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 Evolutionary History of Metastatic Pancreatic Neuroendocrine Tumours Reveals a Therapy Driven Route to High-Grade Transformation.

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.

Pan-genomic characterization of high-risk pediatric papillary thyroid carcinoma.

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.

Somatic mutations of GNA11 and GNAQ in CTNNB1-mutant aldosterone-producing adenomas presenting in puberty, pregnancy or menopause.

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.

Whole genome sequencing of apparently mutation-negative MEN1 patients.

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 1q 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 disease-causing 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.

RNA Sequencing Provides Novel Insights into the Transcriptome of Aldosterone Producing Adenomas.

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.

Epigenetics of pheochromocytoma and paraganglioma.

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.

Comprehensive analysis of CTNNB1 in adrenocortical carcinomas: Identification of novel mutations and correlation to survival.

The Wnt/ß-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/ß-Catenin signaling pathway by analyzing the expression level of ß-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 ß-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 ß-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/ß-Catenin pathway via alterations in CTNNB1 occurs frequently in ACCs. We also confirm that alterations in Wnt/ß-Catenin signaling pathway members have a negative effect on overall survival of patients.

RNA-Sequencing Analysis of Adrenocortical Carcinoma, Pheochromocytoma and Paraganglioma from a Pan-Cancer Perspective.

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.

Detection of Somatic Mutations in Gastroenteropancreatic Neuroendocrine Tumors Using Targeted Deep Sequencing.

Mutations affecting the mechanistic target of rapamycin (MTOR) signalling pathway are frequent in human cancer and have been identified in up to 15% of pancreatic neuroendocrine tumours (NETs). Grade A evidence supports the efficacy of MTOR inhibition with everolimus in pancreatic NETs. Although a significant proportion of patients experience disease stabilization, only a minority will show objective tumour responses. It has been proposed that genomic mutations resulting in activation of MTOR signalling could be used to predict sensitivity to everolimus. PATIENTS AND METHODS: Patients with NETs that underwent treatment with everolimus at our Institution were identified and those with available tumour tissue were selected for further analysis. Targeted next-generation sequencing (NGS) was used to re-sequence 22 genes that were selected on the basis of documented involvement in the MTOR signalling pathway or in the tumourigenesis of gastroenterpancreatic NETs. Radiological responses were documented using Response Evaluation Criteria in Solid Tumours. RESULTS: Six patients were identified, one had a partial response and four had stable disease. Sequencing of tumour tissue resulted in a median sequence depth of 667.1 (range=404-1301) with 1-fold coverage of 95.9-96.5% and 10-fold coverage of 87.6-92.2%. A total of 494 genetic variants were discovered, four of which were identified as pathogenic. All pathogenic variants were validated using Sanger sequencing and were found exclusively in menin 1 (MEN1) and death domain associated protein (DAXX) genes. No mutations in the MTOR pathway-related genes were observed. CONCLUSION: Targeted NGS is a feasible method with high diagnostic yield for genetic characterization of pancreatic NETs. A potential association between mutations in NETs and response to everolimus should be investigated by future studies.

Global DNA Methylation Analysis Identifies Two Discrete clusters of Pheochromocytoma with Distinct Genomic and Genetic Alterations.

Pheochromocytomas and paragangliomas (PPGLs) are rare and frequently heritable neural-crest derived tumours arising from the adrenal medulla or extra-adrenal chromaffin cells respectively. The majority of PPGL tumours are benign and do not recur with distant metastases. However, a sizeable fraction of these tumours secrete vasoactive catecholamines into the circulation causing a variety of symptoms including hypertension, palpitations and diaphoresis. The genetic landscape of PPGL has been well characterized and more than a dozen genes have been described as recurrently mutated. Recent studies of DNA-methylation have revealed distinct clusters of PPGL that share DNA methylation patterns and driver mutations, as well as identified potential biomarkers for malignancy. However, these findings have not been adequately validated in independent cohorts. In this study we use an array-based genome-wide approach to study the methylome of 39 PPGL and 4 normal adrenal medullae. We identified two distinct clusters of tumours characterized by different methylation patterns and different driver mutations. Moreover, we identify genes that are differentially methylated between tumour subcategories, and between tumours and normal tissue.

Spatiotemporal Heterogeneity Characterizes the Genetic Landscape of Pheochromocytoma and Defines Early Events in Tumorigenesis.

PURPOSE: Pheochromocytoma and paraganglioma (PPGL) patients display heterogeneity in the clinical presentation and underlying genetic cause. The degree of inter- and intratumor genetic heterogeneity has not yet been defined. EXPERIMENTAL DESIGN: In PPGLs from 94 patients, we analyzed LOH, copy-number variations, and mutation status of SDHA, SDHB, SDHC, SDHD, SDHAF2, VHL, EPAS1, NF1, RET, TMEM127, MAX, and HRAS using high-density SNP array and targeted deep sequencing, respectively. Genetic heterogeneity was determined through (i) bioinformatics analysis of individual samples that estimated absolute purity and ploidy from SNP array data and (ii) comparison of paired tumor samples that allowed reconstruction of phylogenetic trees. RESULTS: Mutations were found in 61% of the tumors and correlated with specific patterns of somatic copy-number aberrations (SCNA) and degree of nontumoral cell admixture. Intratumor genetic heterogeneity was observed in 74 of 136 samples using absolute bioinformatics estimations and in 22 of 24 patients by comparison of paired samples. In addition, a low genetic concordance was observed between paired primary tumors and distant metastases. This allowed for reconstructing the life history of individual tumors, identifying somatic mutations as well as copy-number loss of 3p and 11p (VHL subgroup), 1p (Cluster 2), and 17q (NF1 subgroup) as early events in PPGL tumorigenesis. CONCLUSIONS: Genomic landscapes of PPGL are specific to mutation subtype and characterized by genetic heterogeneity both within and between tumor lesions of the same patient.

Novel somatic mutations and distinct molecular signature in aldosterone-producing adenomas.

Aldosterone-producing adenomas (APAs) are found in 1.5-3.0% of hypertensive patients in primary care and can be cured by surgery. Elucidation of genetic events may improve our understanding of these tumors and ultimately improve patient care. Approximately 40% of APAs harbor a missense mutation in the KCNJ5 gene. More recently, somatic mutations in CACNA1D, ATP1A1 and ATP2B3, also important for membrane potential/intracellular Ca(2) (+) regulation, were observed in APAs. In this study, we analyzed 165 APAs for mutations in selected regions of these genes. We then correlated mutational findings with clinical and molecular phenotype using transcriptome analysis, immunohistochemistry and semiquantitative PCR. Somatic mutations in CACNA1D in 3.0% (one novel mutation), ATP1A1 in 6.1% (six novel mutations) and ATP2B3 in 3.0% (two novel mutations) were detected. All observed mutations were located in previously described hotspot regions. Patients with tumors harboring mutations in CACNA1D, ATP1A1 and ATP2B3 were operated at an older age, were more often male and had tumors that were smaller than those in patients with KCNJ5 mutated tumors. Microarray transcriptome analysis segregated KCNJ5 mutated tumors from ATP1A1/ATP2B3 mutated tumors and those without mutation. We observed significant transcription upregulation of CYP11B2, as well as the previously described glomerulosa-specific gene NPNT, in ATP1A1/ATP2B3 mutated tumors compared to KCNJ5 mutated tumors. In summary, we describe novel somatic mutations in proteins regulating the membrane potential/intracellular Ca(2) (+) levels, and also a distinct mRNA and clinical signature, dependent on genetic alteration.