The KIT/c-KIT proto-oncogene is frequently over-expressed in Merkel cell carcinoma (MCC), an aggressive skin cancer commonly caused by Merkel cell polyomavirus (MCPyV). Here, we demonstrated that truncated MCPyV-encoded large T-antigen (LT) suppressed macroautophagy/autophagy by stabilizing and sequestering KIT in the paranuclear compartment via binding VPS39. KIT engaged with phosphorylated BECN1, thereby enhancing its association with BCL2 while diminishing its interaction with the PIK3C3 complex. This process ultimately resulted in the suppression of autophagy. Depletion of KIT triggered both autophagy and apoptosis, and decreased LT expression. Conversely, blocking autophagy in KIT-depleted cells restored LT levels and rescued apoptosis. Additionally, stimulating autophagy efficiently increased cell death and inhibited tumor growth of MCC xenografts in mice. These insights into the interplay between MCPyV LT and autophagy regulation reveal important mechanisms by which viral oncoproteins are essential for MCC cell viability. Thus, autophagy-inducing agents represent a therapeutic strategy in advanced MCPyV-associated MCC.Abbreviation: 3-MA, 3-methyladenine; AL, autolysosome; AP, autophagosome; Baf-A1, bafilomycin A1; BARA, β-α repeated autophagy specific domain; BH3, BCL2 homology 3 domain; CCD, coiled-coil domain; CHX, cycloheximide; Co-IP, co-immunoprecipitation; CQ, chloroquine; CTR, control; DAPI, 4',6-diamidino-2-phenylindole; EBSS, Earle's balanced salt solution; ECD, evolutionarily conserved domain; EEE, three-tyrosine phosphomimetic mutations Y229E Y233E Y352E; ER, endoplasmic reticulum; FFF, three-tyrosine non-phosphomimetic mutations; FFPE, formalin-fixed paraffin-embedded; FL, full-length; GIST, gastrointestinal stromal tumor; IB, immunoblotting; IHC, immunohistochemistry; KIT-HEK293, KIT stably expressing HEK293 cells; KRT20/CK20, keratin 20; LT, large T-antigen; LT339, MCPyV truncated LT antigen; LTco, codon-optimized MCPyV LT antigen; MCC, Merkel cell carcinoma; MCPyV-, MCPyV-negative; MCPyV, Merkel cell polyomavirus; MCPyV+, MCPyV-positive; PARP1, poly(ADP-ribose) polymerase 1; PCI, pan-caspase inhibitor; PI, propidium iodide; PtdIns3K, class III phosphatidylinositol 3-kinase; PtdIns3P, phosphatidylinositol-3-phosphate; RB1, RB transcriptional corepressor 1; RTKs, receptor tyrosine kinases; KITLG/SCF, KIT ligand; sT, small T-antigen; sTco, codon-optimized MCPyV sT antigen; T-B, Tat-BECN1; T-S, Tat-scrambled; TEM, transmission electron microscopy.

Merkel cell carcinoma (MCC) is an aggressive skin cancer with frequent metastasis; however, effective treatment options for advanced disease are often lacking. In this study, we investigated the clinical significance and functional impact of IGF2 mRNA-binding protein 3 (IGF2BP3) in MCC. Our results revealed elevated IGF2BP3 expression in metastases compared to that in primary tumors. High IGF2BP3 levels in primary MCCs were associated with shorter disease-specific survival rates. In an MCC xenograft model, the lung metastases exhibited increased IGF2BP3 expression. Functional studies showed that IGF2BP3 primarily regulates MCC cell migration and invasion. We identified 281 direct RNA targets of IGF2BP3 with enriched functions linked to metastasis-related processes, and several targets overlapped with genes differentially expressed between MCC primary tumors and metastases, implying that IGF2BP3 and its targets contribute to tumor progression. Inhibition or silencing of bromodomain-containing protein 4 reduced IGF2BP3 expression, suggesting that bromodomain-containing protein 4 is a potential regulator of IGF2BP3. Our study underscores the role of IGF2BP3 in MCC metastasis and its potential as a prognostic biomarker.

Papillary thyroid cancer is associated with lymph node metastasis and tumor recurrence that need repeated surgery, entailing surgical challenges with a high risk of complications such as nerve damage and bleeding. Magseed is a metal coil used to detect non-palpable lesions and is an established modality in breast cancer surgery. In this case series, we explore the feasibility of Magseed in metastasis surgery of papillary thyroid cancer. Under the guidance of ultrasonography, Magseed is injected into the target tissue and intraoperatively detected with a handheld magnetometer probe, Sentimag®. Five patients with recurrence of papillary thyroid cancer were operated on with focused Magseed-guided localization. All patients had repeated surgery and radioiodine treatments. Four of the patients had lymph node metastasis hidden in the fibrosis, and one patient had recurrent tumor tissue on the left side of the larynx. Magseed was easy to use, safe, and precise in detecting the target tissue.

Supplementary Figure 1 from CREB3L2-PPARγ Fusion Mutation Identifies a Thyroid Signaling Pathway Regulated by Intramembrane Proteolysis

Supplemental Table 2. (A) Single tandem repeat (STR) analysis of the LUTC-1, LUTC-2, LUTC-8, LUTC-10, LUTC-12, LUTC-14 and K562 cell line. STR analysis of the LUTC cell lines was performed on January 31 2013 and the K562 cell line was authenticated by STR analysis on November 28 2013.

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 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.

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.

Supplementary Figure 1 from CREB3L2-PPARγ Fusion Mutation Identifies a Thyroid Signaling Pathway Regulated by Intramembrane Proteolysis

Abnormalities of the insulin-like 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 IGF2-H19 locus using PCR-based 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 miR-483-3p and miR-483-5p hosted by IGF2. The downregulated expression of H19 in ACC compared to ACA correlated with miR-675 expression hosted by H19. Several proteins exhibited an inverse correlation in expression and were predicted as targets of miR-483-3p, miR-483-5p or miR-675. 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 miR-483-5p 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 IGF2-H19 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.

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.

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.

AIMS:Upregulation 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.METHODS:We 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).RESULTS:TERT 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 p<0.001). Strikingly, TERT 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.CONCLUSIONS:TERT 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.

Viruses can inhibit host autophagy through multiple mechanisms, and evasion of autophagy plays an important role in immune suppression and viral oncogenesis. Merkel cell polyomavirus (MCPyV) T‐antigens are expressed and involved in the pathogenesis of a large proportion of Merkel cell carcinoma (MCC). Yet, how MCPyV induces tumorigenesis is not fully understood. Herein, we show that MCPyV T‐antigens induce miR‐375, miR‐30a‐3p and miR‐30a‐5p expressions, which target multiple key genes involved in autophagy, including ATG7, SQSTM1 (p62) and BECN1. In MCC tumors, low expression of ATG7 and p62 are associated with MCPyV‐positive tumors. Ectopic expression of MCPyV small T‐antigen and truncated large T‐antigen (LT), but not the wild‐type LT, resulted in autophagy suppression, suggesting the importance of autophagy evasion in MCPyV‐mediated tumorigenesis. Torin‐1 treatment induced cell death, which was attenuated by autophagy inhibitor, but not pan‐caspase inhibitor, suggesting a potential role of autophagy in promoting cell death in MCC. Conceptually, our study shows that MCPyV oncoproteins suppress autophagy to protect cancer cells from cell death, which contribute to a better understanding of MCPyV‐mediated tumorigenesis and potential MCC treatment.

Parathyroid lipoadenomas (PLAs) are rare tumors, and case descriptions are limited, < 80 to date. As a consequence, scarce information regarding the etiology of these enigmatic lesions is available. We searched our departmental pathology database for PLAs diagnosed between 1992 and 2020, reexamined the histology of each case, and recorded clinical parameters from the patients' medical charts. As the diagnostic criteria of this lesion vary over this time period, we defined PLA as an enlarged parathyroid gland with > 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.

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 < 0.001). We conclude that parafibromin immunohistochemistry is a useful marker in the clinical routine when applied on a pre-selected material of cases, with positive immunoreactivity as a confident rule out marker of malignancy.

Abstract Background Follicular thyroid carcinoma (FTC) is a neoplasm that presents with a micro-follicular growth pattern and a neutrally stained cytoplasm. Seldom, FTCs display unusual morphological characteristics – but given the rarity of these histological subtypes, little is known regarding the underlying genetics and the coupling to patient outcome. Case presentation We present two extremely rare cases of minimally invasive FTC with signet ring cell morphology (SRC-FTC) and describe the cytological, microscopic, immunohistochemical and molecular features for both tumors. Both were male patients, age 71 and 51 respectively. The preoperative cytology for both cases could not pinpoint a clear-cut signet ring cell morphology, but a tendency towards nuclear marginalization was seen. The tumors were 38 mm and 22 mm respectively and displayed evident signet ring cell features in subsets of tumor cells as well as degenerative stromal changes. The tumor cells were positive for TTF1, PAX8 and thyroglobulin, and the proliferation indexes were 4% and 1,9% respectively. Both tumors displayed capsular invasion, but not lymphovascular invasion. The tumors were sequenced for mutations in the TERT promoter and 22 additional cancer-related genes, interestingly; one patient was shown to carry a deleterious intronic variant in PTEN, a tumor suppressor gene coupled to thyroid tumorigenesis and Cowden syndrome. Both patients are alive and well awaiting postoperative radioiodine treatment. Conclusions The SRC-FTCs described herein were small, TERT promoter wildtype tumors exhibiting low proliferation, thereby suggesting that these exceedingly rare lesions probably carry a favorable prognosis – although the scarce availability regarding descriptions of this tumor entity nevertheless might justify careful clinical monitoring and mandate investigations in larger case series.

Clear cell change is a rare observation in thyroid cancer, resulting from aberrant cytoplasmic accumulation of lipids, glycogen, or thyroglobulin in the tumor cells. The phenomenon is most common for follicular thyroid neoplasia, with no definite coupling to patient outcome. The clear cell variant of papillary thyroid carcinoma (ccPTC) is even more infrequent—making conclusions regarding prognosis difficult. Single reports describe distant metastases of ccPTCs as well as co-occurrence with anaplastic thyroid carcinoma (ATC). In this report, a case of a therapy-resistant ccPTC dedifferentiating into an ATC is characterized from morphological and immunohistochemical standpoints. The patient was a 79-year-old female presenting with a 45-mm nodule in her right thyroid lobe. A first round of cytology raised the suspicion of PTC, but a repeated biopsy verified an ATC diagnosis. Neoadjuvant doxorubicin and external irradiation therapy was administered, and the patient developed lung metastases concomitantly. A palliative lobectomy was performed, and the final diagnosis was a ccPTC with focal dedifferentiation into an ATC. Intriguingly, the ccPTC component was viable and dominated the lesion. The clear cell morphology stemmed from an accumulation of glycogen, while the anaplastic component was devoid of evident clear cell changes. The case is one of exceedingly few descriptions of a ccPTC that dedifferentiates to an ATC, suggesting that this PTC subtype is not without potential for development of a highly lethal tumor component. Moreover, the partial lack of response to neoadjuvant therapy suggests a possible underlying resistance to aggressive treatment modalities in this particular case.

Mutations of the Telomerase reverse transcriptase (TERT) gene promoter are recurrently found in follicular thyroid carcinoma (FTC) and follicular tumors of uncertain malignant potential (FT-UMP), but nearly never in follicular thyroid adenoma (FTA). We, therefore, believe these mutations could signify malignant potential. At our department, postoperative TERT promoter mutational testing of FT-UMPs was implemented in 2014, with a positive mutation screening leading to vigilant follow-up and sometimes adjuvant treatment. To date, we screened 51 FT-UMPs and compared outcomes to 40 minimally invasive FTCs (miFTCs) with known TERT genotypes. Eight FT-UMPs (16%) displayed TERT promoter mutations, of which four cases underwent a completion lobectomy at the discretion of the patient, and a single patient also opted in for radioiodine (RAI) treatment. Three mutation-positive patients developed distant metastases, registered in one patient receiving a completion lobectomy and in two patients with no additional treatment. Three out of four patients who received additional surgery, including the RAI-treated patient, are still without metastatic disease. We conclude that FT-UMPs with TERT promoter mutations harbor malignant potential and exhibit at least similar recurrence rates to TERT-promoter-mutated miFTCs. Mutational screening should constitute a cornerstone analysis in the histopathological work-up of FT-UMPs.