Aberrant splicing in neuroblastoma generates RNA-fusion transcripts and provides vulnerability to spliceosome inhibitors.

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.

EglN3 hydroxylase stabilizes BIM-EL linking VHL type 2C mutations to pheochromocytoma pathogenesis and chemotherapy resistance.

Despite the discovery of the oxygen-sensitive regulation of HIFα by the von Hippel-Lindau (VHL) protein, the mechanisms underlying the complex genotype/phenotype correlations in VHL disease remain unknown. Some germline VHL mutations cause familial pheochromocytoma and encode proteins that preserve their ability to down-regulate HIFα. While type 1, 2A, and 2B VHL mutants are defective in regulating HIFα, type 2C mutants encode proteins that preserve their ability to down-regulate HIFα. Here, we identified an oxygen-sensitive function of VHL that is abolished by VHL type 2C mutations. We found that BIM-EL, a proapoptotic BH3-only protein, is hydroxylated by EglN3 and subsequently bound by VHL. VHL mutants fail to bind hydroxylated BIM-EL, regardless of whether they have the ability to bind hydroxylated HIFα or not. VHL binding inhibits BIM-EL phosphorylation by extracellular signal-related kinase (ERK) on serine 69. This causes BIM-EL to escape from proteasomal degradation, allowing it to enhance EglN3-induced apoptosis. BIM-EL was rapidly degraded in cells lacking wild-type VHL or in which EglN3 was inactivated genetically or by lack of oxygen, leading to enhanced cell survival and chemotherapy resistance. Combination therapy using ERK inhibitors, however, resensitizes VHL- and EglN3-deficient cells that are otherwise cisplatin-resistant.

Macrofollicular variant follicular thyroid tumors are DICER1 mutated and exhibit distinct histological features.

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.

Whole-genome sequencing of synchronous thyroid carcinomas identifies aberrant DNA repair in thyroid cancer dedifferentiation.

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.

The Clinical Significance of Lymph Node Ratio and Ki-67 Expression in Papillary Thyroid Cancer.

BACKGROUND: The N stage in papillary thyroid cancer (PTC) is an important prognostic factor based on anatomical localization of cervical lymph nodes (LNs) only and not the extent of lymphatic metastasis. In this retrospective study, the clinical significance of lymph node ratio (LNR) and tumor cell proliferation in relation to the conventional classification of PTC was explored. METHODS: Patients diagnosed with PTC at the Karolinska University Hospital in Stockholm, Sweden, during the years 2009-2011 were included. The LNR, defined as the number of metastatic LNs divided by the total number of LNs investigated, and the Ki-67 index were analyzed in relation to clinical data. RESULTS: The median number of LN removed was 16 with the following N stage distribution: N0 (26%), N1a (45%), and N1b (29%). A Ki-67 index of ≥3% was significantly correlated with the presence of metastases and tumor recurrence with a sensitivity of 50% and specificity of 80% (p = 0.015). Lymph node ratio ≥21% was related to tumor recurrence with sensitivity of 89% and specificity of 70% (p = 0.006). Patients with LN metastases in the lateral cervical compartment only had significantly lower LNR (14.5%) compared to those with both central and lateral cervical metastases (39.5%) (p = 0.004) and exhibited no tumor recurrence. Increased Ki-67 index was significantly related to LNR ≥21% (p = 0.023) but was not associated with N stage. CONCLUSIONS: The Ki-67 proliferation index and LNR may better reflect the malignant behavior of PTC compared to the anatomical classification of LN metastases solely.

Parafibromin immunostainings of parathyroid tumors in clinical routine: a near-decade experience from a tertiary center.

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.

Papillary thyroid carcinoma with pleomorphic tumor giant cells in a pregnant woman - a case report.

BACKGROUND: Papillary thyroid carcinoma with pleomorphic tumor giant cells (PTC-PC) is characterized by the occurrence of bizarre, pleomorphic cells within a small area of a conventional PTC. The histologic distinction between PTC-PC and PTC's with a focal anaplastic thyroid cancer (ATC) component (denoted in the 2004 WHO classification as "papillary thyroid carcinoma with spindle and giant cell carcinoma", PTC-SGC) is debated, however the prognosis is thought to be different (excellent for PTC-PC, poor for PTC-SGC). Therefore, this diagnostic challenge is significant for any endocrine pathologist to recognize. Herein, we report the histological and clinical workup of a PTC-PC case, with particular focus on the molecular analyses that facilitated the establishment of the final diagnosis. CASE PRESENTATION: The patient was a pregnant, 28-year-old female presenting with a 30 mm conventional PTC, with focal areas with undifferentiated cells exhibiting exaggerated nuclear pleomorphism. No foci of extrathyroidal extension, angioinvasion or lymph node engagement were seen. Immunohistochemical analyses revealed the pleomorphic cells exhibiting retained differentiation. Molecular genetic analyses demonstrated a codon V600 missense mutation of the BRAF gene, but no TP53 or TERT promoter mutations. The absence of an aggressive phenotype in addition to the lack of mutations in two major ATC-related genes led to the diagnosis of a PTC-PC. Postoperative MRI showed no evidence of metastatic disease. Radioiodine ablation was performed seven months post-operatively, and a SPECT-CT imaging did not show signs of residual tissue. She is well and without signs of disease 16 months post-operatively. CONCLUSIONS: PTC-PC is a differential diagnosis to PTC-SGC that mandates careful considerations. Taken together with previous publications, PTC-PC seems to be histologically similar to PTC-SGC, but clinically distinct. Even so, the distinction is not easily made given the different therapeutic consequences for each individual patient. This is the first report that includes molecular genetics to aid in finalizing the diagnosis. Exclusion of mutations in TP53 and the TERT promoter could be considered as an adjunct tool when assessing papillary thyroid cancer with focal pleomorphism.

Characterization of the mutational landscape of anaplastic thyroid cancer via whole-exome sequencing.

Anaplastic thyroid carcinoma (ATC) is a frequently lethal malignancy that is often unresponsive to available therapeutic strategies. The tumorigenesis of ATC and its relationship to the widely prevalent well-differentiated thyroid carcinomas are unclear. We have analyzed 22 cases of ATC as well as 4 established ATC cell lines using whole-exome sequencing. A total of 2674 somatic mutations (121/sample) were detected. Ontology analysis revealed that the majority of variants aggregated in the MAPK, ErbB and RAS signaling pathways. Mutations in genes related to malignancy not previously associated with thyroid tumorigenesis were observed, including mTOR, NF1, NF2, MLH1, MLH3, MSH5, MSH6, ERBB2, EIF1AX and USH2A; some of which were recurrent and were investigated in 24 additional ATC cases and 8 ATC cell lines. Somatic mutations in established thyroid cancer genes were detected in 14 of 22 (64%) tumors and included recurrent mutations in BRAF, TP53 and RAS-family genes (6 cases each), as well as PIK3CA (2 cases) and single cases of CDKN1B, CDKN2C, CTNNB1 and RET mutations. BRAF V600E and RAS mutations were mutually exclusive; all ATC cell lines exhibited a combination of mutations in either BRAF and TP53 or NRAS and TP53. A hypermutator phenotype in two cases with >8 times higher mutational burden than the remaining mean was identified; both cases harbored unique somatic mutations in MLH mismatch-repair genes. This first comprehensive exome-wide analysis of the mutational landscape of ATC identifies novel genes potentially associated with ATC tumorigenesis, some of which may be targets for future therapeutic intervention.

A novel FOXO1-mediated dedifferentiation blocking role for DKK3 in adrenocortical carcinogenesis.

BACKGROUND: Dysregulated WNT signaling dominates adrenocortical malignancies. This study investigates whether silencing of the WNT negative regulator DKK3 (Dickkopf-related protein 3), an implicated adrenocortical differentiation marker and an established tumor suppressor in multiple cancers, allows dedifferentiation of the adrenal cortex. METHODS: We analyzed the expression and regulation of DKK3 in human adrenocortical carcinoma (ACC) by qRT-PCR, immunofluorescence, promoter methylation assay, and copy number analysis. We also conducted functional studies on ACC cell lines, NCI-H295R and SW-13, using siRNAs and enforced DKK3 expression to test DKK3's role in blocking dedifferentiation of adrenal cortex. RESULTS: While robust expression was observed in normal adrenal cortex, DKK3 was down-regulated in the majority (>75%) of adrenocortical carcinomas (ACC) tested. Both genetic (gene copy loss) and epigenetic (promoter methylation) events were found to play significant roles in DKK3 down-regulation in ACCs. While NCI-H295R cells harboring ß-catenin activating mutations failed to respond to DKK3 silencing, SW-13 cells showed increased motility and reduced clonal growth. Conversely, exogenously added DKK3 also increased motility of SW-13 cells without influencing their growth. Enforced over-expression of DKK3 in SW-13 cells resulted in slower cell growth by an extension of G1 phase, promoted survival of microcolonies, and resulted in significant impairment of migratory and invasive behaviors, largely attributable to modified cell adhesions and adhesion kinetics. DKK3-over-expressing cells also showed increased expression of Forkhead Box Protein O1 (FOXO1) transcription factor, RNAi silencing of which partially restored the migratory proficiency of cells without interfering with their viability. CONCLUSIONS: DKK3 suppression observed in ACCs and the effects of manipulation of DKK3 expression in ACC cell lines suggest a FOXO1-mediated differentiation-promoting role for DKK3 in the adrenal cortex, silencing of which may allow adrenocortical dedifferentiation and malignancy.

HRAS mutation prevalence and associated expression patterns in pheochromocytoma.

Pheochromocytomas (PCC) and abdominal paragangliomas (PGL) display a highly diverse genetic background and recent gene expression profiling studies have shown that PCC and PGL (together PPGL) alter either kinase signaling pathways or the pseudo-hypoxia response pathway dependent of the genetic composition. Recurrent mutations in the Harvey rat sarcoma viral oncogene homolog (HRAS) have recently been verified in sporadic PPGLs. In order to further establish the HRAS mutation frequency and to characterize the associated expression profiles of HRAS mutated tumors, 156 PPGLs for exon 2 and 3 hotspot mutations in the HRAS gene was screened, and compared with microarray-based gene expression profiles for 93 of the cases. The activating HRAS mutations G13R, Q61R, and Q61K were found in 10/142 PCC (7.0%) and a Q61L mutation was revealed in 1/14 PGL (7.1%). All HRAS mutated cases included in the mRNA expression profiling grouped in Cluster 2, and 21 transcripts were identified as altered when comparing the mutated tumors with 91 HRAS wild-type PPGL. Somatic HRAS mutations were not revealed in cases with known PPGL susceptibility gene mutations and all HRAS mutated cases were benign. The HRAS mutation prevalence of all PPGL published up to date is 5.2% (49/950), and 8.8% (48/548) among cases without a known PPGL susceptibility gene mutation. The findings support a role of HRAS mutations as a somatic driver event in benign PPGL without other known susceptibility gene mutations. HRAS mutated PPGL cluster together with NF1- and RET-mutated tumors associated with activation of kinase-signaling pathways.

Shifting patterns of genomic variation in the somatic evolution of papillary thyroid carcinoma.

BACKGROUND: Cancer is increasingly understood to arise in the context of dynamically evolving genomes with continuously generated variants subject to selective pressures. Diverse mutations have been identified in papillary thyroid carcinoma (PTC), but unifying theories underlying genomic change are lacking. Applying a framework of somatic evolution, we sought to broaden understanding of the PTC genome through identification of global trends that help explain risk of tumorigenesis. METHODS: Exome sequencing was performed on 53 PTC and matched adjacent non-tumor thyroid tissues (ANT). Single nucleotide substitution (SNS) signatures from each sample pair were divided into three subsets based on their presence in tumor, non-tumor thyroid, or both. Nine matched blood samples were sequenced and SNS signatures intersected with these three subsets. The intersected genomic signatures were used to define branch-points in the evolution of the tumor genome, distinguishing variants present in the tissues' common ancestor cells from those unique to each tissue type and therefore acquired after genomic divergence of the tumor, non-tumor, and blood samples. RESULTS: Single nucleotide substitutions shared by the tumor and the non-tumor thyroid were dominated by C-to-T transitions, whereas those unique to either tissue type were enriched for C-to-A transversions encoding non-synonymous, predicted-deleterious variants. On average, SNSs of matched blood samples were 81 % identical to those shared by tumor and non-tumor thyroid, but only 12.5 % identical to those unique to either tissue. Older age and BRAF mutation were associated with increased SNS burden. CONCLUSIONS: The current study demonstrates novel patterns of genomic change in PTC, supporting a theory of somatic evolution in which the zygote's germline genome undergoes continuous remodeling to produce progressively differentiated, tissue-specific signatures. Late somatic events in thyroid tissue demonstrate shifted mutational spectra compared to earlier polymorphisms. These late events are enriched for predicted-deleterious variants, suggesting a mechanism of genomic instability in PTC tumorigenesis.

Whole-exome sequencing defines the mutational landscape of pheochromocytoma and identifies KMT2D as a recurrently mutated gene.

As subsets of pheochromocytomas (PCCs) lack a defined molecular etiology, we sought to characterize the mutational landscape of PCCs to identify novel gene candidates involved in disease development. A discovery cohort of 15 PCCs wild type for mutations in PCC susceptibility genes underwent whole-exome sequencing, and an additional 83 PCCs served as a verification cohort for targeted sequencing of candidate mutations. A low rate of nonsilent single nucleotide variants (SNVs) was detected (6.1/sample). Somatic HRAS and EPAS1 mutations were observed in one case each, whereas the remaining 13 cases did not exhibit variants in established PCC genes. SNVs aggregated in apoptosis-related pathways, and mutations in COSMIC genes not previously reported in PCCs included ZAN, MITF, WDTC1, and CAMTA1. Two somatic mutations and one constitutional variant in the well-established cancer gene lysine (K)-specific methyltransferase 2D (KMT2D, MLL2) were discovered in one sample each, prompting KMT2D screening using focused exome-sequencing in the verification cohort. An additional 11 PCCs displayed KMT2D variants, of which two were recurrent. In total, missense KMT2D variants were found in 14 (11 somatic, two constitutional, one undetermined) of 99 PCCs (14%). Five cases displayed somatic mutations in the functional FYR/SET domains of KMT2D, constituting 36% of all KMT2D-mutated PCCs. KMT2D expression was upregulated in PCCs compared to normal adrenals, and KMT2D overexpression positively affected cell migration in a PCC cell line. We conclude that KMT2D represents a recurrently mutated gene with potential implication for PCC development.

TERT promoter mutation as an early genetic event activating telomerase in follicular thyroid adenoma (FTA) and atypical FTA.

BACKGROUND: The telomerase reverse transcriptase (TERT) promoter mutations C228T and C250T have been found in many malignancies, including in thyroid carcinomas. However, it is unclear how early these mutations occur in thyroid tumorigenesis. METHODS: The study included primary tumors from 58 patients initially diagnosed with follicular thyroid adenoma (FTA), a benign entity, 18 with atypical FTA (AFTA) having an uncertain malignant potential, and 52 with follicular thyroid carcinoma (FTC). Sanger sequencing was used to investigate the mutational status of the TERT promoter. Telomere length and TERT messenger RNA (mRNA) expression were determined using quantitative polymerase chain reaction (PCR). Telomerase activity was assessed using a Telomerase PCR enzyme-linked immunosorbent assay kit. RESULTS: The C228T mutation was identified in 1 of 58 FTA (2%) and 3 of 18 AFTA (17%) samples. These 4 tumors all expressed TERT mRNA and telomerase activity, whereas the majority of C228T-negative adenomas lacked TERT expression (C228T versus wild-type, P = .008). The C228T mutation was associated with NRAS gene mutations (P = .016). The patient with C228T-mutated FTA later developed a scar recurrence and died of FTC, whereas none of the remaining 57 patients with FTA had recurrence. No recurrence occurred in 3 patients with AFTA who carried C228T during the follow-up period (36-285 months). Nine of the 52 FTCs (17%) exhibited the TERT mutation (8 of 9 C228T and 1 of 9 C250T), and the presence of the mutation was associated with shorter patient survival. CONCLUSIONS: TERT promoter mutations may occur as an early genetic event in thyroid follicular tumors that have not developed malignant features on routine histopathological workup.

Adrenal tumors in patients with neuroendocrine neoplasms.

PURPOSE: To study the prevalence of primary adrenal tumors and adrenal metastases in patients with neuroendocrine neoplasms (NENs) and describe these in detail. NENs can be further divided into neuroendocrine tumor (NET) and neuroendocrine carcinoma (NEC). METHODS: A review of medical files was conducted for all patients who underwent a 68Gallium-DOTATOC-PET/CT during 2010-2023 or adrenalectomy during 1999-2023 at the Karolinska University Hospital. RESULTS: In total, 68Gallium-DOTATOC-PET/CT was performed on 1750 individuals with NEN, among whom 12 (0.69%) had adrenal tumors. Of these, 9 (0.51%) were NEN metastases. Out of 1072 adrenalectomies, 4 (0.37%) showed evidence of NEN metastases. Thus, 16 patients with NEN exhibited adrenal tumors. The adrenal tumors were found on average 5 years after the NEN diagnosis and 19% of the adrenal tumors with simultaneous NEN were benign. Few had all adrenal hormones measured. None had an adrenal insufficiency nor an adrenal biopsy. Another synchronous metastasis was found in 69% at the time of the adrenal tumor discovery. During the median 2-year follow-up, 38% of the subjects had deceased (with the exclusion of individuals presenting supposedly benign adrenal tumors 31%) all due to tumor complications. A comparison between individuals identified through 68Gallium-DOTATOC-PET/CT and those who underwent adrenalectomy revealed a higher prevalence of NETs in the former group and NECs in the latter group. CONCLUSION: Adrenal primary tumors and adrenal metastases are infrequent occurrences in patients with NEN. Most cases involved the presence of NEN metastasis upon the initial discovery of adrenal tumors. The overall prognosis was found to be favorable.

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

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

A clinical perspective on ectopic Cushing's syndrome.

Cushing's syndrome (CS) refers to the clinical features of prolonged pathological glucocorticoid excess. About 10-20% of individuals with CS have ectopic CS (ECS), that is, an adrenocorticotropin (ACTH)-producing tumour outside the pituitary gland. ACTH-secreting neuroendocrine neoplasia (NENs) can arise from many organs, although bronchial NEN, small cell lung cancer (SCLC), pancreatic NEN, thymic NEN, medullary thyroid cancer (MTC), and pheochromocytoma are the most common. Patients with ECS frequently present with severe hypercortisolism. The risk of life-threatening complications is high in severe cases, unless the hypercortisolism is effectively treated. A good outcome in ECS requires a methodical approach, incorporating prompt diagnosis, tumour localization, control of cortisol excess, and resection of the primary tumour when possible.

Atrophic changes in thyroid tumors are strong indicators of underlying DICER1 mutations: a bi-institutional genotype-phenotype correlation study.

Somatic and biallelic DICER1 mutations are reported in subsets of thyroid tumors, supporting the role of this gene in thyroid tumor development. As recent studies have brought attention to macrofollicular patterns, atrophic changes, and papillary structures as being associated with DICER1 mutations, we sought to explore these observations in a bi-institutional cohort. A total of 61 thyroid lesions (54 tumors and 7 cases of thyroid follicular nodular disease; TFND), including 26 DICER1 mutated and 35 DICER1 wildtype controls were subjected to histological re-investigation and clinical follow-up. DICER1-mutated lesions showed a statistically significant association with younger age at surgery (29.2 ± 12.5 versus 51.3 ± 18.8, p = 0.0001), a predominant macrofollicular growth pattern (20/26 mutated cases versus 18/35 wildtype; p = 0.01) and atrophic changes (20/26 mutated cases versus 2/35 wildtype; p = 0.0001). Similar results were obtained when excluding TFND cases. We also present clinical and histological triaging criteria for DICER1 sequencing of thyroid lesions, which led to the identification of DICER1 variants in 16 out of 26 cases (62%) when followed. Among these, 3 out of 12 cases with available data were found to carry a constitutional DICER1 mutation. This observation suggests that the majority of DICER1 mutations are somatic-however implies that sequencing of constitutional tissues could be clinically motivated. We conclude that DICER1 mutations are amassed in younger patients with macrofollicular-patterned tumors and, most strikingly, atrophic changes. Given the rate of constitutional involvement, our findings could be of clinical value, allowing the pathologist to triage cases for genetic testing based on histological findings.

Prevalence, Molecular Landscape, and Clinical Impact of DICER1 and DGCR8 Mutated Follicular-Patterned Thyroid Nodules.

BACKGROUND: Mutations in micro-RNA (miRNA) regulators DICER1 and DGCR8 have recently been uncovered, revealing a potential novel mechanism driving thyroid tumor development. However, the true frequency of these hotspot mutations in follicular-patterned thyroid tumors (FTs) and their relation to established driver gene events remain elusive. METHODS: A total of 440 FTs from 2 institutions were interrogated for DICER1, DGCR8, and RAS family hotspot mutations using Sanger sequencing. Whole-exome sequencing was also performed to identify additional driver gene aberrations in DICER1/DGCR8-mutant cases. Subsets of cases were further analyzed using miRNA expression profiling, and key dysregulated miRNAs were validated as markers of DICER1 mutations using quantitative RT-PCR analysis. The Cancer Genome Atlas (TCGA) database was also probed for DICER1/DGCR8 mutations and miRNA dysregulation. RESULTS: Fourteen (3.2%) and 4 (1%) FTs harbored DICER1 and DGCR8 hotspot mutations, respectively, in the combined cohort, and no cases with normal tissue available were found to exhibit a constitutional variant. Two DGCR8-mutant cases also harbored oncogenic RAS mutations. Whole-exome sequencing analysis did not identify additional driver gene events in DICER1/DGCR8-positive cases. Comprehensive miRNA expression profiling revealed a unique pattern of dysregulated miRNAs in DICER1/DGCR8-mutant cases compared with wild-type lesions. Moreover, DICER1-mutant cases showed a remarkable reduction of 5' arm miRNAs, findings corroborated in the TCGA cohort. CONCLUSION: DICER1 and DGCR8 hotspot mutations are rare in unselected cohorts of FTs, and mutated cases exhibit a specific miRNA profile. Although DGCR8 mutations may coexist with established RAS gene alterations, FTs with DICER1 variants were devoid of other driver gene events.

Transplantation and Noninvasive Longitudinal In Vivo Imaging of Parathyroid Cells: A Proof-of-Concept Study.

The parathyroid cell is a vital regulator of extracellular calcium levels, operating through the secretion of parathyroid hormone (PTH). Despite its importance, the regulation of PTH secretion remains complex and not fully understood, representing a unique interplay between extracellular and intracellular calcium, and hormone secretion. One significant challenge in parathyroid research has been the difficulty in maintaining cells ex vivo for in-depth cellular investigations. To address this issue, we introduce a novel platform for parathyroid cell transplantation and noninvasive in vivo imaging using the anterior chamber of the eye as a transplantation site. We found that parathyroid adenoma tissue transplanted into the mouse eye engrafted onto the iris, became vascularized, and retained cellular composition. Transplanted animals exhibited elevated PTH levels, indicating a functional graft. With in vivo confocal microscopy, we were able to repetitively monitor parathyroid graft morphology and vascularization. In summary, there is a pressing need for new methods to study complex cellular processes in parathyroid cells. Our study provides a novel approach for noninvasive in vivo investigations that can be applied to understand parathyroid physiology and pathology under physiological and pathological conditions. This innovative strategy can deepen our knowledge on parathyroid function and disease.

Genetic variants and down-regulation of CACNA1H in pheochromocytoma.

Pheochromocytoma (PCC) and abdominal paraganglioma (aPGL) (together abbreviated PPGL) frequently present with an underlying genetic event in a PPGL driver gene, and additional susceptibility genes are anticipated. Here, we re-analyzed whole-exome sequencing data for PCC patients and identified two patients with rare missense variants in the calcium voltage-gated channel subunit 1H gene (CACNA1H). CACNA1H variants were also found in the clinical setting in PCC patients using targeted sequencing and from analysis of The Cancer Genome Atlas database. In total, CACNA1H variants were found in six PCC cases. Three of these were constitutional, and two are known to have functional consequences on hormone production and gene expression in primary aldosteronism and aldosterone-producing adrenocortical adenoma. In general, PPGL exhibited reduced CACNA1H mRNA expression as compared to normal adrenal. Immunohistochemistry showed strong CACNA1H (CaV3.2) staining in adrenal medulla while PPGL typically had weak or negative staining. Reduced CACNA1H gene expression was especially pronounced in PCC compared to aPGL and in PPGL with cluster 2 kinase signaling phenotype. Furthermore, CACNA1H levels correlated with HIF1A and HIF2A. Moreover, TCGA data revealed a correlation between CACNA1H methylation density and gene expression. Expression of rCacna1h in PC12 cells induced differential protein expression profiles, determined by mass spectrometry, as well as a shift in the membrane potential where maximum calcium currents were observed, as determined by electrophysiology. The findings suggest the involvement of CACNA1H/CaV3.2 in pheochromocytoma development and establish a potential link between the etiology of adrenomedullary and adrenocortical tumor development.