BRAFV600E and BRAF-WT Specific Antitumor Immunity in Papillary Thyroid Cancer.

One feature of papillary thyroid cancer (PTC) is the frequently present somatic BRAFV600E mutation. PTCs are also characterized by a lymphocytic infiltration, which may correlate with an improved clinical outcome. The objective of the study was the characterization of BRAFV600E specific anti-immunity in PTC patients and correlation analyses with the clinical outcome. Fourteen HLA A2 positive PTC patients were included into the study of whom tumor tissue samples were also available. Of those, 8 PTC patients revealed a somatic BRAFV600E mutation. All PTC patients were also MHC class II typed. Tetramer analyses for detection of MHC class I and MHC class II-restricted, BRAFV600E epitope-specific T cells using unstimulated and peptide-stimulated T cells were performed; correlation analyses between MHC phenotypes, T cell immunity, and the clinical course were performed. In regard to unstimulated T cells, a significantly higher amount of BRAFV600E epitope specific T cells was detected compared to a control tetramer. Importantly, after overnight peptide stimulation a significantly higher number of BRAFV600E positive and BRAF WT epitope-specific T cells could be seen. In regard to the clinical course, however, no significant differences were seen, neither in the context of the initial tumor size, nor in the context of lymph node metastases or peripheral metastastic spread. In conclusion, we clearly demonstrated a BRAF-specific tumor immunity in PTC-patients which is, however, independent of a BRAFV600E status of the PTC patients.

DHEAS and Differential Blood Counts as Indirect Signs of Glucocorticoid Excess in Adrenal Non-Producing Adenomas.

The majority of incidentally discovered adrenal tumors are later characterized as non-producing adrenocortical adenomas (NPA). We asked whether laboratory abnormalities in parameters that reflect glucocorticoid action can be found in patients with NPA despite their nature of being clinically unapparent. Since glucocorticoids are potent immunosuppressants we studied blood counts and differential blood counts along with corticotropin and dehydroepiandrostenedione sulfate (DHEAS) blood concentrations, as well as cortisol values before and after an overnight 1 mg dexamethasone suppression test. We compared the results of normal individuals, of patients with adrenal adenomas and normal hormone profiles and with subclinical autonomous glucocorticoid hypersecretion, as well as overt cortisol excess. We found that almost all indices of the blood counts were significantly different between the patients groups. In particular, patients with adrenal non-producing adenomas already showed signs of glucocorticoid excess, including relative lymphocytopenia, lowered DHEAS, and ACTH concentrations than control individuals. We also found that the extent of lymphocytopenia correlated with the concentrations of DHEAS and ACTH, and DHEAS correlated well with ACTH. We conclude that the basal ACTH and DHEAS values along with the differential blood counts give good information on the extent of glucocorticoid excess and that silent adrenal adenomas seem to oversecrete glucocorticoids at concentrations that already alter these parameters.

Radioiodine therapy reduces the frequency of circulating tumour cells in patients with differentiated thyroid cancer.

OBJECTIVE: The aim of the study was the quantification of circulating tumour cells (CTCs) in differentiated thyroid cancer (DTC) patients before and 6 weeks after radioiodine therapy (RIT). CONTEXT: Circulating tumour cells (CTCs) were described more recently in cancer patients, mostly correlating with poor outcome and advanced metastases. DESIGN: Peripheral blood for identification and quantification of CTC before RIT or/and 6 weeks after RIT was provided by 55 DTC patients that received RIT for remnant tissue ablation. PATIENTS: 13 follicular thyroid cancer (FTC) patients, 31 papillary thyroid cancer (PTC) patients and 11 patients having the follicular variant PTC (FV-PTC) were included. MEASUREMENTS: Peripheral blood mononuclear cells (PBMCs) were isolated and EpCAM-positive CTCs were counted by immune fluorescent staining. RESULTS: A CTC positivity of 31.8% before RIT could be observed. Six weeks after RIT, the CTC positivity was reduced to 13.6%. Paired data at both time points of blood sampling could be gathered for n = 33 DTC patients. These patients had significantly higher CTC numbers before RIT than 6 weeks afterwards (0.27 ± 0.47 vs 0.05 ± 0.15, P = .0215). Additionally, significantly reduced CTC numbers were also demonstrated in pre-RIT CTC-positive patients (0.88 ± 0.43 vs 0.05 ± 0.16, P = .0039). CONCLUSION: Our results indicate a reducing effect on the number of CTCs by RIT. Therefore, CTC enumeration should be considered as efficient tool for treatment monitoring during RIT.

Update: Selective adrenal venous sampling (AVS) - Indication, technique, and significance. / Update: Selektive Nebennierenvenenblutentnahme (NNVBE) ­ Indikation, Technik und Stellenwert.

BACKGROUND: Primary aldosteronism (PA) is the most common detectable cause of secondary hypertension. The majority of patients have either an adrenal aldosterone-producing adenoma (APA) or bilateral adrenal hyperplasia (BAH) demanding different therapeutic approaches. Screening tests and imaging cannot reliably distinguish between a unilateral or bilateral PA. METHODS: This review article gives an overview concerning etiology, diagnostics, and therapeutic options of PA, and reviews the indication, the technique, and relevance of selective adrenal venous sampling (AVS) in the context of the current literature and the authors' experience. RESULTS: AVS can verify or exclude a unilaterally dominated secretion with a high success rate. Patients with PA and a unilateral APA can be treated curatively by adrenalectomy. CONCLUSIONS: AVS is an established diagnostic examination for differentiation of unilateral from bilateral adrenal disease in patients with PA. KEY POINTS: · Selective adrenal venous sampling (AVS) is a safe, reliable, and minimally invasive method to detect a unilateral or bilateral adrenal adrenal gland disease.. · Verification of lateralization by AVS has direct therapeutic relevance for patients with primary aldosteronism (PA).. · AVS can be performed with low radiation exposure, without contrast medium, and with a high success rate when performed by an experienced interventional radiologist.. CITATION FORMAT: · Loberg C, Antoch G, Stegbauer J et al. Update: Selective adrenal venous sampling (AVS) - Indication, technique, and significance. Fortschr Röntgenstr 2021; 193: 658 - 666.

Aldosterone-Mediated Sodium Retention Is Reflected by the Serum Sodium to Urinary Sodium to (Serum Potassium)2 to Urinary Potassium (SUSPPUP) Index.

The serum sodium to urinary sodium ratio divided by the (serum potassium)2 to urinary potassium ratio (SUSPPUP formula) reflects aldosterone action. We here prospectively investigated into the usefulness of the SUSPPUP ratio as a diagnostic tool in primary hyperaldosteronism. Parallel measurements of serum and urinary sodium and potassium concentrations (given in mmol/L) in the fasting state were done in 225 patients. Of them, 69 were diagnosed with primary aldosteronism (PA), 102 with essential hypertension (EH), 26 with adrenal insufficiency (AI) and 28 did not suffer from the above-mentioned disorders and were assigned to the reference group (REF). The result of the SUSPPUP formula was highest in the PA group (7.4, 4.2-12.3 L/mmol), followed by EH (3.2, 2.3-4.3 L/mmol), PA after surgery (3.9, 3.0-6.0 L/mmol), REF (3.4 ± 1.4 L/mmol) and AI (2.9 +/- 1.2 L/mmol). The best sensitivity in distinguishing PA from EH was reached by multiplication of the aldosterone to renin-ratio (ARR) with the SUSPPUP formula (92.7% at a cut off > 110 L/mmol), highest specificity was reached by the SUSPPUP determinations (87.2%). The integration of the SUSPPUP ratio into the ARR helps to improve the diagnosis of hyperaldosteronism substantially.

Clinical spectrum of primary adrenal lymphoma: results of a multicenter cohort study.

Purpose: We sought to refine the clinical picture of primary adrenal lymphoma (PAL), a rare lymphoid malignancy with predominant adrenal manifestation and risk of adrenal insufficiency. Methods: Ninety-seven patients from 14 centers in Europe, Canada and the United States were included in this retrospective analysis between 1994 and 2017. Results: Of the 81 patients with imaging data, 19 (23%) had isolated adrenal involvement (iPAL), while 62 (77%) had additional extra-adrenal involvement (PAL+). Among patients who had both CT and PET scans, 18FDG-PET revealed extra-adrenal involvement not detected by CT scan in 9/18 cases (50%). The most common clinical manifestations were B symptoms (55%), fatigue (45%), and abdominal pain (35%). Endocrinological assessment was often inadequate. With a median follow-up of 41.6 months, 3-year progression-free (PFS) and overall (OS) survival rates in the entire cohort were 35.5% and 39.4%, respectively. The hazard ratios of iPAL for PFS and OS were 40.1 (95% CI: 2.63-613.7, P = 0.008) and 2.69 (95% CI: 0.61-11.89, P = 0.191), respectively. PFS was much shorter in iPAL vs PAL+ (median 4 months vs not reached, P = 0.006), and OS also appeared to be shorter (median 16 months vs not reached), but the difference did not reach statistical significance (P = 0.16). Isolated PAL was more frequent in females (OR = 3.81; P = 0.01) and less frequently associated with B symptoms (OR = 0.159; P = 0.004). Conclusion: We found unexpected heterogeneity in the clinical spectrum of PAL. Further studies are needed to clarify whether clinical distinction between iPAL and PAL+ is corroborated by differences in molecular biology.

Subcellular localization of fibroblast growth factor receptor type 2 and correlation with CTNNB1 genotype in adrenocortical carcinoma.

OBJECTIVE: Fibroblast growth factor receptor (FGFR) 2 regulates the development of the adrenal gland in mice. In addition, FGFR2-mediated signalling has been shown to prevent apoptosis and to enhance proliferation in adrenocortical precursor cells. The activation of the Wingless/Int-1 (WNT)/beta catenin pathway as a key mechanism of adrenocortical tumourigenesis has been linked to FGFR2 signalling in other cell types. Therefore we hypothesised that FGFR2 expression may also play a role in adrenocortical carcinoma (ACC). We conducted a pilot study and analysed protein expression of FGFR2 in 26 ACCs using immunohistochemistry technique. Data on the CTNNB1 mutation status and clinical data were correlated to the expression of FGFR2. RESULTS: We observed a high variability in FGFR2 expression between the different tumour samples. There was a subset of ACC with comparatively high nuclear expression of FGFR2. We did not find a clear association between the CTNNB1 mutational status or clinical features and the FGFR2 expression. We conclude that FGFR signalling plays a role in adrenocortical carcinoma. Our data encourages further investigations of FGFR signalling in ACC, especially since new inhibitors of FGFR signalling are already entering clinical trials for the treatment of other cancer types.

The Pain Paradox of Borderline Personality and Total Knee Arthroplasty (TKA): Recruiting Borderline Personality Organization to Predict the One-Year Postoperative Outcome.

BACKGROUND: TKA is a common treatment for arthropathies of the knee; however, its results are compromised by psychosocial equivalents of pain: prior research suggests persistent pain and dysfunction after TKA not only to be linked to psychological symptoms such as depression or anxiety but also to psychodynamic determinants of borderline personality, namely borderline personality organization. Osteoarthritis (OA) and Rheumatoid arthritis (RA), the main indications for TKA, are themselves linked to personality factors and disorders, e.g. borderline. The present study investigates the influence of borderline personality organization (BPO) on the outcomes of TKA one year postoperatively. METHODS: We studied 144 patients scheduled for primary TKA before and after the operation using the IPO-16 and the WOMAC for the assessment of knee pain and function. RESULTS: Non-parametric correlations were found between primitive defenses and knee-pain, not function. Linear regression showed prediction of knee pain and knee function by the preoperative WOMAC scores (p<0.01), whereas there was additional prediction of knee-pain by gender (p=0.03) and primitive defenses (p=0.04). DISCUSSION: The results suggest a psychodynamic mechanism of maladaptation after TKA apparently representing the bodily manifestations of fundamental psychic defenses.

Increased Numbers of Circulating Tumor Cells in Thyroid Cancer Patients.

Circulating tumor cells (CTCs) have been shown to be a valuable prognostic marker for different solid cancers. Within the present study we quantified CTCs in thyroid cancer (TC) patients. Special focus was given to disease-free PTC patients with undetectable serum thyroglobulin (Tg) levels. Altogether, 67 TC patients (33 papillary, 20 follicular, 14 medullary) were included in the study. CTC numbers, which were normalized to 3.3×105 peripheral blood mononuclear cells, were correlated with clinical outcome. TC patients had significantly higher CTC numbers compared to controls. The number of CTCs correlated to the initial tumor stage. Importantly, in comparison to controls, differentiated TC patients with serum Tg levels<0.3 ng/ml (no evidence of tumor recurrence) revealed a significantly higher amount of CTCs, also associated to their former tumor stage. Regarding the tumor-free papillary TC (PTC) patients the number of CTCs additionally correlated to the time point of radioiodine (RI) therapy: PTC patients with RI therapies>8 years before CTC measurement had significantly higher CTC numbers compared to those with RI therapy<8 years ago. We found a clear correlation between the number of CTCs and the tumor stage. Importantly, PTC patients who are in remission may still have increased numbers of CTCs. Follow-up analyses in these patients will reveal whether these data will have a clinical impact.

Measurement of Basal Serum Calcitonin for the Diagnosis of Medullary Thyroid Cancer.

Calcitonin (CT), a tumor marker for medullary thyroid cancer (MTC), can be stimulated with pentagastrin or calcium. Because of the unavailability of pentagastrin, basal CT measurement is frequently used for the preoperative diagnosis of MTC. The aim of the study was to define basal serum calcitonin (bCT) cut-off thresholds for diagnosing MTC. Within a retrospective analysis, 114 patients (51 males) were included fulfilling the criteria of an increased preoperative bCT level (>10 pg/ml) and the criteria of an available postoperative histology analysis. Based on a ROC plot analysis, the cut-off values for the diagnosis of MTC vs. non-malignancy (C cell hyperplasia and goiter) were identified. The most precise bCT thresholds for the identification of MTC were ≥46 pg/ml for males (sensitivity: 93.6%, specificity: 95.0%, PPV: 97%, NPV: 90%) and ≥35 pg/ml for females (sensitivity: 87.3%, specificity: 87.5%, PPV: 98%, NPV: 50%). Using these cut-offs, only 6% of male patients were not identified of having MTC, whereas 5% were false positive (having instead C cell hyperplasia). In females, the discrepancy was higher since 13% of female MTC patients were false negative by using the cut-off of ≥35 pg/ml, and 13% had false positive results (suffering from C cell hyperplasia). Gender-specific bCT cut-offs for the identification of MTC vs. C cell hyperplasia and non-malignancy were defined, which can be used in clinical routine. In female patients, however, the accuracy is much lower compared to males.

Excessive Catecholamine Secretion and the Activation of the Renin-Angiotensin-Aldosterone-System in Patients with Pheochromocytoma: A Single Center Experience and Overview of the Literature.

Catecholamines stimulate renin-secretion in the juxtaglomerular cells of the kidney and a number of case reports suggest an association between pheochromocytoma and activation of the RAAS. Therefore, it could be asked whether patients suffering from pheochromocytoma with high concentrations of circulating catecholamines present with oversecretion of renin and aldosterone. We identified twelve patients with excessive catecholamine secretion due to pheochromocytoma and compared them to a group of twelve patients with essential hypertension (EH) with regard to the activation of the renin-angiotensin-aldosterone-system (RAAS). The PubMed database was screened for studies that investigate the association between pheochromocytoma and activation of the RAAS. The plasma concentrations of metanephrines (19.9-fold) and normetanephrines (29.5-fold) were significantly higher in the pheochromocytoma group than in the EH group. Renin and aldosterone levels were 1.3-fold and 1.6-fold higher, respectively, as compared to the EH group, whereas the differences were not statistically significant. There was no significant correlation between plasma metanephrine or normetanephrine levels and the plasma renin concentration (rs=0.077, rs=0.049, respectively) in our patients. The data from our institution and from review of literature suggest that an association between pheochromocytoma in the context of high plasma catecholamine levels and activation of the RAAS is present. However, results have not been consistent. Thus, other causes of RAAS-activation should be considered also in the presence of pheochromocytoma or reinvestigation for aldosteronism should be offered to such patients after removal of the catecholamine-producing tumour.

Epitope-Specific Antitumor Immunity Suppresses Tumor Spread in Papillary Thyroid Cancer.

Context: Papillary thyroid cancer (PTC) is characterized by a lymphocytic infiltration. PTC patients with lymphocytic infiltration may have a better clinical outcome. Objective: Characterization of tumor epitope-specific immunity and correlation analyses with the clinical outcome. Patients: 150 PTC patients; 40 Hashimoto thyroiditis (HT) patients; 21 healthy controls; 27,239 healthy whites (for HLA typing). Main Outcome Measures: HLA class I restricted thyroperoxidase (TPO) and thyroglobulin (Tg) epitope-specific T cells (tetramer analyses), correlation analyses between HLA class II phenotypes, T cell immunity, and the clinical course. Results: The frequency of TPO- and Tg-specific CD8+ T cells in PTC patients was largely increased compared with healthy controls (TPO and Tg, P < 0.005 and P < 0.005) and was similar to those in HT patients. HLA-DQB1*03-positive PTC patients had a significantly lower risk [risk ratio (RR), 0.170; 95% confidence interval (CI), 0.037 to 0.755; P < 0.05] and HLA-DRB1*03-positive and HLA-DQB1*02-positive PTC patients a significantly higher risk (HLA-DRB1*03: RR, 4.400; 95% CI, 1.378 to 14.05; P < 0.05; HLA-DQB1*02: RR, 3.692; 95% CI, 1.102 to 12.38; P < 0.05) for distant metastases, compared with patients with other haplotypes. HLA-DQB1*03-positive PTC patients revealed an increased responsiveness of tumor epitopes in vitro. These tumor epitope-specific CD8+ T cells were also found in lymph node metastases of HLA-DQB1*03-positive PTC patients. Conclusion: We demonstrate a tumor epitope-specific immunity in PTC patients and the protective role of HLA-DQB1*03 against metastatic spread. These results have direct implications for new treatment options with immune checkpoint inhibitors.

Endothelial cells regulate ß-catenin activity in adrenocortical cells via secretion of basic fibroblast growth factor.

Endothelial cell-derived products influence the synthesis of aldosterone and cortisol in human adrenocortical cells by modulating proteins such as steroidogenic acute-regulatory (StAR) protein, steroidogenic factor (SF)-1 and CITED2. However, the potential endothelial cell-derived factors that mediate this effect are still unknown. The current study was perfomed to look into the control of ß-catenin activity by endothelial cell-derived factors and to identify a mechanism by which they affect ß-catenin activity in adrenocortical NCIH295R cells. Using reporter gene assays and Western blotting, we found that endothelial cell-conditioned medium (ECCM) led to nuclear translocation of ß-catenin and an increase in ß-catenin-dependent transcription that could be blocked by U0126, an inhibitor of the mitogen-activated protein kinase pathway. Furthermore, we found that a receptor tyrosin kinase (RTK) was involved in ECCM-induced ß-catenin-dependent transcription. Through selective inhibition of RTK using Su5402, it was shown that receptors responding to basic fibroblast growth factor (bFGF) mediate the action of ECCM. Adrenocortical cells treated with bFGF showed a significant greater level of bFGF mRNA. In addition, HUVECs secrete bFGF in a density-dependent manner. In conclusion, the data suggest that endothelial cells regulate ß-catenin activity in adrenocortical cells also via secretion of basic fibroblast growth factor.

Mass Spectrometry-Based Adrenal and Peripheral Venous Steroid Profiling for Subtyping Primary Aldosteronism.

BACKGROUND: Differentiating patients with primary aldosteronism caused by aldosterone-producing adenomas (APAs) from those with bilateral adrenal hyperplasia (BAH), which is essential for choice of therapeutic intervention, relies on adrenal venous sampling (AVS)-based measurements of aldosterone and cortisol. We assessed the utility of LC-MS/MS-based steroid profiling to stratify patients with primary aldosteronism. METHODS: Fifteen adrenal steroids were measured by LC-MS/MS in peripheral and adrenal venous plasma from AVS studies for 216 patients with primary aldosteronism at 3 tertiary referral centers. Ninety patients were diagnosed with BAH and 126 with APAs on the basis of immunoassay-derived adrenal venous aldosterone lateralization ratios. RESULTS: Among 119 patients confirmed to have APAs at follow-up, LC-MS/MS-derived lateralization ratios of aldosterone normalized to cortisol, dehydroepiandrosterone, and androstenedione were all higher (P < 0.0001) than immunoassay-derived ratios. The hybrid steroids, 18-oxocortisol and 18-hydroxycortisol, also showed lateralized secretion in 76% and 35% of patients with APAs. Adrenal venous concentrations of glucocorticoids and androgens were bilaterally higher in patients with BAH than in those with APAs. Consequently, peripheral plasma concentrations of 18-oxocortisol were 8.5-fold higher, whereas concentrations of cortisol, corticosterone, and dehydroepiandrosterone were lower in patients with APAs than in those with BAH. Correct classification of 80% of cases of APAs vs BAH was thereby possible by use of a combination of steroids in peripheral plasma. CONCLUSIONS: LC-MS/MS-based steroid profiling during AVS achieves higher aldosterone lateralization ratios in patients with APAs than immunoassay. LC-MS/MS also enables multiple measures for discriminating unilateral from bilateral aldosterone excess, with potential use of peripheral plasma for subtype classification.

PRKACA mutations in cortisol-producing adenomas and adrenal hyperplasia: a single-center study of 60 cases.

OBJECTIVE: Cortisol excess due to adrenal adenomas or hyperplasia causes Cushing's syndrome. Recent genetic studies have identified a somatic PRKACA(L206R) mutation as a cause of cortisol-producing adenomas. We aimed to compare the clinical features of PRKACA-mutant lesions with those of CTNNB1 mutations, and to search for similar mutations in unilateral hyperplasia or tumors co-secreting aldosterone. DESIGN, PATIENTS, AND METHODS: In this study, 60 patients with cortisol excess who had adrenalectomies at our institution between 1992 and 2013 were assessed, and somatic mutations were determined by Sanger sequencing. A total of 36 patients had overt Cushing's syndrome, the remainder were subclinical: 59 cases were adenomas (three bilateral) and one was classified as hyperplasia. Four tumors had proven co-secretion of aldosterone. RESULTS: Among cortisol-secreting unilateral lesions without evidence of co-secretion (n=52), we identified somatic mutations in PRKACA (L206R) in 23.1%, CTNNB1 (S45P, S45F) in 23.1%, GNAS (R201C) in 5.8%, and CTNNB1+GNAS (S45P, R201H) in 1.9%. PRKACA and GNAS mutations were mutually exclusive. Of the co-secreting tumors, two (50%) had mutations in KCNJ5 (G151R and L168R). The hyperplastic gland showed a PRKACA(L206R) mutation, while patients with bilateral adenomas did not have known somatic mutations. PRKACA-mutant lesions were associated with younger age, overt Cushing's syndrome, and higher cortisol levels vs non-PRKACA-mutant or CTNNB1-mutant lesions. CTNNB1 mutations were more significantly associated with right than left lesions. CONCLUSIONS: PRKACA(L206R) is present not only in adenomas, but also in unilateral hyperplasia and is associated with more severe autonomous cortisol secretion. Bilateral adenomas may be caused by yet-unknown germline mutations.

An LC-MS/MS method for steroid profiling during adrenal venous sampling for investigation of primary aldosteronism.

BACKGROUND: Steroid profiling for diagnosis of endocrine disorders featuring disordered production of steroid hormones is now possible from advances in liquid chromatography with tandem mass spectrometry (LC-MS/MS). Adrenal venous (AV) measurements of aldosterone and cortisol are a standard practice in the clinical work-up of primary aldosteronism, but do not yet take advantage of steroid profiling. METHODS: A novel LC-MS/MS based method was developed for simultaneous measurement of 15 adrenal steroids: aldosterone, corticosterone, 11-deoxycorticosterone, progesterone, pregnenolone, cortisone, cortisol, 11-deoxycortisol, 17-hydroxyprogesterone, androstenedione, dehydroepiandrosterone, dehydroepiandrosterone-sulfate, 21-deoxycortisol, 18-oxocortisol and 18-hydroxycortisol. These were compared in peripheral venous (pV) and AV plasma from 70 patients undergoing AV sampling with and without cosyntropin stimulation. Aldosterone and cortisol levels measured by LC-MS/MS were compared with those measured by immunoassay. RESULTS: Reproducibility of measurements with coefficients of variation ≤10% as well as analytical sensitivity sufficient to measure low pV levels particularly of aldosterone demonstrate the utility of the assay for profiling adrenal steroids in primary aldosteronism. Method comparisons indicated assay and concentration dependent differences of cortisol and aldosterone concentrations measured by immunoassay and LC-MS/MS. Median AV/pV ratios of 11-deoxycortisol (53.0), 17-hydroxyprogesterone (33.4), pregnenolone (62.4), androstenedione (40.6) and dehydroepiandrosterone (33.3) were 2.9- to, 5.4-fold larger than those for cortisol (11.6), with additionally generally larger increases than for cortisol with than without cosyntropin stimulation. CONCLUSION: Our LC-MS/MS assay, in addition to improvements over existing immunoassay measurements of aldosterone and cortisol, offers profiling of 13 other adrenal steroids, providing a potentially useful method for the clinical work-up of patients with primary aldosteronism. In particular, the larger AV/pV ratios of several steroids compared to cortisol suggest more sensitive alternatives to the latter for assessing positioning of AV sampling catheters.

Whole-exome sequencing characterizes the landscape of somatic mutations and copy number alterations in adrenocortical carcinoma.

CONTEXT: Adrenocortical carcinoma (ACC) is a rare and lethal malignancy with a poorly defined etiology, and the molecular genetics of ACC are incompletely understood. OBJECTIVE: To utilize whole-exome sequencing for genetic characterization of the underlying somatic mutations and copy number alterations present in ACC. DESIGN: Screening for somatic mutation events and copy number alterations (CNAs) was performed by comparative analysis of tumors and matched normal samples from 41 patients with ACC. RESULTS: In total, 966 nonsynonymous somatic mutations were detected, including 40 tumors with a mean of 16 mutations per sample and one tumor with 314 mutations. Somatic mutations in ACC-associated genes included TP53 (8/41 tumors, 19.5%) and CTNNB1 (4/41, 9.8%). Genes with potential disease-causing mutations included GNAS, NF2, and RB1, and recurrently mutated genes with unknown roles in tumorigenesis comprised CDC27, SCN7A, and SDK1. Recurrent CNAs included amplification at 5p15.33 including TERT (6/41, 14.6%) and homozygous deletion at 22q12.1 including the Wnt repressors ZNRF3 and KREMEN1 (4/41 9.8% and 3/41, 7.3%, respectively). Somatic mutations in ACC-established genes and recurrent ZNRF3 and TERT loci CNAs were mutually exclusive in the majority of cases. Moreover, gene ontology identified Wnt signaling as the most frequently mutated pathway in ACCs. CONCLUSIONS: These findings highlight the importance of Wnt pathway dysregulation in ACC and corroborate the finding of homozygous deletion of Wnt repressors ZNRF3 and KREMEN1. Overall, mutations in either TP53 or CTNNB1 as well as focal CNAs at the ZNRF3 or TERT loci denote mutually exclusive events, suggesting separate mechanisms underlying the development of these tumors.

Outcome of adrenal vein sampling performed during concurrent mineralocorticoid receptor antagonist therapy.

CONTEXT: Pharmacological inhibition of mineralocorticoid receptor (MR) signaling in patients with primary aldosteronism (PA) reestablishes aldosterone synthesis by nondiseased zona glomerulosa cells through activation of the renin-angiotensin-aldosterone system. In this context, current guidelines recommend discontinuing MR blockade for diagnostic procedures, including adrenal vein sampling (AVS). Discontinuation of MR blockade in high-risk patients may be harmful because of uncontrolled hypertension and severe hypokalemia. We hypothesize that MR antagonist therapy can be continued during AVS as long as renin levels remain suppressed. OBJECTIVE: The objective of this study was to assess the validity of AVS results in the context of MR antagonistic therapy. DESIGN: We retrospectively analyzed all AVS studies in Munich (since 2008) and Düsseldorf (since 2011) and identified four of 237 (1.7%) patients with PA who underwent AVS while treated with an MR antagonist. Adrenalectomy was recommended based on the results of AVS in all four patients. After adrenalectomy, follow-up data were obtained to confirm improvement or remission of PA. Main outcome measures included blood pressure values, daily defined doses of antihypertensive medication, as well as levels of aldosterone, renin, and potassium, and the aldosterone/renin ratio. RESULTS: In all patients, renin remained low or suppressed during AVS despite MR antagonist treatment. AVS clearly demonstrated unilateral aldosterone excess in each case. After adrenalectomy, all patients showed remission of PA as demonstrated by blood pressure values, potassium levels, and the aldosterone/renin ratio. CONCLUSION: In selected cases of PA, MR antagonist therapy might be continued during AVS, provided that renin values remain low.

Recurrent activating mutation in PRKACA in cortisol-producing adrenal tumors.

Adrenal tumors autonomously producing cortisol cause Cushing's 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 (ß-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 Cushing's 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.

Enhanced iodine supplementation alters the immune process in a transgenic mouse model for autoimmune thyroiditis.

BACKGROUND: The impact of excessive iodine intake on the development of autoimmune thyroiditis (AIT) is still under debate. Transgenic, antibody-devoid TAZ10 mice spontaneously develop AIT due to autoreactive thyroperoxidase-specific T cells. In this model, development of AIT is determined by a T cell infiltration of the thyroid gland leading to an elevation of serum thyrotropin (TSH) levels and significant weight gain. In the present study we investigated the impact of moderate and high iodine supplementation on the course of disease in these mice, which are immunologically prone to AIT. METHODS: In addition to normal nutrition, mice were supplemented for 20 weeks with 2.5 µg versus 5 µg iodine per milliliter drinking water, which corresponds to a human daily iodine supplementation of 150 µg, 315 µg, and 615 µg iodine. AIT-defining parameters (weight gain, elevation of serum TSH levels, cellular infiltration of the thyroid) and immunologic effects were analyzed. RESULTS: No significant differences were displayed when comparing weight and serum TSH levels in the iodine-supplemented versus control groups. Increased thyroid infiltrates with CD8⁺ T cells were detected by fluorescein-activated cell sorter (FACS) and immunofluorescence staining in mice supplemented with elevated iodine amounts (315 µg and 615 µg iodine per day, respectively). Immunologic monitoring revealed selective changes in immune cell frequencies (CD8⁺ and regulatory T cells, natural killer [NK] cells) and cytokine production (interferon-γ, interleukin-1α, and interleukin-17), however, without affecting the overall immune balance. CONCLUSION: Our results demonstrate that elevated iodine supplementation has no physical impact on the course of disease in transgenic, antibody-devoid TAZ10 mice, which are immunologically prone to AIT.