Peter Stålberg

Recurrent activating mutation in PRKACA in cortisol-producing adrenal tumors

Adrenal tumors autonomously producing cortisol cause Cushings syndrome. We performed exome sequencing of 25 tumor-normal pairs and identified 2 subgroups. Eight tumors (including three carcinomas) had many somatic copy number variants (CNVs) with frequent deletion of CDC42 and CDKN2A, amplification of 5q31.2 and protein-altering mutations in TP53 and RB1. Seventeen tumors (all adenomas) had no somatic CNVs or TP53 or RB1 mutations. Six of these had known gain-of-function mutations in CTNNB1 (β-catenin) or GNAS (Gαs). Six others had somatic mutations in PRKACA (protein kinase A (PKA) catalytic subunit) resulting in a p.Leu206Arg substitution. Further sequencing identified this mutation in 13 of 63 tumors (35% of adenomas with overt Cushings syndrome). PRKACA, GNAS and CTNNB1 mutations were mutually exclusive. Leu206 directly interacts with the regulatory subunit of PKA, PRKAR1A. Leu206Arg PRKACA loses PRKAR1A binding, increasing the phosphorylation of downstream targets. PKA activity induces cortisol production and cell proliferation, providing a mechanism for tumor development. These findings define distinct mechanisms underlying adrenal cortisol-producing tumors.

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.

High Success Rate of Parathyroid Reoperation may be Achieved with Improved Localization Diagnosis

IntroductionBecause of the difficulty of reoperative parathyroid surgery, preoperative imaging studies have been increasingly adopted. We report the use of consistently applied localization diagnosis to yield high success rates in parathyroid reoperations.MethodsParathyroid reoperation was performed after previous parathyroid surgery in 144 patients with nonmalignant hyperparathyroidism (HPT) between 1962 and 2007. From the year 2000, 46 patients who underwent parathyroid reoperation and 14 patients who were subjected to thyroid surgery before primary parathyroid operation were investigated with sestamibi scintigraphy (MIBI), 11C-methionine PET/CT (met-PET), surgeon-performed ultrasound (US), US-guided fine-needle aspiration biopsy (US-FNA), and selective venous sampling (SVS) with rapid PTH (Q-PTH) analyses. When imaging was considered adequate, additional studies were generally not obtained.ResultsReversal of hypercalcemia was achieved by reoperation in 134 of 144 (93%) of all patients with previous parathyroid surgery. In patients operated from year 2000, MIBI had 90% sensitivity and 88% predictive value, met-PET 79% sensitivity and 87% predictive value, and US 72% sensitivity and 93% predictive value. SVS with Q-PTH analyses provided accurate localization or regionalization in 11 of 11 recently selected patients. Q-PTH analyses in fine-needle aspirations verified parathyroid origin of excised specimens, and intraoperative Q-PTH helped decide when operations could be terminated. In patients subjected to the algorithm of imaging procedures, reversal of hypercalcemia and apparent cure was obtained after the reoperation in 45 of 46 patients with previous parathyroid surgery, implying a success rate of 98%, and in all patients with previous thyroid surgery.ConclusionsReoperative parathyroid surgery is challenging. Results can be improved by consistently applied sensitive methods of preoperative imaging, and reoperative procedures may then achieve nearly the same success rates as primary operations.

Surgical Treatment of Graves’ Disease: Evidence-Based Approach

BackgroundThe optimal treatment of Graves´ disease (GD) is still controversial. Surgery is one treatment option along with radioactive iodine (RAI) and antithyroid medication. In this evidence-based review, we examine four issues: (1) Is surgery better than RAI or long-term antithyroid medication? (2) What is the recommended surgical approach? (3) How does the presence of Graves’ ophthalmopathy (GO) influence the role of surgery? (4) What is the role of surgery in children with GD?MethodsWe conducted a systematic review of the literature using evidence-based criteria regarding these four issues. Results(1) There are no recommendations reaching any grade of evidence for which treatment to choose for adults with GD. (2) Total thyroidectomy has complication rates equal to those seen with lesser resections but it has higher cure rates and negligible recurrence rates (Level I–IV data leading to a grade A recommendation). (3) Data support surgery when severe GO is present, but RAI combined with glucocorticoids may be equally safe (Level II–IV data, grade B recommendation). The extent of thyroid resection does not influence the outcome of GO (Level II data, grade B recommendation). (4) Based on the available data, definitive treatment can be advocated for children (Level IV data, grade C recommendation) using either RAI or surgery. No recommendation can be given as to whether RAI or surgery is preferred owing to the lack of studies addressing this issue. Increased cancer risk with RAI in children below the age of 5 years supports surgery in this setting (Level I data, grade A recommendation).ConclusionIf surgery is considered for definitive management, evidence-based criteria support total thyroidectomy as the surgical technique of choice for GD. Available evidence also supports surgery in the presence of severe endocrine GO. Children with GD should be treated with an ablative strategy. Whether this is achieved by total thyroidectomy or RAI may still be debatable. Data on long-term cancer risk are missing or conflicting; and until RAI has proven harmless in children, we continue to recommend surgery in this group.

Nerve stimulation in thyroid surgery : is it really useful?

Background:  Monitoring of the recurrent laryngeal nerve (RLN) has been claimed in some studies to reduce rates of nerve injury during thyroid surgery compared with anatomical dissection and visual identification of the RLN alone, whereas other studies have found no benefit. Continuous monitoring with endotracheal electrodes is expensive whereas discontinuous monitoring by laryngeal palpation with nerve stimulation is a simple and inexpensive technique. This study aimed to assess the value of nerve stimulation with laryngeal palpation as a means of identifying and assessing the function of the RLN and external branch of the superior laryngeal nerve (EBSLN) during thyroid surgery.

MicroRNA profiling of benign and malignant pheochromocytomas identifies novel diagnostic and therapeutic targets

MicroRNAs (miRNAs) are small RNAs ( approximately 22 bp) that post-transcriptionally regulate protein expression and are found to be differentially expressed in a number of human cancers. There is increasing evidence to suggest that miRNAs could be useful in cancer diagnosis, prognosis, and therapy. We performed miRNA microarray expression profiling on a cohort of 12 benign and 12 malignant pheochromocytomas and identified a number of differentially expressed miRNAs. These results were validated in a separate cohort of ten benign and ten malignant samples using real-time quantitative RT-PCR; benign samples had a minimum follow-up of at least 2 years. It was found that IGF2 as well as its intronic miR-483-5p was over-expressed, while miR-15a and miR-16 were under-expressed in malignant tumours compared with benign tumours. These miRNAs were found to be diagnostic and prognostic markers for malignant pheochromocytoma. The functional role of miR-15a and miR-16 was investigated in vitro in the rat PC12 pheochromocytoma cell line, and these miRNAs were found to regulate cell proliferation via their effect on cyclin D1 and apoptosis. These data indicate that miRNAs play a pivotal role in the biology of malignant pheochromocytoma, and represent an important class of diagnostic and prognostic biomarkers and therapeutic targets warranting further investigation.

Minimally invasive parathyroidectomy using the lateral focused mini-incision technique without intraoperative parathyroid hormone monitoring

Minimally invasive parathyroidectomy (MIP) involves scan‐directed removal of a single adenoma through a 2·0‐cm mini‐incision without intraoperative monitoring. The aim of this study was to analyse the outcomes of MIP using such a simplified technique.

Activating mutations in CTNNB1 in aldosterone producing adenomas

Primary aldosteronism (PA) is the most common cause of secondary hypertension with a prevalence of 5–10% in unreferred hypertensive patients. Aldosterone producing adenomas (APAs) constitute a large proportion of PA cases and represent a surgically correctable form of the disease. The WNT signaling pathway is activated in APAs. In other tumors, a frequent cause of aberrant WNT signaling is mutation in the CTNNB1 gene coding for β-catenin. Our objective was to screen for CTNNB1 mutations in a well-characterized cohort of 198 APAs. Somatic CTNNB1 mutations were detected in 5.1% of the tumors, occurring mutually exclusive from mutations in KCNJ5, ATP1A1, ATP2B3 and CACNA1D. All of the observed mutations altered serine/threonine residues in the GSK3β binding domain in exon 3. The mutations were associated with stabilized β-catenin and increased AXIN2 expression, suggesting activation of WNT signaling. By CYP11B2 mRNA expression, CYP11B2 protein expression, and direct measurement of aldosterone in tumor tissue, we confirmed the ability for aldosterone production. This report provides compelling evidence that aberrant WNT signaling caused by mutations in CTNNB1 occur in APAs. This also suggests that other mechanisms that constitutively activate the WNT pathway may be important in APA formation.

Targeted disruption of the mouse phospholipase C β3 gene results in early embryonic lethality

In order to investigate the biological function of phosphatidylinositol‐specific phospholipase C (PLC) we generated mutant mice by gene targeting. Homozygous inactivation of PLCβ3 is lethal at embryonic day 2.5. These mutants show poor embryonic organization as well as reduced numbers of cells. Identical phenotypes were recorded in homozygous mutants generated from two independently targeted embryonic stem cell clones. Heterozygous mutant mice, however, are viable and fertile for at least two generations. We also showed that mouse PLCβ3 is expressed in unfertilized eggs, 3‐cell and egg cylinder stages of embryos. In conclusion, these results indicate that PLCβ3 expression is essential for early mouse embryonic development.