of symptoms. Conclusions This analysis is the largest and most comprehensive to date of RT and provides clinicians with vital information on the common presentation features that may alert to the diagnosis and highlight management options.

Rarely, salivary gland tumors such as mucoepidermoid carcinoma, mammary analogue secretory carcinoma and mucinous carcinoma arise as primary tumors from ectopic or metaplastic salivary gland tissue adjacent to or within the thyroid gland. We report for the first time a case of primary salivary acinic cell carcinoma (AcCC) adjacent to the thyroid gland in a 71-year-old female patient with Crohns disease and a previous history of malignant melanoma. Following the development of a nodule adjacent to the left thyroid lobe, a fine-needle aspiration biopsy was reported as consistent with a follicular lesion of undetermined significance (Bethesda III). A left-sided hemithyroidectomy was performed. A circumscribed lesion measuring 33 mm was noted adjacent to the thyroid and trapping parathyroid, it was composed of solid nests and glands with microcystic and follicular patterns. The tumor was negative for thyroid, parathyroid and paraganglioma markers, but positive for pan-cytokeratins, CK7, CD10, CD117, androgen receptor and HNF-beta. A metastasis of a thyroid-like renal cell carcinoma was suspected but ruled out, and the patient had no evident lesions on extensive radiology of the urogenital, pulmonary and GI tracts. Based on the morphology, a diagnosis of AcCC was suggested, and confirmed with DOG1 and PAS-diastase staining. Molecular analyses pinpointed a constitutional ASXL1 variant of uncertain significance, but no fusion events. The patient had no radiological or clinical evidence of parotid, submandibular or sublingual tumors postoperatively, and the excised lesion was therefore assumed to be a primary tumor. We here detail the morphological and immunophenotypic profile of this previously undescribed perithyroidal tumor.

Parathyroid lipoadenomas (PLAs) are rare tumors, and case descriptions are limited, 50% fat on histologic examination with preoperative evidence of primary hyperparathyroidism (PHPT). A total of 8 bona fide PLA cases and 2 equivocal PLAs (close to 50% fat) were included. As approximately 4000 conventional parathyroid adenomas were diagnosed at our department during the same time interval, the prevalence of PLA was 0.20%. PLA patients were predominately female (63%) and presented with classical PHPT-related symptoms. Majority of cases were successfully located preoperatively and had an average tumor weight of 445 mg. Histologically, all PLAs consisted of > 50% mature adipose tissue, except a single case with brown fat. Of note, PLA patients exhibited a body mass index in line with PHPT patients in general, but a relatively high, near-significant prevalence of arterial hypertension was observed when compared to tumors with less fat (P = 0.0584). Future studies on this finding might be warranted. To summarize, we present one of the largest institutional PLA case series to date, and conclude that PLAs are rare, sporadic tumors mirroring many clinical aspects of conventional adenomas—with a potential coupling to hypertension worthy of follow-up studies. Electronic supplementary material The online version of this article (10.1007/s12022-020-09616-3) contains supplementary material, which is available to authorized users.

Anaplastic thyroid carcinoma (ATC) exhibits an exceedingly poor prognosis, and the current treatment options are, for most cases, palliative by nature. Few reports of long-time survivors exist, although in these patients, tumors often were limited to the thyroid and/or regional lymph nodes. We describe a 64-year-old male who developed a rapidly growing mass in the left thyroid lobe. A fine-needle aspiration biopsy (FNAB) was consistent with ATC, and the patient underwent preoperative combined chemo- and radiotherapy followed by a hemithyroidectomy. The ensuing histopathological investigation was consistent with ATC adjoined by an oxyphilic well-differentiated lesion, likely a Hürthle cell carcinoma. Tumor margins were negative, and no extrathyroidal extension was noted. Focused next-generation sequencing analysis of the primary tumor tissue identified a TP53 gene mutation but could not identify any potential druggable targets. Additional Sanger sequencing detected a C228T TERT promoter mutation. The tumor was found to be microsatellite stable and displayed PDL1 expression in 80% of tumor cells. Following a CT scan 1 month postoperatively, metastatic deposits were suspected in the lung as well as in the left adrenal gland, of which FNAB verified the latter. Remarkably, upon radiological follow-up, the disease had gone into apparent complete remission. The patient is alive and well with no signs of residual disease after 12 months of follow-up. We here summarize the clinical, histological, and molecular data of this highly interesting patient case and review the literature for possible common denominators with other patients with disseminated ATC.

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/β-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.

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.

Summary: Merkel cell carcinoma is an aggressive skin malignancy, mostly caused by Merkel cell polyomavirus (MCPyV). MCPyV T-antigens can induce mature microRNA expressions through the DnaJ domain, but its underlying mechanism is still unknown. Here, we report that the T-antigens induce protein expression and mRNA stability of DICER1, a key factor in microRNA biogenesis, through heat shock cognate 70 (HSC70). HSC70 directly interacts with the AU-rich elements (ARE) of DICER1 mRNA in both coding and 3′ untranslated region in the presence of MCPyV T-antigen. The T-antigen/HSC70 interaction could induce luciferase activity of synthetic ARE-containing reporter, as well as the stability of ARE-containing mRNAs, suggesting a broader role of MCPyV T-antigens in regulating multiple mRNAs via HSC70. These findings highlight a new role for the interaction of HSC70 and MCPyV T-antigens in mRNA regulation and an undescribed regulatory mechanism of DICER1 mRNA stability and translation through its direct interaction with HSC70.

Childhood neuroblastoma has a remarkable variability in outcome. Age at diagnosis is one of the most important prognostic factors, with children less than 1 year old having favorable outcomes. Here we study single-cell and single-nuclei transcriptomes of neuroblastoma with different clinical risk groups and stages, including healthy adrenal gland. We compare tumor cell populations with embryonic mouse sympatho-adrenal derivatives, and post-natal human adrenal gland. We provide evidence that low and high-risk neuroblastoma have different cell identities, representing two disease entities. Low-risk neuroblastoma presents a transcriptome that resembles sympatho- and chromaffin cells, whereas malignant cells enriched in high-risk neuroblastoma resembles a subtype of TRKB+ cholinergic progenitor population identified in human post-natal gland. Analyses of these populations reveal different gene expression programs for worst and better survival in correlation with age at diagnosis. Our findings reveal two cellular identities and a composition of human neuroblastoma tumors reflecting clinical heterogeneity and outcome., Childhood neuroblastoma can be separated into high and low risk groups, with prognosis depending on age at diagnosis. Here, the authors show that low and high risk neuroblastoma tumours are composed of different cell types with different malignancy potential.

Background Thyroid cancer dedifferentiation is an unusual observation among young patients and is poorly understood, although a recent correlation to DICER1 gene mutations has been proposed. Case presentation A 28-year old patient presented with a sub-centimeter cytology-verified primary papillary thyroid carcinoma (PTC) and a synchronous lateral lymph node metastasis. Following surgery, histopathology confirmed a 9 mm oxyphilic PTC and a synchronous metastasis of poorly differentiated thyroid carcinoma (PDTC). Extensive molecular examinations of both lesions revealed wildtype DICER1 sequences, but identified a somatic ETV6-NTRK3 gene fusion and a MET germline variant (c.1076G > A, p.Arg359Gln). MET is an established oncogene known to be overexpressed in thyroid cancer, and this specific alteration was not reported as a single nucleotide polymorphism (SNP), suggestive of a mutation. Both the primary PTC and the metastatic PDTC displayed strong MET immunoreactivity. A validation cohort of 50 PTCs from young patients were analyzed using quantitative real-time PCR, revealing significantly higher MET gene expression in tumors than normal thyroid controls, a finding which was particularly pronounced in BRAF V600E mutated cases. No additional tumors apart from the index case harbored the p.Arg359Gln MET mutation. Transfecting PTC cell lines MDA-T32 and MDA-T41 with a p.Arg359Gln MET plasmid construct revealed no obvious effects on cellular migratory or invasive properties, whereas overexpression of wildtype MET stimulated invasion. Conclusions The question of whether the observed MET mutation in any way influenced the dedifferentiation of a primary PTC into a PDTC metastasis remains to be established. Moreover, our data corroborate earlier studies, indicating that MET is aberrantly expressed in PTC and may influence the invasive behavior of these tumors.

The cell division cycle 73 gene is mutated in familial and sporadic forms of primary hyperparathyroidism, and the corresponding protein product parafibromin has been proposed as an adjunct immunohistochemical marker for the identification of cell division cycle 73 mutations and parathyroid carcinoma. Here, we present data from our experiences using parafibromin immunohistochemistry in parathyroid tumors since the marker was implemented in clinical routine in 2010. A total of 2019 parathyroid adenomas, atypical adenomas, and carcinomas were diagnosed in our department, and parafibromin staining was ordered for 297 cases with an initial suspicion of malignant potential to avoid excessive numbers of false positives. The most common inclusion criteria for immunohistochemistry were marked tumor weight (146 cases) and/or fibrosis (77 cases) and/or marked pleomorphism (58 cases). In total, 238 cases were informatively stained, and partial or complete loss of nuclear parafibromin immunoreactivity was noted in 40 cases; 10 out of 182 adenomas (5%), 27 out of 46 atypical adenomas (59%), and 7 out of 10 carcinomas (70%), with positive and negative predictive values of 85 and 90%, respectively for the detection of atypical adenomas/carcinomas versus adenomas, and 18 and 98%, respectively for carcinomas versus atypical adenomas/adenomas. Male patients with high-proliferative tumors were overrepresented among cases with aberrant parafibromin immunohistochemistry, and carcinomas more frequently harbored parafibromin aberrancies than atypical adenomas and adenomas (p

Telomere maintenance is a critical requirement for enabling replicative immortality and tumour development. Here, telomerase expression and activity, telomere length (TL) and potential regulatory factors that can underlie telomerase machinery alterations in small intestinal neuroendocrine tumours (SI-NETs) were analyzed. Telomerase activity assessed by TRAP assay was increased in SI-NETs compared to normal ileum (P < 0.001). The telomerase reverse transcriptase gene (TERT) was over-expressed in SI-NETs vs. normal ileal samples (P = 0.01). Furthermore, relative TL assessed by qPCR was found shorter in tumours compared with normal ileum (P = 0.02) and in distant metastasis samples compared to primary tumours and local metastases (P= 0.02). TERT promoter hotspot mutations were not present and TERT copy number gain was only observed in 3/70 tumour samples. TERT or chromosome 18 copy number alterations were not associated with telomerase expression and activity or TL. However, hypermethylation of TERT promoter in Region B - in the proximity of the transcription start site - was inversely correlated with TERT expression and telomerase activity and positively correlated with TL. Global LINE1 methylation was positively correlated with TERT promoter Region B methylation and was inversely correlated with telomerase activity, TERT expression and the upstream Region A methylation. The results show that telomerase activation, TERT expression and shorter telomeres are commonly found in SI-NETs. Aberrant DNA methylation of TERT promoter and of LINE1 can be implicated in abnormal regulation of TERT in SI-NETs.

Context Information about adrenal medullary hyperplasia (AMH) is scarce. Objective To study a large cohort of AMHs. Design, Setting, and Participants Nineteen AMH cases were compared with 95 pheochromocytomas (PCCs) without AMH. AMH without (n = 7) and with PCC (n = 12) were analyzed separately. Results Of 936 adrenalectomies, 2.1% had AMH. Mean age was 47.2 ± 15.1 years. Only two (11%) AMHs had no concurrent PCC or adrenocortical adenoma. In AMHs, a genetic syndrome was present in 58% vs 4% in PCCs (P < 0.001). The noradrenaline/metanephrine levels were lower in AMHs, whereas suppression of dexamethasone was less than in PCCs. Cushing syndrome was found in 11% of AMHs. More AMHs were found during screening and less as incidentalomas. PCC symptoms were less prevalent in AMHs. Surgical management was similar; however, fewer of the AMHs were pretreated with alpha-blockers. Adrenalectomy improved blood pressure slightly less in AMHs. The disappearance of glycemic disturbances was similar to the PPCs. During a period of 11.2 ± 9.4 years, a new PCC developed in 32% of patients with AMH, 11% died, but no PCC metastasis occurred (PCCs: 4%, P < 0.001; 14% and 5%). AMHs without PCC had milder symptoms but more often Cushing disease than patients with PCC, whereas AMH with PCC more often displayed a familiar syndrome with more PCC recurrences. Conclusion A total of 2.1% of all adrenalectomies displayed AMH. AMH seemed to be a PCC precursor. The symptoms and signs were milder than PCCs. AMHs were mainly found due to screening. Outcomes seemed favorable, but new PCCs developed in many during follow-up.

Pediatric papillary thyroid carcinomas (pPTCs) are often indolent tumors with excellent long-term outcome, although subsets of cases are clinically troublesome and recur. Although it is generally thought to exhibit similar molecular aberrancies as their counterpart tumors in adults, the pan-genomic landscape of clinically aggressive pPTCs has not been previously described. In this study, five pairs of primary and synchronously metastatic pPTC from patients with high-risk phenotypes were characterized using parallel whole-genome and -transcriptome sequencing. Primary tumors and their metastatic components displayed an exceedingly low number of coding somatic mutations and gross chromosomal alterations overall, with surprisingly few shared mutational events. Two cases exhibited one established gene fusion event each (SQSTM1-NTRK3 and NCOA4-RET) in both primary and metastatic tissues, and one case each was positive for a BRAF V600E mutation and a germline truncating CHEK2 mutation, respectively. One single case was without apparent driver events and was considered as a genetic orphan. Non-coding mutations in cancer-associated regions were generally not present. By expressional analyses, fusion-driven primary and metastatic pPTC clustered separately from the mutation-driven cases and the sole genetic orphan. We conclude that pPTCs are genetically indolent tumors with exceedingly stable genomes. Several mutations found exclusively in the metastatic samples which may represent novel genetic events that drive the metastatic behavior, and the differences in mutational compositions suggest early clonal divergence between primary tumors and metastases. Moreover, an overrepresentation of mutational and expressional dysregulation of immune regulatory pathways was noted among fusion-positive pPTC metastases, suggesting that these tumors might facilitate spread through immune evasive mechanisms.

Mitochondria are the main consumers of oxygen within the cell. How mitochondria sense oxygen levels remains unknown. Here we show an oxygen-sensitive regulation of TFAM, an activator of mitochondrial transcription and replication, whose alteration is linked to tumours arising in the von Hippel–Lindau syndrome. TFAM is hydroxylated by EGLN3 and subsequently bound by the von Hippel–Lindau tumour-suppressor protein, which stabilizes TFAM by preventing mitochondrial proteolysis. Cells lacking wild-type VHL or in which EGLN3 is inactivated have reduced mitochondrial mass. Tumorigenic VHL variants leading to different clinical manifestations fail to bind hydroxylated TFAM. In contrast, cells harbouring the Chuvash polycythaemia VHLR200W mutation, involved in hypoxia-sensing disorders without tumour development, are capable of binding hydroxylated TFAM. Accordingly, VHL-related tumours, such as pheochromocytoma and renal cell carcinoma cells, display low mitochondrial content, suggesting that impaired mitochondrial biogenesis is linked to VHL tumorigenesis. Finally, inhibiting proteolysis by targeting LONP1 increases mitochondrial content in VHL-deficient cells and sensitizes therapy-resistant tumours to sorafenib treatment. Our results offer pharmacological avenues to sensitize therapy-resistant VHL tumours by focusing on the mitochondria.

The paucity of recurrent mutations has hampered efforts to understand and treat neuroblastoma. Alternative splicing and splicing-dependent RNA-fusions represent mechanisms able to increase the gene product repertoire but their role in neuroblastoma remains largely unexplored. Here we investigate the presence and possible roles of aberrant splicing and splicing-dependent RNA-fusion transcripts in neuroblastoma. In addition, we attend to establish whether the spliceosome can be targeted to treat neuroblastoma. Through analysis of RNA-sequenced neuroblastoma we show that elevated expression of splicing factors is a strong predictor of poor clinical outcome. Furthermore, we identified >900 primarily intrachromosomal fusions containing canonical splicing sites. Fusions included transcripts from well-known oncogenes, were enriched for proximal genes and in chromosomal regions commonly gained or lost in neuroblastoma. As a proof-of-principle that these fusions can generate altered gene products, we characterized a ZNF451-BAG2 fusion, producing a truncated BAG2-protein which inhibited retinoic acid induced differentiation. Spliceosome inhibition impeded neuroblastoma fusion expression, induced apoptosis and inhibited xenograft tumor growth. Our findings elucidate a splicing-dependent mechanism generating altered gene products in neuroblastoma and show that the spliceosome is a potential target for clinical intervention.