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Free access

Matthias Kroiss, Dietmar Plonné, Sabine Kendl, Diana Schirmer, Cristina L Ronchi, Andreas Schirbel, Martina Zink, Constantin Lapa, Hartwig Klinker, Martin Fassnacht, Werner Heinz, and Silviu Sbiera

Objective

Oral mitotane (o,p′-DDD) is a cornerstone of medical treatment for adrenocortical carcinoma (ACC).

Aim

Serum mitotane concentrations >14 mg/l are targeted for improved efficacy but not achieved in about half of patients. Here we aimed at a better understanding of intestinal absorption and lipoprotein association of mitotane and metabolites o,p′-dichlorodiphenylacetic acid (o,p′-DDA) and o,p′-dichlorodiphenyldichloroethane (o,p′-DDE).

Design

Lipoproteins were isolated by ultracentrifugation from the chyle of a 29-year-old patient and serum from additional 14 ACC patients treated with mitotane. HPLC was applied for quantification of mitotane and metabolites. We assessed NCI–H295 cell viability, cortisol production, and expression of endoplasmic reticulum (ER) stress marker genes to study the functional consequences of mitotane binding to lipoproteins.

Results

Chyle of the index patient contained 197 mg/ml mitotane, 53 mg/ml o,p′-DDA, and 51 mg/l o,p′-DDE. Of the total mitotane in serum, lipoprotein fractions contained 21.7±21.4% (VLDL), 1.9±0.8% (IDL), 8.9±5.5% (LDL1), 18.9±9.6% (LDL2), 10.1±4.0% (LDL3), and 26.3±13.0% (HDL2). Only 12.3±5.5% were in the lipoprotein-depleted fraction.

Discussion

Mitotane content of lipoproteins directly correlated with their triglyceride and cholesterol content. O,p′-DDE was similarly distributed, but 87.9±4.2% of o,p′-DDA found in the HDL2 and lipoprotein-depleted fractions. Binding of mitotane to human lipoproteins blunted its anti-proliferative and anti-hormonal effects on NCI–H295 cells and reduced ER stress marker gene expression.

Conclusion

Mitotane absorption involves chylomicron binding. High concentrations of o,p′-DDA and o,p′-DDE in chyle suggest intestinal mitotane metabolism. In serum, the majority of mitotane is bound to lipoproteins. In vitro, lipoprotein binding inhibits activity of mitotane suggesting that lipoprotein-free mitotane is the therapeutically active fraction.

Free access

Dirk Weismann, Mirko Peitzsch, Anna Raida, Aleksander Prejbisz, Maria Gosk, Anna Riester, Holger S Willenberg, Reiner Klemm, Georg Manz, Timo Deutschbein, Matthias Kroiss, Roland Därr, Martin Bidlingmaier, Andrzej Januszewicz, Graeme Eisenhofer, and Martin Fassnacht

Background

Reports conflict concerning measurements of plasma metanephrines (MNs) for diagnosis of pheochromocytomas/paragangliomas (PPGLs) by immunoassays compared with other methods. We aimed to compare the performance of a commercially available enzyme-linked immunoassay (EIA) kit with liquid chromatography–tandem mass spectrometric (LC–MS/MS) measurements of MNs to diagnose PPGLs.

Methods

In a substudy of a prospective, multicenter trial to study the biochemical profiles of monoamine-producing tumors, we included 341 patients (174 males and 167 females) with suspected PPGLs (median age 54 years), of whom 54 had confirmed PPGLs. Plasma MNs were measured by EIA and LC–MS/MS, each in a specialized laboratory.

Results

Plasma normetanephrine (NMN) and MN were measured 60 and 39% lower by EIA than by LC–MS/MS. Using upper cut-offs stipulated for the EIA, diagnostic sensitivity was only 74.1% at a specificity of 99.3%. In contrast, use of similar cut-offs for MN and overall lower age-adjusted cut-offs for NMN measured by LC–MS/MS returned a diagnostic sensitivity and specificity of 98.1 and 99.7%. Areas under receiver-operating characteristic curves, nevertheless, indicated comparable diagnostic performance of the EIA (0.993) and LC–MS/MS (0.985). Diagnostic sensitivity for the EIA increased to 96.2% with a minimal loss in specificity (95.1%) following use of cut-offs for the EIA adapted to correct for the negative bias.

Conclusions

The EIA underestimates plasma MNs and diagnostic sensitivity is poor using commonly stipulated cut-offs, resulting in a high risk for missing patients with PPGLs. Correction of this shortcoming can be achieved by appropriately determined cut-offs resulting in comparable diagnostic performance of EIA and LC–MS/MS assays.

Free access

Cristina L Ronchi, Erika Peverelli, Sabine Herterich, Isabel Weigand, Giovanna Mantovani, Thomas Schwarzmayr, Silviu Sbiera, Bruno Allolio, Jürgen Honegger, Silke Appenzeller, Andrea G Lania, Martin Reincke, Davide Calebiro, Anna Spada, Michael Buchfelder, Joerg Flitsch, Tim M Strom, and Martin Fassnacht

Context

Alterations in the cAMP signaling pathway are common in hormonally active endocrine tumors. Somatic mutations at GNAS are causative in 30–40% of GH-secreting adenomas. Recently, mutations affecting the USP8 and PRKACA gene have been reported in ACTH-secreting pituitary adenomas and cortisol-secreting adrenocortical adenomas respectively. However, the pathogenesis of many GH-secreting adenomas remains unclear.

Aim

Comprehensive genetic characterization of sporadic GH-secreting adenomas and identification of new driver mutations.

Design

Screening for somatic mutations was performed in 67 GH-secreting adenomas by targeted sequencing for GNAS, PRKACA, and USP8 mutations (n=31) and next-generation exome sequencing (n=36).

Results

By targeted sequencing, known activating mutations in GNAS were detected in five cases (16.1%), while no somatic mutations were observed in both PRKACA and USP8. Whole-exome sequencing identified 132 protein-altering somatic mutations in 31/36 tumors with a median of three mutations per sample (range: 1–13). The only recurrent mutations have been observed in GNAS (31.4% of cases). However, seven genes involved in cAMP signaling pathway were affected in 14 of 36 samples and eight samples harbored variants in genes involved in the calcium signaling or metabolism. At the enrichment analysis, several altered genes resulted to be associated with developmental processes. No significant correlation between genetic alterations and the clinical data was observed.

Conclusion

This study provides a comprehensive analysis of somatic mutations in a large series of GH-secreting adenomas. No novel recurrent genetic alterations have been observed, but the data suggest that beside cAMP pathway, calcium signaling might be involved in the pathogenesis of these tumors.

Open access

Carmina Teresa Fuss, Katharina Brohm, Max Kurlbaum, Anke Hannemann, Sabine Kendl, Martin Fassnacht, Timo Deutschbein, Stefanie Hahner, and Matthias Kroiss

Objective

Saline infusion testing (SIT) for confirmation of primary aldosteronism (PA) is based on impaired aldosterone suppression in PA compared to essential hypertension (EH). In the past, aldosterone was quantified using immunoassays (IA). Liquid chromatography tandem mass spectrometry (LC-MS/MS) is increasingly used in clinical routine. We aimed at a method-specific aldosterone threshold for the diagnosis of PA during SIT and explored the diagnostic utility of steroid panel analysis.

Design

Retrospective cohort study of 187 paired SIT samples (2009–2018). Diagnosis of PA (n = 103) and EH (n = 84) was established based on clinical routine workup without using LC-MS/MS values.

Setting

Tertiary care center.

Methods

LC-MS/MS using a commercial steroid panel. Receiver operator characteristics analysis was used to determine method-specific cut-offs using a positive predictive value (PPV) of 90% as criterion.

Results

Aldosterone measured by IA was on average 31 ng/L higher than with LC-MS/MS. The cut-offs for PA confirmation were 54 ng/L for IA (sensitivity: 95%, 95% CI: 89.0–98.4; specificity: 87%, 95% CI: 77.8–93.3; area under the curve (AUC): 0.955, 95% CI: 0.924–0.986; PPV: 90%, 95% CI: 83.7–93.9) and 69 ng/L for LC-MS/MS (79%, 95% CI: 69.5–86.1; 89%, 95% CI: 80.6–95.0; 0.902, 95% CI: 0.857–0.947; 90%, 95% CI: 82.8–94.4). Other steroids did not improve SIT.

Conclusions

Aldosterone quantification with LC-MS/MS and IA yields comparable SIT-cut-offs. Lower AUC for LC-MS/MS is likely due to the spectrum of disease in PA and previous decision making based on IA results. Until data of a prospective trial with clinical endpoints are available, the suggested cut-off can be used in clinical routine.

Restricted access

Laura-Sophie Landwehr, Jochen Schreiner, Silke Appenzeller, Stefan Kircher, Sabine Herterich, Silviu Sbiera, Martin Fassnacht, Matthias Kroiss, and Isabel Weigand

Background

The response of advanced adrenocortical carcinoma (ACC) to current chemotherapies is unsatisfactory and a limited rate of response to immunotherapy was observed in clinical trials. High tumour mutational burden (TMB) and the presence of a specific DNA signature are characteristic features of tumours with mutations in the gene MUTYH encoding the mutY DNA glycosylase. Both have been shown to potentially predict the response to immunotherapy. High TMB in an ACC cell line model has not been reported yet.

Design and methods

The JIL-2266 cell line was established from a primary ACC tumour, comprehensively characterised and oxidative damage, caused by a dysfunctional mutY DNA glycosylase, confirmed.

Results

Here, we characterise the novel patient-derived ACC cell line JIL-2266, which is deficient in mutY-dependent DNA repair. JIL-2266 cells have a consistent STR marker profile that confirmed congruousness with primary ACC tumour. Cells proliferate with a doubling time of 41 ± 13 h. Immunohistochemistry revealed positivity for steroidogenic factor-1. Mass spectrometry did not demonstrate significant steroid hormone synthesis. JIL-2266 have hemizygous mutations in the tumour suppressor gene TP53 (c.859G>T:p.E287X) and MUTYH (c.316C>T:p.R106W). Exome sequencing showed 683 single nucleotide variants and 4 insertions/deletions. We found increased oxidative DNA damage in the cell line and the corresponding primary tumour caused by impaired mutY DNA glycosylase function and accumulation of 8-oxoguanine.

Conclusion

This model will be valuable as a pre-clinical ACC cell model with high TMB and a tool to study oxidative DNA damage in the adrenal gland.

Open access

Wiebke Schloetelburg, Ines Ebert, Bernhard Petritsch, Andreas Max Weng, Ulrich Dischinger, Stefan Kircher, Andreas Konrad Buck, Thorsten Alexander Bley, Timo Deutschbein, and Martin Fassnacht

Objective

Reliable results of wash-out CT in the diagnostic workup of adrenal incidentalomas are scarce. Thus, we evaluated the diagnostic accuracy of delayed wash-out CT and determined thresholds to accurately differentiate adrenal masses.

Design

Retrospective, single-center cohort study including 216 patients with 252 adrenal lesions who underwent delayed wash-out CT. Definitive diagnoses based on histopathology (n = 92) or comprehensive follow-up.

Methods

Size, average attenuation values of the adrenal lesions in all CT scan phases, and absolute and relative percentage wash-out (APW/RPW) were determined by an expert radiologist blinded for clinical data. Adrenal lesions with unenhanced attenuation values >10 Hounsfield units (HU) built a subgroup (n = 142). Diagnostic accuracy was calculated.

Results

The study group consisted of 171 adenomas, 32 other benign tumors, 11 pheochromocytomas, 9 adrenocortical carcinomas, and 29 other malignant tumors. All (potentially) malignant and 46% of benign lesions showed unenhanced attenuation values >10 HU. In this most relevant subgroup, the established thresholds of 60% for APW and 40% for RPW misclassified 35.9 and 35.2% of the masses, respectively. When we applied optimized cutoffs (APW >83%; RPW >58%) and excluded pheochromocytomas, we missed only one malignant tumor by APW and none by RPW. However, only 11 and 15% of the benign tumors were correctly identified.

Conclusions

Wash-out CT with the established thresholds for APW and RPW is insufficient to reliably diagnose adrenal masses. Using the proposed cutoff of 58% for RPW, malignant tumors will be correctly identified, but the added value is limited, namely 15% of patients with benign tumors can be prevented from additional imaging or even unnecessary surgery.

Free access

Mariko Sue, Victoria Martucci, Florina Frey, Jacques W M Lenders, Henri J Timmers, Mariola Pęczkowska, Aleksander Prejbisz, Brede Swantje, Stefan R Bornstein, Wiebke Arlt, Martin Fassnacht, Felix Beuschlein, Mercedes Robledo, Karel Pacak, and Graeme Eisenhofer

Objective

Testing for succinate dehydrogenase subunit B (SDHB) mutations is recommended in all patients with metastatic phaeochromocytomas and paragangliomas (PPGLs), but may not be required when metastatic disease is accompanied by adrenaline production. This retrospective cohort study aimed to establish the prevalence of SDHB mutations among patients with metastatic PPGLs, characterised by production of adrenaline compared with those without production of adrenaline, and to establish genotype–phenotype features of metastatic PPGLs according to underlying gene mutations.

Design and methods

Presence of SDHB mutations or deletions was tested in 205 patients (114 males) aged 42±16 years (range 9–86 years) at diagnosis of metastatic PPGLs with and without adrenaline production.

Results

Twenty-three of the 205 patients (11%) with metastatic PPGLs had disease characterised by production of adrenaline, as defined by increased plasma concentrations of metanephrine larger than 5% of the combined increase in both normetanephrine and metanephrine. None of these 23 patients had SDHB mutations. Of the other 182 patients with no tumoural adrenaline production, 51% had SDHB mutations. Metastases in bone were 36–41% more prevalent among patients with SDHB mutations or extra-adrenal primary tumours than those without mutations or with adrenal primary tumours. Liver metastases were 81% more prevalent among patients with adrenal than extra-adrenal primary tumours.

Conclusion

SDHB mutation testing has no utility among patients with adrenaline-producing metastatic PPGLs, but is indicated in other patients with metastatic disease. Our study also reveals novel associations of metastatic spread with primary tumour location and presence of SDHB mutations.

Free access

Julie Refardt, Clara Odilia Sailer, Irina Chifu, Bettina Winzeler, Ingeborg Schnyder, Martin Fassnacht, Wiebke Fenske, Mirjam Christ-Crain, and the CODDI-Investigators

Background

Diagnosis and treatment of dysnatremia is challenging and further complicated by the pitfalls of different sodium measurement methods. Routinely used sodium measurements are the indirect (plasma/serum) and direct (whole blood) ion-selective electrode (ISE) method, showing discrepant results especially in the setting of acute illness. Few clinicians are aware of the differences between the methods in clinically stable patients or healthy volunteers.

Methods

Data of 140 patients and 91 healthy volunteers undergoing osmotic stimulation with hypertonic saline infusion were analyzed. Sodium levels were measured simultaneously by indirect and direct ISE method before and at different time points during osmotic stimulation up to a sodium threshold of ≥150 mmol/L. The primary outcome was the difference in sodium levels between the indirect and direct ISE method.

Results

878 sodium measurements were analyzed. Mean (s.d.) sodium levels ranged from 141 mmol/L (2.9) to 151 mmol/L (2.1) by the indirect ISE compared to 140 mmol/L (3) to 149 mmol/L (2.8) by the direct ISE method. The interclass correlation coefficient between the two methods was 0.844 (95% CI: 0.823–0.863). On average, measurements by the indirect ISE were 1.9 mmol/L (95% CI limits: −3.2 to 6.9) higher than those by the direct ISE method (P < 0.001). The tendency of the indirect ISE method resulting in higher levels increased with increasing sodium levels.

Conclusion

Intra-individual sodium levels differ significantly between the indirect and direct ISE method also in the absence of acute illness. It is therefore crucial to adhere to the same method in critical situations to avoid false decisions due to measurement differences.

Free access

Valeria Laufs, Barbara Altieri, Silviu Sbiera, Stefan Kircher, Sonja Steinhauer, Felix Beuschlein, Marcus Quinkler, Holger S Willenberg, Andreas Rosenwald, Martin Fassnacht, and Cristina L Ronchi

Objective

Platinum-based chemotherapy (PBC) is the most effective cytotoxic treatment for advanced adrenocortical carcinoma (ACC). Excision repair cross complementing group 1 (ERCC1) plays a critical role in the repair of platinum-induced DNA damage. Two studies investigating the role of ERCC1 immunostaining as a predictive marker for the response to PBC in ACC had reported conflicting results. Both studies used the ERCC1-antibody clone 8F1 that later turned out to be not specific. The aim of this study was to evaluate the predictive role of ERCC1 with a new specific antibody in a larger series of ACC.

Design and methods

146 ACC patients with available FFPE slides were investigated. All patients underwent PBC (median cycles = 6), including cisplatin (n = 131) or carboplatin (n = 15), in most cases combined with etoposide (n = 144), doxorubicin (n = 131) and mitotane (n = 131). Immunostaining was performed with the novel ERCC1-antibody clone 4F9. The relationship between ERCC1 expression and clinicopathological parameters, as well as best objective response to therapy and progression-free survival (PFS) during PBC was evaluated.

Results

High ERCC1 expression was observed in 66% of ACC samples. During PBC, 43 patients experienced objective response (29.5%), 49 stable disease (33.6%), 8 mixed response (5.5%) and 46 progressive disease (31.5%) without any relationship with the ERCC1 immunostaining. No significant correlation was also found between ERCC1 expression and progression-free survival (median 6.5 vs 6 months, P=0.33, HR = 1.23, 95% CI = 0.82–2.0).

Conclusion

ERCC1 expression is not directly associated with sensitivity to PBC in ACC. Thus, other predictive biomarkers are required to support treatment decisions in patients with ACC.

Free access

Sophie Schweitzer, Meik Kunz, Max Kurlbaum, Johannes Vey, Sabine Kendl, Timo Deutschbein, Stefanie Hahner, Martin Fassnacht, Thomas Dandekar, and Matthias Kroiss

Objective

Current workup for the pre-operative distinction between frequent adrenocortical adenomas (ACAs) and rare but aggressive adrenocortical carcinomas (ACCs) combines imaging and biochemical testing. We here investigated the potential of plasma steroid hormone profiling by liquid chromatography tandem mass spectrometry (LC-MS/MS) for the diagnosis of malignancy in adrenocortical tumors.

Design

Retrospective cohort study of prospectively collected EDTA-plasma samples in a single tertiary reference center.

Methods

Steroid hormone profiling by liquid chromatography tandem mass spectrometry (LC-MS/MS) in random plasma samples and logistic regression modeling.

Results

Fifteen steroid hormones were quantified in 66 ACAs (29 males; M) and 42 ACC (15 M) plasma samples. Significantly higher abundances in ACC vs ACA were observed for 11-deoxycorticosterone, progesterone, 17-hydroxyprogesterone, 11-deoxycortisol, DHEA, DHEAS and estradiol (all P < 0.05). Maximal areas under the curve (AUC) for discrimination between ACA and ACC for single analytes were only 0.76 (estradiol) and 0.77 (progesterone), respectively. Logistic regression modeling enabled the discovery of diagnostic signatures composed of six specific steroids for male and female patients with AUC of 0.95 and 0.94, respectively. Positive predictive values in males and females were 92 and 96%, negative predictive values 90 and 86%, respectively.

Conclusion

This study in a large adrenal tumor patient cohort demonstrates the value of plasma steroid hormone profiling for diagnosis of ACC. Application of LC-MS/MS analysis and of our model may facilitate diagnosis of malignancy in non-expert centers. We propose to continuously evaluate and improve diagnostic accuracy of LC-MS/MS profiling by applying machine-learning algorithms to prospectively obtained steroid hormone profiles.