Several studies have attempted to find the initiating drivers of small intestinal neuroendocrine tumors (SI-NET) development and the molecular mechanisms driving progression and metastatic spread. For gene expression studies using bulk microarrays and RNA sequencing, researchers commonly use normal intestinal mucosa as a control. The intestine is made up of several different cell types, and using bulk RNA-seq may generate findings that reflect factors other than the tumor transformation. This could potentially contribute to the lack of discoverable treatments and prevention strategies for SI-NETs. We performed scRNA-seq on tissue from two patients that had tumor resection surgery and separated the EC cells from the normal intestinal mucosa and used specific markers to compare them against SI-NET tumors cells. We pinpointed new genes associated with chr18 haploinsufficiency but also others with loss or gain of expression that have not previously been associated with SI-NETs, and which could be potential targets for further functional genomic studies. ### Competing Interest Statement The authors have declared no competing interest.

Tumour evolution with acquisition of more aggressive disease characteristics is a hallmark of disseminated cancer. Metastatic pancreatic neuroendocrine tumours (PanNETs) in particular, show frequent progression from a low/intermediate to a high-grade disease. To understand the molecular mechanisms underlying this phenomenon, we performed multi-omics analysis of 32 longitudinal samples from six metastatic PanNET patients. Following MEN1 inactivation, PanNETs exhibit genetic heterogeneity on both spatial and temporal dimensions with parallel and convergent tumuor evolution involving the ATRX/DAXX and mTOR pathways. Following alkylating chemotherapy treatment, some PanNETs develop mismatch repair deficiency and acquire a hypermutator phenotype. This DNA hypermutation phenotype was only found in cases that also showed transformation into a high-grade PanNET. Overall, our findings contribute to broaden the understanding of metastatic PanNET, and suggests that therapy driven disease evolution is an important hallmark of this disease.

OBJECTIVE:Hereditary pheochromocytoma (hPCC) commonly develops bilaterally, causing adrenal insufficiency when standard treatment, radical adrenalectomy (RA), is performed. Partial adrenalectomy (PA) aims to preserve adrenal function, but with higher recurrence rates. This study compares outcomes of PA versus RA in hPCC. METHODS:Patients with hPCC due to pathogenic variants in RET, VHL, NF1, MAX, and TMEM127 from 12 European centers (1974-2023) were studied retrospectively. Stratified analysis based on surgery type and initial presentation was conducted. The main outcomes included recurrence, adrenal insufficiency, metastasis, and mortality. RESULTS:The study included 256 patients (223 RA, 33 PA). Ipsilateral recurrence rates were 9/223 (4%) after RA versus 5/33 (15%) after PA (P = 0.02). Metastasis and mortality did not differ between groups. Overall, 103 patients (40%) underwent bilateral adrenalectomy either synchronously or metachronously (75 RA, 28 PA). Of these, 46% developed adrenal insufficiency after PA.In total, 191 patients presented with initial unilateral disease, of whom 50 (26%) developed metachronous contralateral disease, most commonly in RET, VHL, and MAX. In patients with metachronous bilateral disease, adrenal insufficiency developed in 3/4 (75%) when PA was performed as the first operation followed by RA, compared to 1/7 (14%) when PA was performed as the second operation after prior RA (P = 0.09). CONCLUSION:In patients with hPCC undergoing PA, local recurrence rates are higher than after RA, but metastasis and disease-specific mortality are similar. Therefore, PA seems a safe method to preserve adrenal function in patients with hPCC, in cases of both synchronous and metachronous bilateral disease, when performed as a second operation.

Supplementary Table 4. A binary distance matrix

Supplementary Table 5. Generated segmentations from SNP array data and subsequent analyses

Supplementary Figures 1-9, Tables 1-3. Supplementary Figure 1 Overview of the workflow executed in the current study. Supplementary Figure 2 Summary of the utilized sequencing workflow. Supplementary Figures 3A-B Sequencing reads and chromatograms from tumor and normal tissue showing (A)validation of H-RAS, RET and VHL mutations. Figure 3B presents data from patient 29 that had previously been determined as wild type in the VHL locus by Sanger Sequencing analysis. Supplementary Figure 4 Validation of NF1 mutations that was determined to cause a splice site disruption by in silico analysis Supplementary Figure 5. Standard curve showing relationship between copy number and Log R ratio generated by all segments included samples and arrays. Supplementary Figure 6 Correlation of study observations and ASCAT analysis in estimating the total fraction of tumor cells within each individual tumor sample. ASCAT could not determine the tumor purity for two samples. Supplementary Figure 7 Correlation of results from histopathological investigation and ASCAT in estimating the total fraction of tumor cells. Pearson correlation test R=0.658. Supplementary Figure 8 Correlation between TAPS and the study workflow in the estimation of clonal fractions for specific segments Supplementary Figure 9A and B. (A) correlation of allele frequencies of germline SNPs between SNP array and deeps sequencing methods. (B) relative difference between SNP array and deep sequencing variant frequencies dependent on deep sequencing read coverage. Supplementary table 1. Sequence coverage of targeted NGS Supplementary table 2. Statistical correlations Supplementary Table 3. Statistics on a tumor lesion bases

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.

Most aldosterone-producing adenomas (APAs) have gain-of-function somatic mutations of ion channels or transporters. However, their frequency in aldosterone-producing cell clusters of normal adrenal gland suggests a requirement for codriver mutations in APAs. Here we identified gain-of-function mutations in both CTNNB1 and GNA11 by whole-exome sequencing of 3/41 APAs. Further sequencing of known CTNNB1-mutant APAs led to a total of 16 of 27 (59%) with a somatic p.Gln209His, p.Gln209Pro or p.Gln209Leu mutation of GNA11 or GNAQ. Solitary GNA11 mutations were found in hyperplastic zona glomerulosa adjacent to double-mutant APAs. Nine of ten patients in our UK/Irish cohort presented in puberty, pregnancy or menopause. Among multiple transcripts upregulated more than tenfold in double-mutant APAs was LHCGR, the receptor for luteinizing or pregnancy hormone (human chorionic gonadotropin). Transfections of adrenocortical cells demonstrated additive effects of GNA11 and CTNNB1 mutations on aldosterone secretion and expression of genes upregulated in double-mutant APAs. In adrenal cortex, GNA11/Q mutations appear clinically silent without a codriver mutation of CTNNB1.

Most aldosterone-producing adenomas (APA) have gain-of-function somatic mutations of ion channels or transporters. However, their frequency in aldosterone-producing cell-clusters of normal adrenals suggests the existence of co-driver mutations which influence the development or phenotype of APAs. Gain-of-function mutations in both CTNNB1 and GNA11 were found by whole exome sequencing in 3 of 41 APAs from a UK/Irish cohort. Targeted sequencing for exon 3 mutations of CTNNB1 and p.Gln209 mutations of either GNA11 or closely homologous GNAQ confirmed these and 7 further double mutant APAs in this discovery cohort. The presence of GNA11/Q p.Gln209 mutations in CTNNB1 mutant APAs were replicated in 2 cohorts from France (n=14) and Sweden (n=3). In total, 16 (59%) of the 27 CTNNB1 mutant APAs investigated had a mutation at p.Gln209 of GNA11 (n=11) or GNAQ (n=5). Interestingly, CTNNB1-mutant APAs were more commonly present in women (23/27), and of these, those with GNA11/Q mutations were all women except for a pubertal boy. To also note, 9 of 10 of the UK/Irish double mutant APAs in the discovery cohort presented in puberty, pregnancy, or menopause. Mutation of p.Gln209, or homologous p.Gln in GNAS, GNA12-14, impair hydrogen bonds between G-protein α and β subunits. Transfection of H295R cells, an immortalised adrenocortical cell line heterozygous for the p.Ser45Pro mutation of CTNNB1 but wild-type for GNA11-14/Q/S, by each of the GNA11/Q mutations increased aldosterone secretion and CYP11B2 expression (encoding aldosterone synthase) by 1.93-6.1-fold and 8.0-9.8-fold respectively, compared to vector or wild-type -transfected cells. In ZG, GNA11/Q mediate the aldosterone response to angiotensin II, via stimulation of intracellular Ca2+ release by inositol trisphosphate. In the mutant-transfected cells, the stimulatory effect of angiotensin II 10 nM was retained. In order to determine whether the p.Gln209 mutations stimulate aldosterone production even in the absence of CTNNB1 activation, the transfections of H295R cells were repeated after either 24-h treatment with the CTNNB1 inhibitor, ICG-001, or silencing of CTNNB1 using the ONTARGETplus SMARTpool SiRNAs (Dharmacon). Both interventions reduced the aldosterone production relative to vehicle/control-treated cells; however neither ICG-001 nor silencing of CTNNB1 blunted the fold-increase in aldosterone secretion seen in mutant-transfected cells compared to wild-type. In summary, we report the discovery of gain-of-function mutations of the G-protein, GNA11, or its close homologue, GNAQ, in multiple APAs which majority presented during periods of high LH/HCG. To date, the mutation is always residue p.Gln209, and associated with a gain-of-function mutation of CTNNB1. These GNA11/Q p.Gln209 mutations increase aldosterone and CYP11B2 production both in the presence and in the absence of CTNNB1 activation.

3D-structures for GNAQ and 600 GNAS Somatic or mosaic mutation of p.Gln inhibits GTPase activity 601 and constitutively activates downstream signalling. We find that p.Gln mutation of GNA11/Q 602 stimulates aldosterone production, and, in the adrenal, always co-exists with somatic mutation in 603 exon 3 of CTNNB1 . This prevents inactivation by phosphorylation (e.g. of p.Ser33, in purple, in the 604 partial 3D sequence). Double-mutation of GNA11/Q and CTNNB1 induces high expression of multiple 605 genes, including LHCGR, the G α s/cyclic AMP coupled receptor of luteinizing and pregnancy 606 hormones. The 3D structures of CTNNB1, GNAS, GNAQ, AT1-receptor, renin, ACE were downloaded from models 6M93, 3C14, 4QJ3, 6YV1, 2V0Z, 1O8A, respectively, at www.rcsb.org/.

Objective: Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant syndrome usually caused by loss-of-function mutations in the MEN1 gene. However, a minority of patients who fulfill the criteria for MEN1 are not found to harbor MEN1 mutations. Besides, some of these individuals, present with a subtly different phenotype suggestive of sporadic disease. The aim of the present study was to investigate the genetic architecture of mutation-negative MEN1. Design: Fourteen patients with a clinical diagnosis (n = 13) or suspicion (n = 1) of MEN1 who had negative genetic screening of the MEN1 gene were included. Methods: Constitutional DNA from the included patients, as well as tumor DNA from six of the patients, was subjected to whole genome sequencing. Constitutional variants were filtered against population databases and somatic variants were studied under a tumor-suppressor model. Results: Three patients carried pathogenic variants (two splice-site variants, one missense variant) in MEN1 that had not been detected during routine clinical sequencing, one patient carried a pathogenic variant in CASR and one patient carried a gross deletion on chromosome 1 q which included the CDC73 gene. Analysis of matched tumor DNA from six patients without mutations did not detect any recurrent genes fulfilling Knudson's two-hit model. Conclusion: These results highlight the possibility of germline mutations being missed in routine screening, the importance of considering phenocopies in atypical or mutation-negative cases. The absence of apparent diseasecausing mutations suggests that a fraction of MEN1 mutation-negative MEN1 cases may be due to the chance occurrence of several endocrine tumors in one patient.

Aldosterone producing adenomas (APAs) occur in the adrenal glands of around 30% of patients with primary aldosteronism, the most common form of secondary hypertension. Somatic mutations in KCNJ5, ATP1A1, ATP2B3, CACNA1D and CTNNB1 have been described in ~60% of these tumours. We subjected 15 aldosterone producing adenomas (13 with known mutations and two without) to RNA Sequencing and Whole Genome Sequencing (n = 2). All known mutations were detected in the RNA-Seq reads, and mutations in ATP2B3 (G123R) and CACNA1D (S410L) were discovered in the tumours without known mutations. Adenomas with CTNNB1 mutations showed a large number of differentially expressed genes (1360 compared to 106 and 75 for KCNJ5 and ATP1A1/ATP2B3 respectively) and clustered together in a hierarchical clustering analysis. RT-PCR in an extended cohort of 49 APAs confirmed higher expression of AFF3 and ISM1 in APAs with CTNNB1 mutations. Investigation of the expression of genes involved in proliferation and apoptosis revealed subtle differences between tumours with and without CTNNB1 mutations. Together our results consolidate the notion that CTNNB1 mutations characterize a distinct subgroup of APAs.

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.

TFF3 in primary tumours has a negative impact on survival in small intestinal neuroendocrine tumours

Adrenocortical carcinoma (ACC) and pheochromocytoma and paraganglioma (PPGL) are defined by clinicopathological criteria and can be further sub-divided based on different molecular features. Whether differences between these molecular subgroups are significant enough to re-challenge their current clinicopathological classification is currently unknown. It is also not fully understood to which other cancers ACC and PPGL show similarity to. To address these questions, we included recent RNA-Seq data from the Cancer Genome Atlas (TCGA) and Therapeutically Applicable Research to Generate Effective Treatments (TARGET) datasets. Two bioinformatics pipelines were used for unsupervised clustering and principal components analysis. Results were validated using consensus clustering model and interpreted according to previous pan-cancer experiments. Two datasets consisting of 3319 tumors from 35 disease categories were studied. Consistent with the current classification, ACCs clustered as a homogenous group in a pan-cancer context. It also clustered close to neural crest derived tumors, including gliomas, neuroblastomas, pancreatic neuroendocrine tumors, and PPGLs. Contrary, some PPGLs mixed with pancreatic neuroendocrine tumors or neuroblastomas. Thus, our unbiased gene-expression analysis of PPGL did not overlap with their current clinicopathological classification. These results emphasize some importances of the shared embryological origin of these tumors, all either related or close to neural crest tumors, and opens for investigation of a complementary categorization based on gene-expression features.

The Wnt/β-Catenin signaling pathway is one of the most frequently altered pathways in adrenocortical carcinomas (ACCs). The aim of this study was to investigate the status of Wnt/β-Catenin signaling pathway by analyzing the expression level of β-Catenin and the mutational status of APC, AXIN2, CTNNB1, and ZNRF3 in ACCs. Mutations in APC, CTNNB1, ZNRF3 and homozygous deletions in ZNRF3 were observed in 3.8% (2/52), 11.5% (6/52), 1.9% (1/52) and 17.3% (9/52) of the cohort respectively. Novel interstitial deletions in CTNNB1 spanning intron 1 to exon 3/intron 3 were also found in 7.7% (4/52) of the tumours. All the observed alterations were mutually exclusive. Nuclear accumulation of β-Catenin, increased expression of Cyclin D1 and significantly higher expression of AXIN2 (p = 0.0039), ZNRF3 (p = 0.0032) and LEF1(p = 0.0090) observed in the tumours harbouring the deletion in comparison to tumours without CTNNB1 mutation demonstrates that the truncated β-Catenin is functionally active and erroneously activates the downstream targets. Significantly lower overall survival rate in patients with tumours harbouring alterations in APC/CTNNB1/ZNRF3 in comparison to those without mutation was observed. In conclusion, the discovery of novel large deletions in addition to the point mutations in CTNNB1 infers that activation of Wnt/β-Catenin pathway via alterations in CTNNB1 occurs frequently in ACCs. We also confirm that alterations in Wnt/β-Catenin signaling pathway members have a negative effect on overall survival of patients.

Pheochromocytomas and paragangliomas (PPGLs) are neuroendocrine tumors arising in the medullae of the adrenal glands or in paraganglia. The knowledge of the tumor biology of these lesions has increased dramatically during the past two decades and more than a dozen recurrently mutated genes have been identified. Different clusters have been described that share epigenetic signatures. Mutations in the succinate dehydrogenase complex subunit genes play a pivotal role in reprogramming the epigenetic state of these tumors by inhibiting epigenetic regulators such as TET enzymes and histone demethylases. Another subgroup of tumors carries hypomethylated genomes, and overexpression of several micro-RNAs has been described. While much remains to be investigated regarding the epigenetics of PPGLs, it is clear that it plays an important role in PPGL biology.

Integrated Molecular Characterization of Benign Cortisol Producing Adenomas Defines Two Distinct Clusters.