Joakim Crona

Comprehensive Re-Sequencing of Adrenal Aldosterone Producing Lesions Reveal Three Somatic Mutations near the KCNJ5 Potassium Channel Selectivity Filter

Background Aldosterone producing lesions are a common cause of hypertension, but genetic alterations for tumorigenesis have been unclear. Recently, either of two recurrent somatic missense mutations (G151R or L168R) was found in the potassium channel KCNJ5 gene in aldosterone producing adenomas. These mutations alter the channel selectivity filter and result in Na+ conductance and cell depolarization, stimulating aldosterone production and cell proliferation. Because a similar mutation occurs in a Mendelian form of primary aldosteronism, these mutations appear to be sufficient for cell proliferation and aldosterone production. The prevalence and spectrum of KCNJ5 mutations in different entities of adrenocortical lesions remain to be defined. Materials and Methods The coding region and flanking intronic segments of KCNJ5 were subjected to Sanger DNA sequencing in 351 aldosterone producing lesions, from patients with primary aldosteronism and 130 other adrenocortical lesions. The specimens had been collected from 10 different worldwide referral centers. Results G151R or L168R somatic mutations were identified in 47% of aldosterone producing adenomas, each with similar frequency. A previously unreported somatic mutation near the selectivity filter, E145Q, was observed twice. Somatic G151R or L168R mutations were also found in 40% of aldosterone producing adenomas associated with marked hyperplasia, but not in specimens with merely unilateral hyperplasia. Mutations were absent in 130 non-aldosterone secreting lesions. KCNJ5 mutations were overrepresented in aldosterone producing adenomas from female compared to male patients (63 vs. 24%). Males with KCNJ5 mutations were significantly younger than those without (45 vs. 54, respectively; p<0.005) and their APAs with KCNJ5 mutations were larger than those without (27.1 mm vs. 17.1 mm; p<0.005). Discussion Either of two somatic KCNJ5 mutations are highly prevalent and specific for aldosterone producing lesions. These findings provide new insight into the pathogenesis of primary aldosteronism.

Somatic Mutations in H-RAS in Sporadic Pheochromocytoma and Paraganglioma Identified by Exome Sequencing

CONTEXT Up to 60% of pheochromocytoma (PCC) and paraganglioma (PGL) are associated with either somatic or germline mutations in established PCC and PGL susceptibility loci. Most unexplained cases are characterized by an increased activity of the RAS/RAF/ERK signaling pathway. Mutations in RAS subtypes H, K, and N are common in human cancers; however, previous studies have been inconsistent regarding the mutational status of RAS in PCC and PGL. OBJECTIVES The aim of this study was to identify novel disease causing genes in PCC and PGL tumors. DESIGN, SETTING, AND PARTICIPANTS Four benign and sporadic PCC and PGL tumors were subjected to whole exome sequencing using the Illumina HiSeq Platform. Sequences were processed by CLC genomics 4.9 bioinformatics software and the acquired list of genetic variants was filtered against the Catalogue of Somatic Mutations in Cancer database. Findings were validated in an additional 78 PCC and PGL tumor lesions. RESULTS Exome sequencing identified 2 cases with somatic mutations in the H-RAS. In total, 6.9% (n = 4/58) of tumors negative for mutations in major PCC and PGL loci had mutations in H-RAS: G13R, Q61K, and Q61R. There were 3 PCC and 1 PGL; all had sporadic presentation with benign tumor characteristics and substantial increases in norepinephrine and/or epinephrine. H-RAS tumors were exclusively found in male patients (P = .007). CONCLUSIONS We identified recurrent somatic H-RAS mutations in pheochromocytoma and paraganglioma. Tumors with H-RAS mutations had activation of the RAS/RAF/ERK signaling pathway and were associated with male PCC patients having benign and sporadic disease characteristics. H-RAS could serve as a prognostic and predictive marker as well as a novel therapeutic target.

Next-generation sequencing in the clinical genetic screening of patients with pheochromocytoma and paraganglioma

Background Recent findings have shown that up to 60% of pheochromocytomas (PCCs) and paragangliomas (PGLs) are caused by germline or somatic mutations in one of the 11 hitherto known susceptibility genes: SDHA, SDHB, SDHC, SDHD, SDHAF2, VHL, HIF2A (EPAS1), RET, NF1, TMEM127 and MAX. This list of genes is constantly growing and the 11 genes together consist of 144 exons. A genetic screening test is extensively time consuming and expensive. Hence, we introduce next-generation sequencing (NGS) as a time-efficient and cost-effective alternative. Methods Tumour lesions from three patients with apparently sporadic PCC were subjected to whole exome sequencing utilizing Agilent Sureselect target enrichment system and Illumina Hi seq platform. Bioinformatics analysis was performed in-house using commercially available software. Variants in PCC and PGL susceptibility genes were identified. Results We have identified 16 unique genetic variants in PCC susceptibility loci in three different PCC, spending less than a 30-min hands-on, in-house time. Two patients had one unique variant each that was classified as probably and possibly pathogenic: NF1 Arg304Ter and RET Tyr791Phe. The RET variant was verified by Sanger sequencing. Conclusions NGS can serve as a fast and cost-effective method in the clinical genetic screening of PCC. The bioinformatics analysis may be performed without expert skills. We identified process optimization, characterization of unknown variants and determination of additive effects of multiple variants as key issues to be addressed by future studies.

Effect of Temozolomide in Patients with Metastatic Bronchial Carcinoids

Introduction: Metastatic bronchial carcinoids are rare neoplasms, where efforts of medical treatment so far have been disappointing. A previous study from our center indicated that temozolomide might be of value. Materials and Methods: All patients with progressive metastatic bronchial carcinoid treated with temozolomide as monotherapy at our center between 2004 and 2010 (n = 31) were included in this retrospective study. 14 tumors were classified as typical and 15 as atypical carcinoids, whereas 2 tumors could not be classified. Temozolomide was given on 5 consecutive days every 4 weeks. Toxicity was evaluable in 28 of 31 patients, and 22 patients were evaluable by RECIST 1.1. Results: There were no complete responses. A partial response was seen in 3 patients (14%), stable disease in 11 (52%) and progressive disease in 7 patients (33%). Median progression-free survival was 5.3 months and median overall survival was 23.2 months from the start of temozolomide. Toxicities grade 3-4 were noted in 4 patients, thrombocytopenia (n = 3) and leukopenia (n = 1). Conclusion: Temozolomide as monotherapy shows activity in metastatic bronchial carcinoids. Regimens combining temozolomide with other agents (e.g. capecitabine and/or bevacizumab, everolimus, radiolabeled somatostatin analogues) should be further studied in these patients.

Treatment, prognostic markers and survival in thymic neuroendocrine tumours. A study from a single tertiary referral centre

Thymic neuroendocrine tumours (TNETs) are uncommon but malignant neoplasms, usually associated with a poor prognosis. The number of cases reported is limited to a few hundreds and there are few prognostic factors available. All 28 patients (22 male, 6 female; median age 46.5 years) with thymic neuroendocrine tumour, treated at the Department of Endocrine Oncology, Uppsala University Hospital, Uppsala, Sweden between 1985 and 2011 were studied. The overall 3, 5 and 10-year survival was 89%, 79% and 41% respectively. Ki67<10% (p=0.018) as well as surgical resection (p=0.001) and macroscopically radical primary surgery (p=0.034) was associated with increased survival. Staging & grading according to Masaoka and ENETS systems did not correlate with survival. However, a modified ENETS grading showed a positive correlation (p=0.015). Median time to progression was 20.5 months with Temozolomide and 18 months with platinum based therapy. Partial responses were noted in three patients (38%) treated with platinum based therapy and in two patients (20%) treated with Temozolomide based therapy. High proliferative rate, measured by Ki67 index, and absence of macroscopically radical primary resection as well as no surgical resection are three negative prognostic factors in patients with TNETs. Temozolomide or Platinum based chemotherapy should be considered as first-line medical therapy in patients with metastatic or non-resectable tumours.

Integrative Genetic Characterization and Phenotype Correlations in Pheochromocytoma and Paraganglioma Tumours

Background About 60% of Pheochromocytoma (PCC) and Paraganglioma (PGL) patients have either germline or somatic mutations in one of the 12 proposed disease causing genes; SDHA, SDHB, SDHC, SDHD, SDHAF2, VHL, EPAS1, RET, NF1, TMEM127, MAX and H-RAS. Selective screening for germline mutations is routinely performed in clinical management of these diseases. Testing for somatic alterations is not performed on a regular basis because of limitations in interpreting the results. Aim The purpose of the study was to investigate genetic events and phenotype correlations in a large cohort of PCC and PGL tumours. Methods A total of 101 tumours from 89 patients with PCC and PGL were re-sequenced for a panel of 10 disease causing genes using automated Sanger sequencing. Selected samples were analysed with Multiplex Ligation-dependent Probe Amplification and/or SNParray. Results Pathogenic genetic variants were found in tumours from 33 individual patients (37%), 14 (16%) were discovered in constitutional DNA and 16 (18%) were confirmed as somatic. Loss of heterozygosity (LOH) was observed in 1/1 SDHB, 11/11 VHL and 3/3 NF1-associated tumours. In patients with somatic mutations there were no recurrences in contrast to carriers of germline mutations (P = 0.022). SDHx/VHL/EPAS1 associated cases had higher norepinephrine output (P = 0.03) and lower epinephrine output (P<0.001) compared to RET/NF1/H-RAS cases. Conclusion Somatic mutations are frequent events in PCC and PGL tumours. Tumour genotype may be further investigated as prognostic factors in these diseases. Growing evidence suggest that analysis of tumour DNA could have an impact on the management of these patients.

Precision medicine in pheochromocytoma and paraganglioma: current and future concepts

Pheochromocytoma and paraganglioma (PPGL) are rare diseases but are also amongst the most characterized tumour types. Hence, patients with PPGL have greatly benefited from precision medicine for more than two decades. According to current molecular biology and genetics‐based taxonomy, PPGL can be divided into three different clusters characterized by: Krebs cycle reprogramming with oncometabolite accumulation or depletion (group 1a); activation of the (pseudo)hypoxia signalling pathway with increased tumour cell proliferation, invasiveness and migration (group 1b); and aberrant kinase signalling causing a pro‐mitogenic and anti‐apoptotic state (group 2). Categorization into these clusters is highly dependent on mutation subtypes. At least 12 different syndromes with distinct genetic causes, phenotypes and outcomes have been described. Genetic screening tests have a documented benefit, as different PPGL syndromes require specific approaches for optimal diagnosis and localization of various syndrome‐related tumours. Genotype‐tailored treatment options, follow‐up and preventive care are being investigated. Future new developments in precision medicine for PPGL will mainly focus on further identification of driver mechanisms behind both disease initiation and malignant progression. Identification of novel druggable targets and prospective validation of treatment options are eagerly awaited. To achieve these goals, we predict that collaborative large‐scale studies will be needed: Pheochromocytoma may provide an example for developing precision medicine in orphan diseases that could ultimately aid in similar efforts for other rare conditions.

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 patterns through an array-based approach in small intestinal neuroendocrine tumors

Endocrine tumors arise from endocrine glands. Most endocrine tumors are benign but malignant variants exist. Several endocrine neoplasms display loss of parts of chromosome 11 or 18, produce hormones and responds poorly to conventional chemotherapeutics. The multiple endocrine neoplasia syndromes are mainly confined to endocrine tumors. This opens the question if there exists a single or several endocrine tumor genes.The aim of the study was to describe genetic derangements in endocrine tumors.Paper I: Investigation of mutational status of SDHAF2 in parathyroid tumors. SDHAF2 is located in the proximity of 11q13, a region that frequently displays loss in parathyroid tumors. We established that mutations in SDHAF2 are infrequent in parathyroid tumors.Paper II: Study of SDHAF2 gene expression in a cohort of benign pheochromocytomas (PCC) (n=40) and malignant PCC (n=10). We discovered a subset of benign PCC (28/40) and all malignant PCC (10/10) with significantly lower SDHAF2 expression. Benign PCC with low SDHAF2 expression and malignant tumors consistently expressing low levels of SDHAF2 were methylated in the promoter region. SDHAF2 expression was restored in vitro after treatment with 5- aza-2-deoxycytidine.Paper III: HumanMethylation27 array (Illumina) covering 27578 CpG sites spanning over 14495 genes were analyzed in a discovery cohort of 10 primary small neuroendocrine tumors (SI-NETs) with matched metastases. 2697 genes showed different methylation pattern between the primary tumor and its metastasis. We identified several hypermethylated genes in key regions. Unsupervised clustering of the tumors identified three distinct clusters, one with a highly malignant behavior.Paper IV: Loss of chromosome 18 is the most frequent genetic aberration in SI-NETs. DNA from SI-NETs were subjected to whole exome capture sequencing and high resolution SNP array. Genomic profiling revealed loss of chromosome 18 in 5 out of 7 SI-NETs. No tumor-specific somatic mutation on chromosome 18 was identified which suggests involvement of other mechanisms than point mutations in SI-NET tumorigenesis.Paper V: The cost for diagnostic genetic screening of common susceptibility genes in PCC is expensive and labor intensive. Three PCC from three patients with no known family history were chosen for exome capture sequencing. We identified three variants in known candidate genes. We suggest that exome-capture sequencing is a quick and cost-effective tool.

Multiple and Secondary Hormone Secretion in Patients With Metastatic Pancreatic Neuroendocrine Tumours

CONTEXT As a group, neuroendocrine tumors (NETs) secrete many different peptide hormones, yet heretofore each NET patient is typically thought to produce at most one hormone that causes a distinct hormonal syndrome. A minority of patients have multiple hormones at diagnosis and may also develop secondary hormone secretion at a later stage. OBJECTIVES The objectives of the study were to determine the frequency and to describe the impact of multiple and secondary hormone secretion in sporadic gasteroenteropancreatic NET patients. DESIGN, SETTING, AND PARTICIPANTS This was a retrospective analysis of patients (n = 972) with gasteroenteropancreatic NET treated at Uppsala University Hospital, Uppsala, Sweden. Patients with the secretion of multiple hormones at diagnosis and/or those developing secondary hormone secretion during the disease course were identified and studied in further detail. RESULTS In pancreatic NETs (PNETs), a total of 19 of 323 patients (6%) had secretion of multiple hormones at diagnosis, and 14 of 323 (4%) had secondary changes during the disease course. These phenomena occurred exclusively in patients with an advanced disease stage, and secondary hormones were detected in a close time span with progressive disease. Patients with secondary insulin hypersecretion had increased morbidity as well as reduced survival (P < .002). In contrast, multiple and secondary hormone secretion was rarely seen in NETs of the small intestine with 0 and 1 of 603 cases, respectively. CONCLUSION Diversity of PNET hormone secretion either at diagnosis or during the disease course occurred in a minority of patients (9.3%). These phenomena had a major impact on patient outcome both through increased morbidity and mortality. Our results support that patients with metastatic PNETs should be monitored for clinical symptoms of secondary hormone secretion during the disease course.