Erythropoiesis in Cushing syndrome: sex-related and subtype-specific differences. Results from a monocentric study.

CONTEXT: Cushing syndrome (CS) is associated with different hematological abnormalities. Nevertheless, conflicting data about erythropoiesis in CS have been reported. Furthermore, it is unclear whether CS sex and subtype-specific alterations in red blood cells (RBC) parameters are present. OBJECTIVE: To investigate sex and subtype-specific changes in RBC in patients with CS at initial diagnosis and after remission. DESIGN: Retrospective, monocentric study including 210 patients with CS (women, n = 162) matched 1:1 for sex and age to patients with pituitary microadenomas or adrenal incidentalomas (both hormonally inactive). RBC parameters were evaluated at initial diagnosis and after remission. RESULTS: Women with CS had higher hematocrit (median 42.2 vs 39.7%), hemoglobin (14.1 vs 13.4 g/dl) and mean corpuscular volume (MCV) (91.2 vs 87.9 fl) compared to the controls (all p < 0.0001). Women with Cushing disease (CD) showed higher hematocrit, RBC and hemoglobin levels than those with ectopic Cushing (ECS) (all p < 0.005). Men with CS had lower hematocrit (42.9 vs 44.7%), RBC count (4.8 vs 5.1n*106/µl) and hemoglobin (14.2 vs 15.4 g/dl), but higher MCV (90.8 vs 87.5 fl) than controls (all p < 0.05). In men with CS, no subtype-specific differences were identified. Three months after remission hemoglobin decreased in both sexes. CONCLUSION: CS is characterized by sexual and subtype-specific differences in RBC parameters. Compared to controls, women with CS showed higher hematocrit/hemoglobin levels, whereas men had lower hematocrit/hemoglobin, which further decreased directly after remission. Therefore, anemia should be considered as complication of CS in men. In women, differences in RBC parameters may help to differentiate CD from ECS.

Corticotropin-releasing hormone test predicts the outcome of unilateral adrenalectomy in primary bilateral macronodular adrenal hyperplasia.

PURPOSE: Primary bilateral adrenal hyperplasia (PBMAH) is associated with hypercortisolism and a heterogeneous clinical expression in terms of cortisol secretion and related comorbidities. Historically, treatment of choice was bilateral adrenalectomy (B-Adx); however, recent data suggest that unilateral adrenalectomy (U-Adx) may be an effective alternative. For the latter, factors predicting the postsurgical outcome (e.g., biochemical control) have not been identified yet. METHODS: PBMAH patients undergoing U-Adx for overt Cushing's syndrome (CS) in two tertiary care centers were retrospectively analysed. Remission was defined as a normalization of urinary free cortisol (UFC) without the need for medical treatment. The potential of hCRH test as a predictor of U-Adx outcome was evaluated in a subgroup. RESULTS: 23 patients were evaluated (69% females, mean age 55 years). Remission rate after U-Adx was 74% at last follow up (median 115 months from UAdx). Before U-Adx, a positive ACTH response to hCRH (Δ%ACTH increase > 50% from baseline) was associated with higher remission rates. CONCLUSIONS: Three of four patients with PBMAH are surgically cured with U-Adx. Pre-operative hCRH testing can be useful to predict long-term remission rates.

Novel AIP mutation in exon 6 causing acromegaly in a German family.

The most frequent genetic alteration of familial isolated growth hormone producing pituitary neuroendocrine tumors is a germline mutation of the aryl hydrocarbon receptor-interacting protein (AIP) gene. Various AIP mutations are already known; however, an AIP mutation in exon 6 (c.811_812del; p.Arg271Glyfs*16) has not been reported yet. Here, we report a German family with two identical twins who were both affected by acromegaly and carried the above-mentioned novel AIP mutation. The father was found to be an unaffected carrier, while the paternal aunt most likely suffered from acromegaly as well and died from metastatic colorectal cancer. Apart from reporting a novel AIP mutation, this study does not only highlight the different clinical and histological features of the AIP mutated growth hormone producing pituitary neuroendocrine tumors but also confirms the poor responsiveness of dopamine agonists in AIP mutated acromegaly. Furthermore, it highlights the increased mortality risk of comorbidities typically associated with acromegaly.

Prognostic factors in stage III-IV adrenocortical carcinomas (ACC): an European Network for the Study of Adrenal Tumor (ENSAT) study.

BACKGROUND: The clinical course of advanced adrenocortical carcinoma (ACC) is heterogeneous. Our study aimed primarily to refine and make headway in the prognostic stratification of advanced ACC. PATIENTS AND METHODS: Patients with advanced ENSAT ACC (stage III or stage IV) at diagnosis registered between 2000 and 2009 in the ENSAT database were enrolled. The primary end point was overall survival (OS). Parameters of potential prognostic relevance were selected. Univariate and multivariate analyses were carried out: model 1 'before surgery'; model 2 'post-surgery'. RESULTS: Four hundred and forty-four patients with advanced ENSAT ACC (stage III: 210; stage IV: 234) were analyzed. After a median follow-up of 55.2 months, the median OS was 24 months. A modified ENSAT (mENSAT) classification was validated: stage III (invasion of surrounding tissues/organs or the vena renalis/cava) and stage IVa, IVb, IVc (2, 3 or >3 metastatic organs, including N, respectively). Two- or 5-year OS was 73%, 46%, 26% and 15% or 50%, 15%, 14% and 2% for stages III, IVa, IVb and IVc, respectively. In the multivariate analysis, mENSAT stages (stages IVa, IVb, or IVc, respectively) were significantly correlated with OS (P < 0.0001), as well as additional parameters: age ≥ 50 years (P < 0.0001), tumor- or hormone-related symptoms (P = 0.01 and 0.03, respectively) in model 1 but also the R status (P = 0.001) and Grade (Weiss >6 and/or Ki67 ≥ 20%, P = 0.06) in model 2. CONCLUSION: The mENSAT classification and GRAS parameters (Grade, R status, Age and Symptoms) were found to best stratify the prognosis of patients with advanced ACC.

Total Testosterone and Calculated Estimates for Free and Bioavailable Testosterone: Influence of Age and Body Mass Index and Establishment of Sex-Specific Reference Ranges.

Measurement of sex steroids is required to evaluate gonadal function, but normative data are lacking (especially for estimates of physiologically active testosterone). Using modern immunoassays, this study established sex-specific reference ranges (2.5% and 97.5% percentiles) for total testosterone (TOT), bioactive testosterone Vermeulen (BTV), free androgen index (FAI), free testosterone Sartorius (FTS), free testosterone Vermeulen (FTV), and sex hormone binding globulin (SHBG). In the comparative study, subjects were grouped by age (18-30; 31-50; >50 years), BMI (<25; 25-30; >30 kg/m(2)), and sex. Study participants were selected in such a way that each group comprised 12 subjects (e.g., 12 males between 18 and 30 years with a BMI of <25 kg/m(2), and so on), resulting in a total of 216 controls (108 males, 108 females; age: 40.3 ± 1.0; BMI: 27.8 ± 0.4). Multiple stepwise regression analyses were performed (covariates: age, BMI, sex), and sex-specific reference ranges were applied to 50 males (age: 46.1 ± 2.3; BMI: 27.4 ± 0.7) with suspected hypogonadism. Regression analysis identified the strongest predictor of each parameter apart from sex, resulting in age-specific (males: FAI, SHBG, BTV, FTV; females: TOT, FTS, SHBG), BMI-specific (males: TOT, FTS; females: FAI, BTV, FTV) and overall cutoffs for both sexes. In male patients, overall agreement between the results derived from the estimates (i.e., BTV, FTS, FTV) was high (with discordant results in only 4%). In summary, if both the endocrine workup and the clinical presentation were taken into account, the newly established reference ranges allowed reliable identification of hypogonadal males.

Screening for Cushing's syndrome: new immunoassays require adequate normative data.

Cushing's syndrome results from chronic inappropriate exposure to excessive glucocorticoid concentrations. Low-dose dexamethasone suppression, late-night salivary cortisol, and 24-h urinary free cortisol are regarded as screening tests of first choice. Consequently, measurement of circulating cortisol (e. g., in serum, saliva, and urine) is mandatory in the diagnostic workup of suspected patients. The particular analytical procedure needs to be chosen carefully. Antibody-based immunoassays offer several potential advantages: they require small volumes and are widely available, relatively cheap, and easy to handle. Modern (ideally automated) systems also have a rapid turnaround time on a large number of samples and demonstrate high analytical accuracy. However, there are some important pitfalls. Inadequate standardization and poor interlaboratory performance remain problematic and precise reference ranges are lacking for some of the newer assays. Immunoassays are also susceptible to error due to cross-reactivity with cortisol metabolites or exogenous glucocorticoids. In contrast, steroid analysis by modern chromatographic and mass spectrometric techniques is largely independent from such interference and is therefore regarded as diagnostic gold standard. To date, however, these procedures are costly, time-consuming, and at least at present restricted to a limited number of specialized centers. This review puts special emphasis on the potential advantages of salivary cortisol analysis by immunoassays. It has been shown in numerous studies that such an approach allows excellent identification of hypercortisolemic states. In this context, use of automated systems may allow for broader use of this diagnostic tool.

Is there a role for laparoscopic adrenalectomy in patients with suspected adrenocortical carcinoma? A critical appraisal of the literature.

Adrenocortical carcinoma (ACC) is a rare endocrine neoplasm and complete resection is the only treatment with curative intent for patients with nonmetastatic disease. It is highly debatable whether minimally invasive surgery is oncologically equal to open procedures in these patients. This review summarizes the current knowledge on the feasibility and oncological effectiveness of laparoscopic surgery for ACC. Using a Pubmed search strategy covering the time period up until July 2012, we identified 568 original articles and reviews with the following search terms: "adrenal gland neoplasms" and "laparoscopy", with restriction to patients over 18 years of age. Finally, 23 publications, including 6 "key studies", became the basis of this review. The key papers described 673 patients with localized ACC, of whom 112 had laparoscopic surgery. Acknowledging the subjectivity of our personal view, we draw the following conclusions: 1) since all available studies are retrospective, a final judgment of laparoscopic surgery in ACC cannot be provided; 2) the surgical treatment of patients with (suspected) ACC should be limited to specialized centers; and 3) For tumors of limited size (<10 cm) without evidence of invasiveness, laparoscopic adrenalectomy does not seem to be oncologically inferior to open surgery when performed in a state of the art manner and when oncological standards (margin-free resection, avoidance of tumor spillage) are respected. However, open adrenalectomy should still be regarded as standard treatment for ACC and laparoscopic surgery should be performed within a clinical trial or at least as an observational study.

Plasma and urinary metanephrines determined by an enzyme immunoassay, but not serum chromogranin A for the diagnosis of pheochromocytoma in patients with adrenal mass.

Adrenal pheochromocytomas are neoplasms characterized by catecholamine excess. Determination of metanephrines by high-pressure liquid chromatography has been well established for the diagnosis of pheochromocytomas, demonstrating high sensitivity and specificity. This study evaluates the diagnostic value of newly available enzyme immunoassays for metanephrines in plasma and urine. Chromogranin A was studied as a potential additional diagnostic tool. Spontaneous blood samples and 24-h urine samples were collected in 149 subjects, including 24 histologically proven pheochromocytomas, 17 aldosterone-secreting and 21 cortisol-secreting adrenal adenomas, 30 nonfunctioning adrenal masses, 15 patients with essential hypertension and 42 healthy normotensive volunteers. Plasma and urinary metanephrine and normetanephrine as well as chromogranin A were determined and putative thresholds were calculated by ROC analysis. Plasma free normetanephrine was found to be the best single parameter with the highest sensitivity (89.5%) and specificity (98.3%) using a threshold of 167 pg/ml. Analysis of the combination of plasma free metanephrines revealed a similar sensitivity with lower specificity of 90.0%. Considering both urinary parameters demonstrated a slightly higher sensitivity (92.9%) with lower specificity (77.6%). ROC analysis revealed a threshold of 215 µg/l for chromogranin A with rather low sensitivity (73.9%) and specificity (74.2%). A weak positive correlation was found between the tumor size of pheochromocytomas and plasma metanephrine (r = 0.53, p ≤ 0.05) as well as chromogranin A (r = 0.60, p ≤ 0.01). In conclusion, plasma free and urinary metanephrines measured by enzyme immunoassays are convenient and reliable parameters for the diagnosis of pheochromocytoma. In contrast, CgA demonstrated poor sensitivity and specificity.

[German adrenocortical carcinoma registry. Surgical therapy results and follow-up treatment]. / Deutsches Nebennierenrindenkarzinom-Register. Chirurgische Therapieergebnisse und Nachbehandlung.

Adrenocortical carcinoma (ACC) is a highly aggressive endocrine disease with an incidence of 1-2 cases per million population per year. Due to the low incidence of ACC knowledge concerning the surgical management is mainly based on retrospective studies or recommendations of isolated experts. Cancer databases, such as the German ACC registry are prerequisite to collect and evaluate clinical data from a large number of patients. For non-metastatic tumor stages, complete tumor resection is the only treatment with curative intent. Open surgery remains the recommended approach for ACC. However, in small tumors with uncertain malignancy a laparoscopic resection by an expert surgeon can be considered. A loco-regional lymphadenectomy should be part of the primary surgical treatment of ACC. Tumor recurrence is common even after an apparently complete primary resection. Therefore, based on the individual risk (tumor size, resection status, proliferation index) adjuvant mitotane treatment is recommended in most patients. Patients with low-risk should be included in the ADIUVO trial. In case of tumor relapse indications for a reoperation should be strongly considered, especially when the time interval since the primary surgery is long (> 12 months) and a complete resection of the recurrent disease seems to be feasible.

Chemotherapy in patients with progressive, undifferentiated neuroendocrine tumors: a single-center experience.

Treatment of patients with undifferentiated and histologically confirmed neuroendocrine tumors (NET) usually includes chemotherapeutic intervention. This retrospective study evaluated the outcome of 2 such chemotherapies. 18 patients (11 males; age 56.2 ± 2.5) with proven progressive disease were enrolled (mean Ki-67 34 ± 5%). Patients were treated from 2005 to 2007 with regimen A (carboplatin, etoposide, paclitaxel), and from 2007 to 2009 with regimen B (cisplatin, etoposide). This change was due to low tolerability of regimen A. The standard imaging procedure was computed tomography. 8 patients underwent treatment with regimen A (mean 3.3 ± 0.7 courses). Due to severe side effects, 3 patients had their therapy prematurely discontinued. The treatment responses of 6 patients who received more than 1 course were: 0% complete response (CR), 17% partial response (PR), 50% stable disease (SD), and 33% progressive disease (PD). The median progression free survival (PFS) was 6.7 months (range 3.2-10.0). In contrast, 12 patients received regimen B (mean 3.8 ± 0.4 courses), and none of them dropped out because of side effects. The overall responses were: 0% CR, 17% PR, 42% SD, and 42% PD. The median PFS was 6.3 months (range 2.8-26.4). The response rates of both regimes were not statistically different. Patients who were treated with regimen B demonstrated comparable PFS and less severe side effects than patients who received regimen A. However, patients need to be aware of the relatively short PFS time. In order to improve therapeutic outcome of patients with progressive undifferentiated NET, new therapeutic approaches and larger multi-center studies are needed.

Measurement of urinary free cortisol by current immunoassays: need for sex-dependent reference ranges to define hypercortisolism.

Urinary free cortisol (UFC) is used to assess disease activity in hypercortisolemic patients. However, reference ranges are often lacking, especially with respect to potential confounding variables. This study analyzed upper limits of normal (ULN, mean + 2 SD) for 2 newer immunoassays, using gas chromatography-mass spectrometry (GC-MS) as reference method. Each 10 healthy subjects were grouped by age (18-29; 30-49; ≥ 50 years), BMI (< 25; ≥ 25 kg/m2), and sex, resulting in a total of 120 controls (60 males; age: 39.3±1.3 years; BMI: 25.9±0.4 kg/m2). ULN were calculated for a radioimmunoassay (RIA, Immunotech) and an electrochemiluminescence immunoassay (ECLIA, Roche) and applied to 12 hypercortisolemic patients (4 males; age: 53.1±3.1 years; BMI: 29.1±1.8 kg/m2). To determine degradation, samples were stored at 4°C (without light) or 22°C (with and without light) for 0, 24, and 72 h. Cortisol concentrations were significantly correlated: r=0.88 for RIA vs. ECLIA, r=0.75 for RIA vs. GC-MS, and r=0.77 for ECLIA vs. GC-MS (always p<0.0001). For each procedure, multiple stepwise regression analysis identified sex as the only significant predictor, resulting in sex-dependent ULN (males vs. females): 294 vs. 208 nmol/24 h (RIA), and 379 vs. 277 nmol/24 h (ECLIA). These ULN classified samples from patients as hypercortisolemic in 100% (RIA) and 95% (ECLIA). Different storage conditions over 72 h did not alter UFC levels significantly. Results of the 3 procedures were well correlated, and the use of assay- and sex-specific ULN allowed excellent identification of hypercortisolic states. UFC is stable over 72 h irrespective of the storage conditions applied.

Treatment of advanced medullary thyroid carcinoma with a combination of cyclophosphamide, vincristine, and dacarbazine: a single-center experience.

OBJECTIVE: Experience with chemotherapy in patients with medullary thyroid carcinomas (MTC) is limited. This retrospective study evaluated the outcome of a combination of cyclophosphamide, vincristine, and dacarbazine ('CVD-regimen'), which has previously been suggested for treatment of malignant pheochromocytomas. METHODS: 9 patients (5 males; age 55.0 ± 4.0 years) with MTC were enrolled. Prior to chemotherapy, progressive disease was established in all patients by use of WHO criteria. On day 1 of each cycle, patients started with cyclophosphamide 750 mg/m(2), vincristine 1.4 mg/m(2), and dacarbazine 600 mg/m(2); on day 2, patients received dacarbazine alone (600 mg/m(2)). Treatment cycles were repeated at 21-day intervals and 6 cycles were planned for each patient. The standard imaging procedure was computed tomography, and the primary end point was the objective tumor response rate. After chemotherapy, patients were followed up until progression. RESULTS: 9 patients underwent a total of 57 cycles (mean 6.3 ± 0.3 cycles). Treatment responses were: 0% complete response, 11% partial response, 56% stable disease, and 33% progressive disease. The median progression free survival was 13.6 months (range 5.8-24.2 months). The median change (baseline vs. end of treatment) of calcitonin was -19% (range -70% to +174%). Reversible myelosuppression and moderate gastrointestinal symptoms were the most common adverse events. CONCLUSION: Although objective tumor response rates were low, the CVD regimen allowed disease stabilization for a substantial period of time and had acceptable toxicity. After initial surgery, chemotherapy may therefore be considered as a medical treatment option.

Monitoring medical treatment in adolescents and young adults with congenital adrenal hyperplasia: utility of salivary 17α-hydroxyprogesterone day profiles.

INTRODUCTION: Efficacy of medical treatment in patients with 21-hydroxylase deficiency is usually monitored by measurement of 17α-hydroxyprogesterone (17OHP). Saliva instead of serum sampling offers some advantages, such as painless handling and measurement of the bioactive free hormone. This study evaluated the diagnostic validity of salivary 17OHP for monitoring medical treatment, with samples collected at 7 time points throughout a day. SUBJECTS AND METHODS: Day profiles were performed in 23 adolescents and young adults with 21-hydroxylase deficiency and 43 healthy volunteers. During each profile, saliva and serum samples for 17OHP were simultaneously collected. RESULTS: With regard to the initial day profiles, samples were pathological in 63% (saliva) and 41% (serum). After the first day profile 14 patients underwent adjustment of medical treatment, either because of highly elevated 17OHP levels or with the aim of dose reduction. When comparing the best with the first day profile fewer samples were pathological (saliva: 32% vs. 71%; p<0.05; serum: 21% vs. 47%; n. s.), while the mean hydrocortisone equivalent dose was significantly reduced (20.09 mg vs. 27.27 mg; p<0.01). In 53% of profiles with controlled salivary 17OHP levels at 0700 h, the necessity for a treatment modification became only apparent when analyzing the whole day profile. CONCLUSION: A single 0700 h value within the reference range does not allow for a reliable assessment of therapeutic efficacy. We therefore suggest 17OHP day profiles for monitoring medical treatment. In this context, saliva analysis appears to be more sensitive in identifying patients who are inadequately treated.

Diagnosis of secondary adrenal insufficiency: unstimulated early morning cortisol in saliva and serum in comparison with the insulin tolerance test.

Unstimulated early morning cortisol has been suggested as a first line parameter to assess adrenal function in patients with suspected secondary adrenal insufficiency. The measurement of basal salivary cortisol (BSaC) instead of basal serum cortisol (BSeC) offers some advantages, such as painless sampling and the determination of the free hormone. The objective of this study was to evaluate the diagnostic value of BSeC and BSaC in comparison to the insulin tolerance test (ITT). Seventy-seven patients with hypothalamic-pituitary disease and 184 healthy controls were enrolled. ITT were performed in patients, and BSeC as well as BSaC levels were measured in patients and controls. Upper and lower thresholds (with >or=95% specificity either for adrenal sufficiency or adrenal insufficiency) were calculated by ROC analysis both for BSeC and BSaC. The ITT identified 41 patients as adrenal insufficient and 36 patients as adrenal sufficient. Upper and lower cutoffs were 470 and 103 nmol/l for BSeC, and 21.1 and 5.0 nmol/l for BSaC, respectively. Thereby, basal cortisol allowed a highly specific diagnosis (i.e., similar to the ITT result) in either 23% (BSeC) or 27% (BSaC) of patients. We suggest the determination of unstimulated early morning cortisol as first-line screening method for the diagnosis of secondary adrenal insufficiency. If upper and lower cutoffs are used, dynamic testing could be obviated in about one fourth of cases. Due to its easy and painless collection BSaC may be preferable to BSeC.

The value of immunoassays for metanephrines in the biochemical diagnosis of pheochromocytomas.

An undiagnosed pheochromocytoma may result in life-threatening consequences. The diagnosis of pheochromocytoma is based on the overproduction of catecholamines. Highly sensitive biochemical assays are essential to avoid false-negative results. Determinations of 24-h urinary epinephrine and norepinephrine levels are established diagnostic tools. However, they may be falsely negative in patients with a biochemically-silent or periodically-secreting pheochromocytoma. Metanephrines, which are metabolites of catecholamines, have been suggested as an alternative diagnostic tool. Urinary metanephrines are determined by high-pressure liquid chromatography (HPLC) in an increasing number of laboratories, whereas plasma metanephrines measured by HPLC are available in specialised centres only. The different HPLC methods may be cost- and time-intensive. Immunoassays such as radio- or enzyme-immunoassays may be alternative procedures. Measurement of metanephrines instead of catecholamines by either technique improved the diagnostic accuracy for the diagnosis of pheochromocytomas. Determination of plasma free metanephrines demonstrated a higher accuracy than their urinary counterparts. The use of immunoassays may be an alternative to the laborious HPLC, although the method needs to be evaluated in more detail.