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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.

AIMS DICER1 germline mutations cause DICER1 syndrome, in which multinodular goitre is a common feature. Recently, somatic DICER1 mutations have been reported in sporadic thyroid carcinomas, of which the newly described macrofollicular variant of follicular thyroid carcinoma (MV-FTC) seems particularly enriched for this aberrancy. We report here histological and genetic findings in five follicular thyroid tumours with macrofollicular architecture (four carcinomas and one adenoma). METHODS AND RESULTS We have diagnosed five cases during a year-long period at the Karolinska University Hospital, a tertiary thyroid cancer center with a catchment area of approximately 2.3 million inhabitants. Tumour DNA was interrogated using a commercially available massive parallel sequencing platform. All cases were female patients, ranging from 13 to 33 years at surgery. A single patient was a DICER1 syndrome carrier; the others were sporadic cases. All tumours displayed a macrofollicular architecture with a broad capsule. The MV-FTCs displayed capsular invasion, but never vascular invasion. Areas with degenerative changes (microinfarctions) were noted in all cases, and focal papillary growth was observed in the majority. The Ki-67 proliferation index was always above 4%. All cases displayed DICER1 gene mutations, of which four of five cases displayed RNase IIIb hot-spot missense mutations adjoined by a second, deleterious variant in three of five tumouurs. CONCLUSIONS Macrofollicular variants of follicular thyroid tumours are predominantly found in younger females and are strongly linked to somatic DICER1 gene mutations. Histological features such as a broad tumorous capsule, focal infarctions and areas with papillae could constitute clues prompting further genetic analyses.

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.

Abnormalities of the insulin‑like growth factor 2

Purpose: TERT promoter mutation (TERTpMut) has a strong association to recurrence and has been suggested to act as a driver mutation for malignant transformation of WHO grade I and II meningiomas. TERTpMut has been investigated in selected high‐grade meningioma samples. The existence of TERTpMut across recurrent tumors in a population‐based cohort needs to be investigated in order to identify when TERTpMut emerges across recurrent samples and to validate prognostic impact among WHO grade III tumors. Methods: We gathered material from a consecutive single‐center cohort of 40 patients with malignant meningioma (WHO grade III) treated between 2000 and 2018, including specimens from primary and secondary malignant meningiomas with the corresponding earlier benign specimens and later malignant recurrences. In total 107 tumor samples were studied by Sanger sequencing for TERT promoter mutational status. Results: Seven of 40 patients (17.5%) harbored TERTpMut thus validating the incidence of TERTpMut in previous non‐population‐based cohorts. In 6/7 patients, the TERTpMut was present at initial surgery (WHO grade I–III) while in one patient the TERTpMut was found de novo when the meningioma became malignant. The incidences were 2/1.000.000/year for TERTpMut WHO grade III meningioma and 8/1.000.000/year for TERTpwt WHO grade III meningioma in our catchment area. We found a 1.7 times higher recurrence rate (CI 95% 0.65–4.44) and a 2.5 higher mortality rate per 10 person‐years (CI 95% 1.01–6.19) for TERTpMut compared to TERTpwt. Conclusion: TERTpMut can occur independently of malignant progression in meningioma and was most often present from the first tumor sample across recurring tumors. TERTpMut in WHO grade III may represent a marker of an aggressive subset of tumors., We investigated the TERT promoter mutation (TERTpMut) in a population‐based, consecutive cohort of 40 malignant meningioma patients with tumor samples available from the corresponding earlier benign specimens and later malignant recurrences. TERTpMut can occur independently of malignant progression in meningioma and was most often present from the first tumor sample across recurring tumors. TERTpMut in WHO grade III may represent a marker of an aggressive subset of tumors.

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.

Purpose Sarcomas of the breast account for about 1% of all breast malignancies. The aim of this national survey was to explore etiologic and prognostic factors. Methods Utilizing national Swedish registers, all patients registered with mesenchymal tumors in the breast during the period 1993–2013 (n = 344) were identified and compared to up to ten age and gender matched controls. Cancer history was retrieved for cases and controls. Conditional Poisson regression models were used for calculation of odds ratios. Results Previous breast cancer was overrepresented among patients with angiosarcoma. The highest risk occurred ≥ 5 years after treatment for breast cancer (OR 73.9, 95% confidence interval, CI, 25.4–215; P P P Conclusions Angiosarcoma increased in incidence and displayed a more severe clinical course, with significantly shorter survival. The strong association between a history of breast cancer 5 years or more prior to the diagnosis of angiosarcoma points to radiotherapy as a contributing factor.

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.

Introduction: Hypoparathyroidism is the most common complication following thyroidectomy, and various algorithms for early detection have been suggested. The aim of this study was to evaluate the predictive value of measuring the parathyroid hormone level 2 h after thyroidectomy and whether determination of the perioperative decline in parathyroid hormone added diagnostic value. Methods: Patients subjected to thyroidectomy for benign thyroid disorders were analyzed in (1) a retrospective register-based study (366 consecutive patients treated during 2015–2016) and (2) a prospective observational study (39 patients treated during 2018). Optimal cut-off values for postoperative parathyroid hormone and perioperative decline (%) in parathyroid hormone were determined by receiver operating characteristics and area under the curve. Sensitivity, specificity, positive and negative predictive values were calculated using cross tabulation. Results: The prevalence of hypoparathyroidism the first day after thyroidectomy was higher among patients treated for hyperthyroidism (30% vs 20%; P = 0.03). The optimal cut-off level for postoperative parathyroid hormone was 1.1 pmol/L (area under the curve = 0.887, 95% confidence interval: 0.839–0.934; positive predictive value: 88%, negative predictive value: 93%) for the entire cohort. When the groups were analyzed separately, the optimal cut-off was 1.05 for the non-hyperfunctioning group and 1.55 pmol/L for the group with hyperthyroidism. Twelve months after thyroidectomy, 3% were defined as having permanent hypoparathyroidism. Measurement of parathyroid hormone decline added diagnostic value for one patient with preoperative parathyroid-hormone-elevation associated with vitamin D deficiency. Conclusions: For patients with vitamin D sufficiency, the diagnostic accuracy of a single measurement of parathyroid hormone 2 h after thyroidectomy is an excellent indicator for predicting transient hypoparathyroidism.

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.

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.

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.

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 genetics underlying thyroid cancer dedifferentiation is only partly understood and has not yet been characterised using comprehensive pan-genomic analyses. We investigated a unique case with synchronous follicular thyroid carcinoma (FTC), poorly differentiated thyroid carcinoma (PDTC), and anaplastic thyroid carcinoma (ATC), as well as regional lymph node metastases from the PDTC and ATC from a single patient using whole-genome sequencing (WGS). The FTC displayed mutations in CALR, RB1, and MSH2, and the PDTC exhibited mutations in TP53, DROSHA, APC, TERT, and additional DNA repair genes - associated with an immense increase in sub-clonal somatic mutations. All components displayed an overrepresentation of C>T transitions with associated microsatellite instability (MSI) in the PDTC and ATC, with borderline MSI in the FTC. Clonality analyses pinpointed a shared ancestral clone enriched for mutations in TP53-associated regulation of DNA repair and identified important sub-clones for each tumour component already present in the corresponding preceding lesion. This genomic characterisation of the natural progression of thyroid cancer reveals several novel genes of interest for future studies. Moreover, the findings support the theory of a stepwise dedifferentiation process and suggest that defects in DNA repair could play an important role in the clonal evolution of thyroid cancer. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Background A pathologic tumor-node-metastasis staging algorithm for pheochromocytoma and sympathetic paraganglioma was introduced recently in the 8th Edition of the cancer staging manual of the American Joint Committee on Cancer. There is no information, however, as to how this staging correlates to well-established clinical cohorts of pheochromocytoma and sympathetic paraganglioma with extensive follow-up. Methods We applied the pathologic tumor-node-metastasis staging retrospectively to a cohort of 118 patients with pheochromocytoma and sympathetic paraganglioma, in which the majority has been characterized for susceptibility gene mutations and global mRNA expressional patterns as well as histologic risk criteria using the pheochromocytoma of the adrenal gland scaled score (PASS). Results The overall tumor stage correlated with the presence of metastases, disease-related death, and PASS scores as well as established mutational and expressional clusters. Conclusion Stage III to IV pheochromocytomas and sympathetic paragangliomas are associated with increased mortality, increased PASS scores, and mutational and expressional aberrancies in the pseudo-hypoxia pathway cluster. These findings validate the stratification proposed by the American Joint Committee on Cancer staging manual by linking malignancy-associated pheno- and genotypes to more advanced stages. Moreover, because few pheochromocytomas and sympathetic paragangliomas are metastatic at the time of the original presentation, the staging relies heavily on identifying histologic signs of extra-adrenal invasion.

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.

Pheochromocytomas (PCCs) and abdominal paragangliomas (PGLs), collectively abbreviated PPGL, are believed to exhibit malignant potential-but only subsets of cases will display full-blown malignant properties. The Pheochromocytoma of the Adrenal Gland Scaled Score (PASS) algorithm is a proposed histologic system to detect potential for aggressive behavior, but little is known regarding the coupling to underlying molecular genetics. In this study, a total of 92 PPGLs, previously characterized for susceptibility gene status and mRNA expressional profiles, were histologically assessed using the PASS criteria. A total of 32/92 PPGLs (35%) exhibited a PASS score ≥4, including all 8 cases with malignant behavior (7 with known metastases and 1 with extensively infiltrative local recurrence). Statistical analyzes between expressional data and clinical parameters as well as individual PASS criteria yielded significant associations to Chromogranin B (CHGB), BRCA2, HIST1H3B, BUB1B, and RET to name a few, and CHGB had the strongest correlation to both PASS and metastasis/local recurrence of all analyzed genes. Evident CHGB downregulation was observed in PPGLs with high PASS and overtly malignant behavior, and was also associated with shorter disease-related survival. This finding was validated using quantitative real-time polymerase chain reaction, in which CHGB expression correlated with both PASS and metastasis/local recurrence with consistent findings obtained in the TCGA cohort. Moreover, immunohistochemical analyses of subsets of tumors showed a correlation between high PASS scores and negative or weak CHGB protein expression. Patients with PPGLs obtaining high PASS scores postoperatively, also exhibited low preoperative plasma levels of CHGB. These data collectively point out CHGB as a possible preoperative and postoperative marker for PPGLs with potential for aggressive behavior.

Upregulation of theWe 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

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.

Introduction Secondary neoplasms in the thyroid are rare. The study aim was to provide an overview of non-thyroid tumours that metastasize to the thyroid through our institutional experience. Materials and methods This study entailed a review of the pathology database searching for patients with metastasis to the thyroid at the Karolinska University Hospital between 1992 and 2019 and review of their medical files. Results Out of 1939 surgical procedures with a histopathological diagnosis of a thyroid malignancy, 31 cases (1.6%, 65% females) with a diagnosis of metastatic epithelial neoplasms to the thyroid gland were identified. The median age at discovery of the thyroid metastasis was 68 years (range 48–85). The most common primary tumours were clear cell renal cell carcinoma (ccRCC) (36%), followed by non-small cell lung cancer (19%), oesophageal cancer (16%), head and neck malignancies (16%), malignant melanoma (10%) and unknown primary tumour (3%). The median time from the diagnosis of the primary tumour to diagnosis of the thyroid metastasis was 20 months (0–232) and was longest for patients with ccRCC (median 107 months). At 12 months after the non-thyroid metastasis diagnosis 48% had died. The longest survival was observed in ccRCC and the shortest in lung cancer. Surgical management of the metastasis was associated with improved survival (25 vs 3.8 months, p = 0.001). Conclusions Non-thyroid metastases to the thyroid were rare but should be suspected in patients with previous history of non-thyroid malignancy and a thyroid nodule. Prognosis was poor, but surgical management was beneficial in selected patients.

Parathyroid lesions exhibiting a water clear cell morphology are exceedingly rare manifestations in primary hyperparathyroidism (PHPT), and the phenomenon has been reported both for uniglandular (water clear cell adenoma; WCCA) and multiglandular disease (water clear cell hyperplasia; WCCH). In all, only 24 previous descriptions of WCCA exist in the literature. Herein, we present seven cases with water clear cell morphology (6 WCCAs and 1 case of WCCH) in an institutional series of approximately 4000 parathyroid lesions spanning 29 years in a tertiary centre setting. Major histological attributes and clinical parameters associated with this morphological subtype were reviewed, and a literature search was conducted. WCCA and WCHH exhibited an institutional prevalence of 0.15% and 0.025%, respectively. All cases displayed histological hallmarks of water clear cell morphology, with cells exhibiting abundant cytoplasm filled with vacuoles. Atypical findings or unequivocal evidence of invasive behaviour were not observed. The gender distribution was 6:1 (F:M), patients were generally symptomatic with mild hypercalcaemia, and the median age at surgery was 53 years (range 38-78). The preoperative localisation was inconclusive in four of seven, and neck exploration of all four glands was undertaken in five cases. The excised WCCAs exhibited an average weight of 1215 mg, markedly higher than conventional adenomas, and all patients were cured of PHPT following parathyroidectomy. Interestingly, previous reports mirror our observations that these lesions often are large, in relation to their sizes biochemically fairly indolent, and indecisively localised using scintigraphy, providing correlations of possible clinical value when pre-operatively assessing these rare lesions.

In thyroid carcinomas, telomerase reverse transcriptase (TERT) promoter mutations C228T and C250T predict an unfavorable clinical outcome. The analysis is particularly valuable when assessing histologically equivocal follicular thyroid tumors of uncertain malignant potential (FT-UMPs). Given recent findings of TERT promoter mutational heterogeneity in thyroid cancer, we determined the frequency of this phenomenon in FT-UMPs and minimally invasive follicular thyroid carcinomas. DNA was extracted from several tissue blocks from 16 FT-UMPs as well as 10 minimally invasive follicular thyroid carcinomas, and interrogated using Sanger sequencing as well as digital droplet PCR (ddPCR). Mutational heterogeneity was observed by Sanger sequencing in four of seven (57%) FT-UMPs. In two FT-UMPs with C228T mutations, analyses of additional blocks gave wild-type results using Sanger sequencing in one or several blocks interrogated, whereas ddPCR found low-frequency C228T mutations in one of these fractions. In two additional FT-UMPs with the C228T and C250T mutation, respectively, sequencing of additional blocks revealed the opposite mutation. Moreover, in the C250T mutated area in one of these tumors, the ddPCR displayed a co-occurring C228T mutation that failed detection through Sanger sequencing. To conclude, most TERT promoter mutated FT-UMPs display mutational heterogeneity when analyzed by Sanger sequencing, thereby emphasizing the importance of the tissue sampling process. The ddPCR technique might overcome this phenomenon because of increased sensitivity and should be considered for clinical screening purposes.