An LC-MS/MS-based method for the simultaneous quantification of melatonin, cortisol and cortisone in saliva.

Disturbances in the diurnal pattern are associated with several clinical and psychological conditions, including depression and fatigue. Salivary sampling for melatonin, cortisol and cortisone provides a non-invasive method for frequent sampling and obtaining biochemical insight into the diurnal pattern of individuals. Therefore, a new liquid chromatography-tandem mass spectrometry-based method for the measurement of salivary melatonin, cortisol and cortisone was developed and validated. The method required 250 µl saliva, used isotope dilution methodology and was based on a liquid-liquid extraction for sample preparation, reversed-phase chromatography and multiple reaction monitoring on a mass spectrometer for quantitation. The lower limits of quantification obtained were 0.010 nmol/L for melatonin, 0.5 nmol/L for cortisol and 1.00 nmol/L for cortisone and the limits of detection were 0.003 nmol/L, 0.15 nmol/L and 0.1 nmol/L respectively. The method imprecision was ≤14% for all measurands, and the method comparison showed highly comparable results with high correlation coefficients (all ≥0.964). Potential interference of cortisol and cortisone by prednisolone was observed and could be detected by chromatogram review. Typical diurnal patterns for melatonin, cortisol and cortisone were observed in the saliva of 20 cancer survivors who collected saliva throughout the day.

Towards deciphering glioblastoma intra-tumoral heterogeneity: The importance of integrating multidimensional models.

Glioblastoma (GBM) is the most common and severe form of brain cancer among adults. Its aggressiveness is largely attributed to its complex and heterogeneous biology that despite maximal surgery and multimodal chemoradiation treatment, inevitably recurs. Traditional large-scale profiling approaches have contributed substantially to the understanding of patient-to-patient inter-tumoral differences in GBM. However, it is now clear that biological differences within an individual (intra-tumoral heterogeneity) are also a prominent factor in treatment resistance and recurrence of GBM and will likely require integration of data from multiple recently developed omics platforms to fully unravel. Here we dissect the growing geospatial model of GBM, which layers intra-tumoral heterogeneity on a GBM stem cell (GSC) precursor, single cell, and spatial level. We discuss potential unique and inter-dependant aspects of the model including potential discordances between observed genotypes and phenotypes in GBM.

Current Routine Testosterone Immunoassays Are Unsuitable for Lowering the General Castration Cutoff Recommendation to <0.7 nmol/l (20 ng/dl).

Testosterone measurements are essential in the management of patients with prostate cancer undergoing castration and androgen deprivation therapy. There has been an ongoing discussion on the testosterone castration cutoff (TCC), with the primary focus on large cohort studies in which the testosterone measurement system was not specified or studies that used individual testosterone measurement systems. Here we present a post hoc analysis of a study comparing testosterone measurement systems in a cohort of 120 castrated patients with prostate cancer. We investigated the suitability of general, measurement system-independent, TCC values recommended in all clinical guidelines. We show that the four testosterone immunoassays commonly used are unsuitable to support lowering of TCC to 0.7 nmol/l (20 ng/dl) testosterone, since testosterone levels are falsely quantified as higher than this cutoff in 4.2-29.2% of the castrated cohort, depending on the testosterone immunoassay used. When using 1.0 nmol/l (30 ng/dl) as the TCC for the Beckman immunoassay, 13.3% of the results were falsely quantified as being higher than this value. The results suggest that the measurement systems used in current practice do not support lowering the TCC to 0.7 nmol/l. Furthermore, a more local, immunoassay-dependent TCC should be considered. Patient summary: Patients with advanced prostate cancer who are treated to reduce their testosterone to a castration level are monitored using testosterone measurements. The testing systems currently used for measurement do not support lowering of the testosterone cutoff value to 0.7 nmol/l. Testosterone cutoff values to define castration status should preferably be based on the measurement system in local use.

Serum testosterone measured by liquid chromatography-tandem mass spectrometry is an independent predictor of response to castration in metastatic hormone-sensitive prostate cancer.

BACKGROUND: Although testosterone levels have been associated with progression-free survival (PFS) in metastatic hormone-sensitive prostate cancer (mHSPC) patients, this has primarily been investigated using inaccurate immunoassays (IA). Here, we investigated whether castrate testosterone levels determined by a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay is an independent risk factor for treatment response in mHSPC. METHODS: In total, 106 mHSPC patients treated with luteinizing-hormone releasing-hormone (LHRH) agonists were retrospectively analyzed between March 2018 and August 2021. Testosterone levels in serum samples were quantitated using an LC-MS/MS assay. In a subset of patients, IA (Roche Cobas Pro) values were compared with LC-MS/MS results. Survival analysis was performed using Kaplan-Meier curves and Cox proportional hazard models. RESULTS: Median PFS was shorter for high testosterone levels (>0.231 nmol/L, 18.4 v. 42.6 months, HR 1.7, p = 0.018). Low testosterone levels and a PSA response below 4 ng/mL was associated with longer median PFS (46.2 months) than the remaining combinations (13.8-19.3 months, 3.4-5.8, overall p < 0.01). In 67 patients, testosterone levels below the median remained associated with longer PFS, whereas IA measurements did not show a similar difference. CONCLUSION: Our results suggest that high castration testosterone levels measured by LC-MS/MS is an independent response predictor for mHSPC patients.

Predictive value of low testosterone concentrations during and prior to enzalutamide treatment in metastatic castration-resistant prostate cancer.

BACKGROUND: Enzalutamide is an effective treatment for metastatic castration-resistant prostate cancer (mCRPC) patients. However, variances in responses are observed and there is a need for biomarkers predicting treatment outcome and selection. In this study, we aimed to explore the predictive value of testosterone for first-line enzalutamide treatment of mCRPC. METHODS: A retrospective analysis of 72 mCRPC patients with no prior abiraterone or docetaxel treatment was performed. Serum testosterone was measured using a liquid chromatography tandem-mass spectrometry method. Association of pre- and during-enzalutimide treatment testosterone levels with progression-free survival (PFS) and failure-free survival (FFS) was investigated using univariate and multivariate Cox models. Testosterone levels were dichotomized into a low (Q1) and high (interquartile range-Q4) group. RESULTS: Median PFS (7.4 v. 20.8 months, P<0.0001) and FFS (6.6 v. 17.7 months, P<0.0001) were shorter for patients with low testosterone levels (<0.217 nmol/L) during enzalutamide treatment. Furthermore, univariate Cox proportional hazards models revealed that low testosterone levels were associated with shorter PFS (HR 3.5, 95%CI 1.9-6.3; P<0.001) and FFS (HR 3.1, 95%CI 1.7-5.5; P<0.001). Pre-treatment testosterone levels were lower than during-treatment levels (P<0.0001) and low pre-treatment testosterone levels (<0.143 nmol/L) were associated with shorter median PFS (12.6 v. 20.5 months, P<0.01) and FFS (12.6 v. 22.5 months, P<0.01). CONCLUSION: The results of this study suggest that low serum testosterone levels during and prior to enzalutamide treatment can predict progression in mCRPC patients and identifies tumors resistant to next-in-line enzalutamide treatment. Validation in a prospective cohort is warranted.

Testosterone analysis in prostate cancer patients.

Testosterone is an essential steroid hormone associated with a wide variety of biological processes in humans. In prostate cancer, androgen signaling is an important driver of tumor cell growth. Depletion of gonadal testosterone, achieved by surgical or chemical castration, prevents androgenic signaling and temporally reduces, stops or reverses tumor growth before inevitable progression to castration-resistant prostate cancer occurs. Additional treatment strategies targeting androgenic signaling have become available, although these are without curative intent. While circulating testosterone is also associated with disease risk and potential clinical utility, the main use in the clinical lab is monitoring adequate castration and subsequent resistance to therapy. Adequate castrate testosterone concentrations are currently based on over 50 year-old double-isotope derivative assays that are disputed in automated immunoassay (IA) analysis. The debate has been further fueled with the introduction of mass spectrometry-based assays for testosterone, offering a substantial increase in sensitivity and specificity. In this review, we discuss testosterone regulation and androgen deprivation therapy in prostate cancer. We provide an overview of the developments in testosterone analysis for monitoring adequate castration and resistance to therapy. Current clinical practice and future clinical utility will be discussed. Finally, clinical and research recommendations will be presented.

Changes in Sex Steroids and Relation With Menopausal Complaints in Women Undergoing Risk-reducing Salpingo-oophorectomy.

Context: Risk-reducing salpingo-oophorectomy (RRSO) is performed in BRCA1 or 2 mutant carriers to minimize ovarian cancer risk. Although studies have been performed investigating sex steroid levels, menopausal complaints, and sexual functioning in relation to RRSO, their exact relationship remains unknown. Objectives: To investigate the impact of RRSO on serum sex steroid levels and their association with menopausal complaints and sexual functioning. Methods: This prospective observational cohort study included 57 premenopausal and 37 postmenopausal women at risk of ovarian cancer and opting for RRSO. Data collection involved validated questionnaires on sexual functioning and menopausal complaints. Testosterone, androstenedione, estradiol, and estrone levels in serum determined by liquid chromatography-tandem mass spectrometry were obtained 1 day before, 6 weeks, and 7 months after RRSO. Results: In premenopausal women, all 4 steroids were decreased both 6 weeks (P < 0.01) and 7 months (P < 0.01) after RRSO. Furthermore, in these women, decreases in estrogens were associated with a decrease in sexual functioning 7 months after RRSO (P < 0.05). In postmenopausal women, only testosterone was decreased 6 weeks and 7 months (P < 0.05) after RRSO, which was associated with an increase in menopausal complaints at 7 months post-RRSO (P < 0.05). Conclusion: Our results suggest that in premenopausal women, decreases in estrogens are related to a decrease in sexual functioning and that in postmenopausal women, testosterone is decreased after RRSO, which indicates that postmenopausal ovaries maintain some testosterone production. Furthermore, in postmenopausal women, a large decrease of testosterone was associated with more menopausal complaints, indicating that future studies investigating testosterone supplementation are warranted.

Retrospective analysis of serum testosterone levels by LC-MS/MS in chemically castrated prostate cancer patients: Biological variation and analytical performance specifications.

BACKGROUND: A sensitive liquid chromatography tandem-mass spectrometry (LC-MS/MS) method was used to monitor serum testosterone levels in castrated prostate cancer patients. We subsequently performed an observational and retrospective study to estimate the within- and between-subject biological variation of these patients. METHODS: In total, 474 samples from 72 prostate cancer patients in the Netherlands receiving either chemical castration (CAS) or castration plus enzalutamide (ENZA) treatment were selected for data analysis. ANOVA was performed to estimate analytical variation (CVA) and within-patient variation (CVI). A nested ANOVA was applied to estimate between-patient variation (CVG). From these data, the reference change value (RCV) and analytical performance specifications (APS) were calculated. RESULTS: Testosterone levels were significantly higher in the ENZA group (0.318 vs. 0.191 nmol/L, p < 0.005) than the CAS group. Overall, variation components were estimated at 6.1%, 24.6% and 60.3% for CVA, CVI and CVG, respectively. Both groups showed high individuality (<0.6). The RCV was 70.3% for all patients. Desirable APS were 12.3% for imprecision, 16.3% for bias and 26.4% for total error. CONCLUSION: The generated APS are valuable for sensitive testosterone assays and the high individuality indicates that castrated testosterone levels can be studied as a predictive or prognostic biomarker in prostate cancer patients.

Simultaneous analysis of E1 and E2 by LC-MS/MS in healthy volunteers: estimation of reference intervals and comparison with a conventional E2 immunoassay.

Monitoring estrogen levels, especially estradiol (E2), is amongst others important for determining menopausal status and guidance of breast cancer treatment. We validated a serum E2 and estrone (E1) liquid chromatography tandem-mass spectrometry assay (LC-MS/MS) suitable for quantitation in human subjects. In addition, we compared our method with an E2 immunoassay (IA) and established preliminary reference values. Validation parameters were within the predetermined acceptance criteria. Assay linearity ranges were 4-1500 pmol/L for E1 and 4-2500 pmol/L for E2. Imprecision ranged from 7.4 to 9.6%. The lower limit of quantitation for E2 (8.0 pmol/L) was 11.4 times lower than the IA. The method comparison revealed differences in E2 quantitation up to 155% between both methods. The method allowed quantitation of E1 in all healthy volunteers, while E2 could not be detected in 95% versus 40% of the post-menopausal women using IA and LC-MS/MS, respectively. Male, pre-, peri- and postmenopausal female reference values were estimated. An LC-MS/MS based method combining E1 and E2 analysis was validated with superior E2 analytical sensitivity when compared to the IA.

Serum Testosterone by Liquid Chromatography Tandem Mass Spectrometry for Routine Clinical Diagnostics.

In clinical diagnostics, samples containing low testosterone cannot be analyzed by random access immunoassays normally available at clinical laboratories. For these samples, sensitive and specific LC-MS/MS-based testosterone methods are required. An LC-MS/MS-based testosterone assay is described that was developed and validated for routine clinical application.