Samuel Backman

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.

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. Clin Cancer Res; 21(19); 4451–60. ©2015 AACR.

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.

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/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.