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.

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.

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.

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.

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.

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.

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.

Hedgehog-signaling is upregulated in non-producing human adrenal adenomas and antagonism of hedgehog-signaling inhibits proliferation of NCI-H295R cells and an immortalized primary human adrenal cell line.

Hedgehog (Hh)-signaling pathway is important in embryonic development. Activation of Hh-signaling is associated with tumorigenesis. Recent studies demonstrate that Hh-signaling is involved in the development of the adrenal gland in mice and is important in regulating adrenal proliferation. We studied the expression of Sonic hedgehog (SHH), Smoothened (SMO), Patched1 (PTCH1) and GLI family zinc finger 1 (GLI1) in human adrenal and in adrenocortical tumors using immunohistochemistry and semi-quantitative reverse transcriptase-polymerase chain reaction. Modulation of GLI1 and SMO messenger ribonucleic acid (mRNA) expression was investigated with forskolin. The role of Hh-signaling was studied in NCI-H295R cells and in an immortalized primary cell line using the Hh-agonist smoothened agonist (SAG) and the Hh-antagonist cyclopamine. The Hh-pathway components SHH, GLI1, PTCH1 and SMO were detectable in all adrenal glands. While in cortisol-producing adenomas (CPA), Hh-signaling expression levels were comparable to that in normal adrenal cortex, a much higher mRNA expression of GLI1, SMO and SHH was observed in non-producing adenomas (NPA). Interestingly, stimulation of cultured adrenal cells with forskolin led to a decrease in expression of GLI1 and SMO mRNAs. Antagonism of Hh-signaling resulted in a lower proliferation rate of adrenocortical cells, while Hh-agonism had no significant effect on adrenal cell proliferation. Our data show Hh-signaling activity in adult adrenal glands. Activation of the PKA pathway results in lower expression of Hh-signaling proteins. This might explain the lower expression of the Hh components GLI1 and SMO in CPA in comparison to the higher expression in NPA. Hh-signaling might be involved in the tumorigenesis of NPA.

Cushing's disease in a patient with steroid 21-hydroxylase deficiency.

Cushing's disease rarely appears as a consequence of hereditary disease. However, familial diseases with diminished glucocorticoid feedback are associated with secondary hypercorticotropinism and have been shown to give rise to pituitary adenomas. We here describe the rare case of a 30-year old female patient with congenital adrenal hyperplasia who also showed clinical signs and a typical history of hypercortisolism that was specified as Cushing's disease. After removal of a pituitary microadenoma, serum-cortisol levels fell below normal and the symptoms improved. However, after four years the menstrual cycle was irregular again and ACTH levels were in the upper range of normal. A corticotropin challenge showed a minor cortisol response but a marked increase in 17-hydroxyprogesterone serum concentrations. Genetic analysis revealed a homozygous mutation in exon 7 of the CYP21A2 gene (CTG>TTG, p.V281L). We conclude that a marked ACTH drive was able to override insufficient 21-hydroxylation and even to cause hypercortisolism. Although we describe a rare case, the impairment of the glucocorticoid feedback system in the context of congenital adrenal hyperplasia and other diseases may contribute to the development of secondary hypercorticotropinism as well as corticotropin producing adenomas.

Evidence for the involvement of endothelial cell products in adrenal CITED2 expression.

The adrenal gland contains a well-organized network of blood vessels, and adrenocortical cells are situated in close proximity to endothelial cells. Recently, several new mechanisms have been characterized that control the release of aldosterone by adrenocortical cells, including the involvement of endothelial-cell-derived factors. Interestingly, a CBP/p300-interacting transactivator with ED-rich tail 2 (CITED2), which is necessary for adrenal development, has been linked to aldosterone synthesis. We have therefore examined the effects of endothelial-cell-conditioned medium (ECCM), as produced during the incubation of human umbilical vein endothelial cells for 24 h, on the promoter activity and mRNA and protein expression of CITED2 in adrenocortical cells as represented by the NCI-H295R cell line. We have found a dose-dependent effect of ECCM on CITED2 promoter activity; this peaks at 480%. Activation of the CITED2 promoter occurs in parallel to an increase in CITED2 messenger RNA (as quantified by real-time polymerase chain reaction) and protein. The stimulatory effect of ECCM can be reversed by blocking mitogen-activated protein kinase activity with the MEK1-inhibitor PD98059. We conclude that products secreted by endothelial cells control not only steroidogenesis, but also factors that are important for adrenocortical development, thereby highlighting the role of cellular interactions within adrenocortical development and physiology.

CITED2 is expressed in human adrenocortical cells and regulated by basic fibroblast growth factor.

CITED2 gene deletion in mice leads to adrenal agenesis. Therefore, we analyzed CITED2, a CBP/p300 interacting transactivator with transforming activity, in the human adrenal gland. In this study, we examined CITED2 expression in human embryonic and adult adrenal glands as well as adrenocortical carcinomas. As ACTH and basic fibroblast growth factor (bFGF) are connected to the physiology and growth of adrenocortical cells we studied the regulation of CITED2 by these factors in the NCI-H295R adrenocortical carcinoma cell line. We found CITED2 expression in the adult adrenal cortex as well in adrenocortical carcinomas. At an early stage of human adrenal organogenesis CITED2 could be located to the definitive zone of the developing adrenal gland using immunohistochemistry. In NCI-H295R cells, stimulation by bFGF led to a dose-dependent increase in CITED2 promotor activity, mRNA and protein expression while ACTH had no significant effect. The stimulatory effect of bFGF could be reduced by blocking mitogen-activated protein kinase activity using the MAPkinase kinase (MEK1)-inhibitor PD98059. CITED2 is expressed in embryonic and adult human adrenal glands as well as in adrenocortical cancer. It is connected to the signaling cascades of bFGF and its expression is modulated by mitogen-activated protein kinases. This suggests a novel role for CITED2 in human adrenal growth and possibly in adrenal tumorigenesis.