Simultaneous Quantification of Aromatase Inhibitors and Estrogens in Postmenopausal Breast Cancer Patients

Context Currently there are no assays that can simultaneously quantify serum levels of the third-generation aromatase inhibitors (AIs): letrozole, anastrozole, and exemestane, and the ultra-low levels of estrogens in postmenopausal breast cancer patients on AI treatment. Such measurements may be pivotal for the determination of optimal and individualized treatment regimens. We aimed at developing a liquid chromatography–tandem mass spectrometry (MS/MS) method for simultaneous assessment of letrozole, anastrozole, exemestane, and 17-hydroxyexemestane as well as subpicomolar levels of estradiol and estrone. Methods Internal standards, calibrators, serum samples, and quality controls were in fully automated steps transferred to a deep-well plate for a 2-step liquid-liquid extraction. The extracts were reconstituted and analytes were separated chromatographically using 2 serially coupled columns, then subject to MS/MS in electrospray ionization mode. The method was thoroughly validated and is traceable to 2 accredited estrogen methods. Results The measurement range for estrone and estradiol was 0.2 to 12 000 pmol/L and 0.8 to 13 000 pmol/L, and covered the expected therapeutic range for the AIs. All analytes had a precision of less than or equal to 13%, and accuracies within 100 ± 8%. As proof of concept, AI and estrogen levels were determined in serum samples from postmenopausal breast cancer patients under treatment. Conclusion We present here an assay suitable for the simultaneous measurement of serum levels of all third-generation AIs and ultra-low levels of estrogens, providing a powerful new tool to study drug efficacy and compliance. The method is highly valuable for postmenopausal patients whose pretreatment estradiol levels are below the threshold of detection for most routine assays, but still require suppression.publishedVersio

Bedtime Salivary Cortisol as a Screening Test for Cushing Syndrome in Children

Background Diagnosing Cushing syndrome (CS) can be challenging. The 24-hour urine free cortisol (UFC) measurement is considered gold standard. This is a laborious test, dependent on correct urine collection. Late-night salivary cortisol is easier and is used as a screening test for CS in adults, but has not been validated for use in children. Objective To define liquid chromatography tandem mass spectrometry (LC-MS/MS)-based cutoff values for bedtime and morning salivary cortisol and cortisone in children, and validate the results in children with and without CS. Methods Bedtime and morning salivary samples were collected from 320 healthy children aged 4 to 16 years. Fifty-four patients from the children’s outpatient obesity clinic and 3 children with pituitary CS were used for validation. Steroid hormones were assayed by LC-MS/MS. Cutoff levels for bedtime salivary cortisol and cortisone were defined by the 97.5% percentile in healthy subjects. Results Bedtime cutoff levels for cortisol and cortisone were 2.4 and 12.0 nmol/L, respectively. Applying these cutoff levels on the verification cohort, 1 child from the obesity clinic had bedtime salivary cortisol exceeding the defined cutoff level, but normal salivary cortisone. All 3 children with pituitary CS had salivary cortisol and cortisone far above the defined bedtime cutoff levels. Healthy subjects showed a significant decrease in salivary cortisol from early morning to bedtime. Conclusions We propose that bedtime salivary cortisol measured by LC-MS/MS with a diagnostic threshold above 2.4 nmol/L can be applied as a screening test for CS in children. Age- and gender-specific cutoff levels are not needed.publishedVersio

Efficacy of adrenal venous sampling is increased by point of care cortisol analysis

Primary aldosteronism (PA) is a common cause of secondary hypertension and is caused by unilateral or bilateral adrenal disease. Treatment options depend on whether the disease is lateralized or not, which is preferably evaluated with selective adrenal venous sampling (AVS). This procedure is technically challenging, and obtaining representative samples from the adrenal veins can prove difficult. Unsuccessful AVS procedures often require reexamination. Analysis of cortisol during the procedure may enhance the success rate. We invited 21 consecutive patients to participate in a study with intra-procedural point of care cortisol analysis. When this assay showed nonrepresentative sampling, new samples were drawn after redirection of the catheter. The study patients were compared using the 21 previous procedures. The intra-procedural cortisol assay increased the success rate from 10/21 patients in the historical cohort to 17/21 patients in the study group. In four of the 17 successful procedures, repeated samples needed to be drawn. Successful sampling at first attempt improved from the first seven to the last seven study patients. Point of care cortisol analysis during AVS improves success rate and reduces the need for reexaminations, in accordance with previous studies. Successful AVS is crucial when deciding which patients with PA will benefit from surgical treatment.publishedVersio

Hormone references for ultrasound breast staging and endocrine profiling to detect female onset of puberty

Context - Application of ultrasound (US) to evaluate attainment and morphology of glandular tissue provides a new rationale for evaluating onset and progression of female puberty, but currently no hormone references complement this method. Furthermore, previous studies have not explored the predictive value of endocrine profiling to determine female puberty onset. Objective - To integrate US breast staging with hypothalamic-pituitary-gonadal hormone references and test the predictive value of an endocrine profile to determine thelarche. Design Setting and Participants - Cross-sectional sample of 601 healthy Norwegian girls, ages 6 to 16 years. Main Outcome Measures - Clinical and ultrasound breast evaluations were performed for all included girls. Blood samples were analyzed by immunoassay and ultrasensitive liquid chromatography–tandem mass spectrometry (LC-MS/MS) to quantify estradiol (E2) and estrone (E1) from the subpicomolar range. Results - References for E2, E1, luteinizing hormone, follicle-stimulating hormone, and sex hormone–binding globulin were constructed in relation to chronological age, Tanner stages, and US breast stages. An endocrine profile index score derived from principal component analysis of these analytes was a better marker of puberty onset than age or any individual hormone, with receiver-operating characteristic area under the curve 0.91 (P < 0.001). Ultrasound detection of nonpalpable glandular tissue in 14 out of 264 (5.3%) girls with clinically prepubertal presentation was associated with significantly higher median serum levels of E2 (12.5 vs 4.9 pmol/L; P < 0.05) and a distinct endocrine profile (arbitrary units; P < 0.001). Conclusions - We provide the first hormone references for use with US breast staging and demonstrate the application of endocrine profiling to improve detection of female puberty onset

Testicular ultrasound to stratify hormone references in a cross-sectional Norwegian study of male puberty

Context: Testicular growth represents the best clinical variable to evaluate male puberty, but current pediatric hormone references are based on chronological age and subjective assessments of discrete puberty development stages. Determination of testicular volume (TV) by ultrasound provides a novel approach to assess puberty progression and stratify hormone reference intervals. Objective: The objective of this article is to establish references for serum testosterone and key hormones of the male pituitary-gonadal signaling pathway in relation to TV determined by ultrasound. Design, Setting, and Participants: Blood samples from 414 healthy Norwegian boys between ages 6 and 16 years were included from the cross-sectional “Bergen Growth Study 2.” Participants underwent testicular ultrasound and clinical assessments, and serum samples were analyzed by liquid chromatography tandem–mass spectrometry and immunoassays. Main Outcome Measures: We present references for circulating levels of total testosterone, luteinizing hormone, follicle-stimulating hormone, and sex hormone–binding globulin in relation to TV, chronological age, and Tanner pubic hair stages. Results: In pubertal boys, TV accounted for more variance in serum testosterone levels than chronological age (Spearman r = 0.753, P < .001 vs r = 0.692, P < .001, respectively). Continuous centile references demonstrate the association between TV and hormone levels during puberty. Hormone reference intervals were stratified by TV during the pubertal transition. Conclusions: Objective ultrasound assessments of TV and stratification of hormone references increase the diagnostic value of traditional references based on chronological age or subjective staging of male puberty.acceptedVersio

Reference curves for pediatric endocrinology: leveraging biomarker z-scores for clinical classifications

Context: Hormone reference intervals in pediatric endocrinology are traditionally partitioned by age and lack the framework for benchmarking individual blood test results as normalized z-scores and plotting sequential measurements onto a chart. Reference curve modeling is applicable to endocrine variables and represents a standardized method to account for variation with gender and age. Objective: We aimed to establish gender-specifc biomarker reference curves for clinical use and benchmark associations between hormones, pubertal phenotype, and body mass index (BMI). Methods: Using cross-sectional population sample data from 2139 healthy Norwegian children and adolescents, we analyzed the pubertal status, ultrasound measures of glandular breast tissue (girls) and testicular volume (boys), BMI, and laboratory measurements of 17 clinical biomarkers modeled using the established “LMS” growth chart algorithm in R. Results: Reference curves for puberty hormones and pertinent biomarkers were modeled to adjust for age and gender. Z-score equivalents of biomarker levels and anthropometric measurements were compiled in a comprehensive beta coeffcient matrix for each gender. Excerpted from this analysis and independently of age, BMI was positively associated with female glandular breast volume (β = 0.5, P < 0.001) and leptin (β = 0.6, P < 0.001), and inversely correlated with serum levels of sex hormone-binding globulin (SHBG) (β = −0.4, P < 0.001). Biomarker z-score profles differed signifcantly between cohort subgroups stratifed by puberty phenotype and BMI weight class. <p<Conclusion: Biomarker reference curves and corresponding z-scores provide an intuitive framework for clinical implementation in pediatric endocrinology and facilitate the application of machine learning classifcation and covariate precision medicine for pediatric patients

Prior Knee Arthroscopy Does Not Influence Long-Term Total Knee Arthroplasty Outcomes and Survivorship

26th AAHKS Annual Meeting, DALLAS, ETATS-UNIS, 10-/11/2016 - 13/11/2016BACKGROUND: Arthroscopic knee surgery frequently precedes total knee arthroplasty (TKA). There have been mixed data on the effect of prior arthroscopic surgery on results of TKA. The purpose of this study was to compare the 10-year Knee Society Score (KSS), survivorship, and complications of TKA in a cohort of patients who had a previous knee arthroscopy to a control cohort. METHODS: A retrospective review of 1315 TKAs who underwent a primary TKA between 2003 and 2004 was performed. Of these, 160 TKAs had previous arthroscopy (excluding ligamentous reconstruction). A matched cohort study 2:1 was carried out with a group of 320 controls (no prior surgery). Outcomes were assessed with the original KSS, range-of-motion, complications, and survivorships. Mean follow-up was 9 years. RESULTS: The mean KSS increased from 36-84 in the arthroscopy group vs 35-86 in the control group (P = .5). The mean preoperative and postoperative range-of-motion was not different between groups (P = .2). The survivorship free of complication at 5 years was similar in both groups (94.3% in arthroscopy group vs. 95.3% in the control; P = .7) with infection in 2 controls and 3 arthroscopy cases (P = .2). The survivorships free of revision for aseptic loosening, revision for any reason, and reoperation were similar at 10 years (96.5%, 94.6%, and 89.2%, respectively, in the arthroscopy group vs 96.2%, 95.9%, and 91.5% in the control group). CONCLUSION: There were no significant differences between both groups. These data are reassuring and valuable in an era in which many candidates for TKA will have had previous arthroscopic knee surgery

Sex Hormones and Adrenal Steroids : Biological Variation Estimated Using Direct and Indirect Methods

Background Biological variation (BV) data may be used to develop analytical performance specifications (APS), reference change values (RCV), and support the applicability of population reference intervals. This study estimates within-subject BV (CVI) for several endocrine biomarkers using 3 different methodological approaches. Methods For the direct method, 30 healthy volunteers were sampled weekly for 10 consecutive weeks. Samples were analyzed in duplicate for 17-hydroxyprogesterone (17-OHP), androstenedione, cortisol, cortisone, estradiol, follicle-stimulating hormone (FSH), luteinizing hormone (LH), sex hormone-binding globulin (SHBG), and testosterone. A CV-ANOVA with outlier removal and a Bayesian model were applied to derive the CVI. For estradiol, FSH and LH, only the male subgroup was included. In the indirect method, using the same analytes and groups, pairs of sequential results were extracted from the laboratory information system. The total result variation for individual pairs was determined by identifying a central gaussian distribution in the ratios of the result pairs. The CVI was then estimated by removing the effect of analytical variation. Results The estimated CVI from the Bayesian model (mu CVP(i)) in the total cohort was: 17-OHP, 23%; androstenedione, 20%; cortisol, 18%; cortisone, 11%; SHBG, 7.4%; testosterone, 16%; and for the sex hormones in men: estradiol, 14%; FSH, 8%; and LH, 26%. CVI-heterogeneity was present for most endocrine markers. Similar CVI data were estimated using the CV-ANOVA and the indirect method. Conclusions Similar CVI data were obtained using 2 different direct and one indirect method. The indirect approach is a low-cost alternative ensuring implementation of CVI data applicable for local conditions.Funding Agencies|Haukeland University Hospital; BritishHeart Foundation [FS/18/78/33902]; Guys and St ThomasCharity (London, UK) [R060701, R100404]; Haukeland University Hospital, Department ofMedical Biochemistry and Pharmacology; University of Oslo</p

Blood steroid levels predict survival in endometrial cancer and reflect tumor estrogen signaling

Objective Blood-based biomarkers are attractive due to ease of sampling and standardized measurement technology, reducing obstacles to clinical implementation. The objective of this study was to evaluate a clinically available method of steroid hormone measurement for its prognostic potential in endometrial cancer. Methods We quantified seven steroid hormones by liquid chromatography-tandem mass spectrometry in 100 endometrial cancer patients from a prospective cohort. Abdominal fat distribution was assessed from abdominal computed tomography (CT) scans. Steroid hormone levels were compared to clinical characteristics, fat distribution and gene expression in primary tumor samples. Results Low levels of 17OH-progesterone, 11-deoxycortisol and androstenedione were associated with aggressive tumor characteristics and poor disease specific survival (p = .003, p = .001 and p = .02 respectively). Adjusting for preoperative risk based on histological type and grade, low 17OH-progesterone and 11-deoxycortisol independently predicted poor outcome with hazard ratios of 2.69 (p = .033, 95%CI: 1.09–6.68) and 3.40 (p = .020, 1.21–9.51), respectively. Tumors from patients with low steroid level displayed increased expression of genes related to mitosis and cell cycle progression, whereas high steroid level was associated with upregulated estrogen signaling and genes associated with inflammation. Estrone and estradiol correlated to abdominal fat volume in all compartments (total, visceral, subcutaneous, p < .001 for all), but not to the visceral fat proportion. Patients with higher levels of circulating estrogens had increased expression of estrogen signaling related genes. Conclusion Low levels of certain endogenous steroids are associated with aggressive tumor traits and poor survival and may provide preoperative information independent of histological biomarkers already in use.publishedVersio