Anastrozole dose escalation for optimal estrogen suppression in postmenopausal early-stage breast cancer: A prospective trial.

BACKGROUND: We previously reported that postmenopausal women with ER+ breast cancer (BC) receiving adjuvant anastrozole 1 mg/day (ANA1) with estrone (E1) ≥1.3 pg/mL and estradiol (E2) ≥0.5 (inadequate estrogen suppression [IES]) had a 3.0-fold increased risk of a BC event. The objective of this study was to determine if increasing anastrozole to 10 mg/day (ANA10) could result in adequate estrogen suppression (AES: E1 <1.3 pg/mL and/or E2 <0.5) among those with IES on ANA1. METHODS: Postmenopausal women with ER+ BC planning to receive adjuvant ANA1 were eligible. E1 and E2 were assessed pre- and post-8-10 weeks of ANA1. Those with IES were switched to 8-10 week cycles of ANA10 followed by letrozole 2.5 mg/day. E1 and E2 were assessed after each cycle. Anastrozole concentrations were measured post-ANA1 and post-ANA10. Primary analyses included patients who documented taking at least 80% of planned treatment (adherent cohort). RESULTS: 132 (84.6%) of 156 eligible patients were ANA1-adherent. IES occurred in 40 (30.3%) adherent patients. 25 (78.1%) of 32 patients who began ANA10 were adherent, and AES was achieved in 19 (76.0%; 90%CI: 58.1-89.0%) patients. Anastrozole concentrations post-ANA1 and post-ANA10 did not differ by estrogen suppression status among adherent patients. AES was maintained/attained in 21 (91.3%) of 23 letrozole-adherent patients. CONCLUSIONS: Approximately 30% of ANA1-adherent patients had IES. Among those who switched to ANA10 and were adherent, 76% had AES. Further studies are required to validate emerging data that ANA1 results in IES for some patients and to determine the clinical benefit of switching to ANA10 or an alternative AI.

Using differential mobility spectrometry to improve the specificity of targeted measurements of 2,3-dinor 11ß-Prostaglandin F2α.

INTRODUCTION: 2,3-dinor 11ß-Prostaglandin F2α (BPG) is an arachidonic acid derivative and the most abundant metabolic byproduct of prostaglandin D2, which is released during mast cell activation. Therefore, measurements of BPG in urine using liquid chromatography-tandem mass spectrometry (LC-MS/MS) provide a noninvasive method for evaluation and management of mast cell disorders. Measurements obtained by LC-MS/MS exhibit a high prevalence of chromatographic interferences resulting in challenges with optimal determination of BGP. In this investigation, differential mobility spectrometry (DMS) is utilized to overcome the limitations of current testing. METHODS: Urine samples were extracted using an automated solid-phase extraction method. Samples were then analyzed with and without DMS devices installed on two commercially available mass spectrometry platforms to assess the benefits of DMS. Following promising results from a preliminary analytical evaluation, LC-DMS-MS/MS measurements of BPG in urine were fully validated to assess the analytical implications of using this technology. RESULTS AND DISCUSSION: The addition of DMS devices to the LC-MS/MS systems evaluated in this investigation significantly reduced interferences observed in the chromatograms. Concomitantly, DMS reduced the number of discordant quantifier/qualifier fragment ion results that significantly exceeded the ± 20 % limits, suggesting greater analytical specificity. The validation studies yielded low interday imprecision, with %CVs less than 6.5 % across 20 replicate measurements. Validation studies assessing other aspects of analytical performance also met acceptance criteria. CONCLUSIONS: Incorporating DMS devices greatly improved the specificity of BPG measurements by LC-MS/MS, as evidenced by the comparison of chromatograms and fragment ion results. Validation studies showed exceptional performance for established analytical metrics, indicating that this technology can be used to minimize the impact of interferences without adversely impacting other aspects of analytical or clinical performance.

Analysis of vitamin D3-sulfate and 25-hydroxyvitamin D3-sulfate in breastmilk by LC-MS/MS.

Sulfated metabolites of vitamin D have been suggested to be in breastmilk, although current methods to measure sulfated vitamin D compounds in breastmilk by liquid chromatography-tandem mass spectrometry (LC-MS/MS) have not adequately accounted for increased aqueous solubility of these sulfated metabolites. The purpose of this study was to generate a method of LC-MS/MS for measuring vitamin D3-3-sulfate (VitD3-S) and 25-hydroxyvitamin D3-3-sulfate (25OHD3-S) specifically in human breastmilk. The resulting method uses methanol to precipitate protein and solid phase extraction to prepare the samples for LC-MS/MS. The limits of quantification for analytes in solvent were 0.23 ng/mL VitD3-S and 0.2 ng/mL 25OHD3-S. Various experiments observed concentrations ranging 0.53 to 1.7 ng/mL VitD3-S and ≤ 0.29 ng/mL 25OHD3-S. Both analytes were present in aqueous skim milk, demonstrating the enhanced aqueous solubility of these vitamin D sulfates. In conclusion, we describe an effective method for measuring VitD3-S and 25OHD3-S in breastmilk by LC-MS/MS.

Analytical Unreliability of 25 Hydroxy Vitamin D Measurements in Pre-Term Neonates.

BACKGROUND: Vitamin D supplementation is common practice for neonates and infants due to limited stores of vitamin D at birth. Although not commonly encountered, vitamin D toxicity can occur due to over-supplementation. However, toxic concentrations are often not included in method validation experiments, and assays often are not validated in the neonatal population. METHODS: We compared serial 25 hydroxy vitamin D [25(OH)D] measurements in pre-term neonates receiving 25(OH)D supplementation and identified 12 patients wherein concentrations of 25(OH)D were above 50 ng/mL (125 nM) that required additional investigations as the 25(OH)D results did not match the clinical picture. Available samples were compared across 4 immunoassay platforms (LIAISON XL, Roche Cobas e602, Abbott Alinity i, and Siemens Centaur XP) and LC-MS/MS. RESULTS: Concentrations of 25(OH)D observed on one individual immunoassay platform (LIAISON XL) fluctuated substantially between subsequent blood draws in select neonates with elevated concentrations. Serum samples from these patients showed variable agreement between LC-MS/MS and other immunoassay platforms. These fluctuations were not explained by the presence of 3-epimer-25(OH)D or 24,25(OH)2D. CONCLUSIONS: Although we were unable to identify a cause for the variable elevated results, our findings suggest that neonatal 25(OH)D measurements alone should not be used for assessment of nutritional monitoring, and that clinical correlation and other laboratory parameters including ionized calcium should be considered.

Changes in serum cortisol levels after 10 days of overfeeding and fasting.

Chronic caloric deprivation and obesity are complicated by hypercortisolemia. The effects of acute overfeeding and fasting on circulating free cortisol levels and conversion of cortisone to free cortisol are unknown. We hypothesized that serum-free cortisol and free cortisol-to-cortisone ratio would increase after both overfeeding and fasting. This is a prospective study of 22 healthy volunteers who completed a 10-day high-calorie protocol followed by a 10-day fast, separated by a 2-wk washout. Morning free and total cortisol and free cortisone levels (LC/MS) were measured at baseline and after 10 days of each intervention. Both high-calorie feeding and fasting increased total and free cortisol and the free cortisol-to-free cortisone ratio (P = 0.001 to P = 0.046). There were sex interactions, with significant effects in men (P < 0.001), but not in women (P = 0.898 and 1.000, respectively) in subset analyses examining the effects of fasting on free cortisol and the free-to-total cortisol ratio. Overfeeding and fasting both increase circulating free cortisol levels and appear to alter the balance between cortisol and its inactive metabolite, cortisone. Further study is warranted to determine whether elevated cortisol levels contribute to complications of starvation and obesity, such as bone fragility.NEW & NOTEWORTHY Overfeeding and fasting both increase circulating free cortisol levels and appear to alter the balance between cortisol and its inactive metabolite, cortisone. The effect of fasting on free cortisol levels is modified by sex. Further study is needed to determine the mechanisms driving the increases in cortisol.

Identification, Measurement, and Assessment of the Clinical Utility of Human Pancreatic Polypeptide by Liquid Chromatography-Tandem Mass Spectrometry.

Human pancreatic polypeptide (HPP) is a 36 amino acid peptide hormone that plays a role in the bidirectional communication between the digestive system and the brain. HPP measurements are used to assess vagal nerve function following sham feeding and to detect gastroenteropancreatic-neuroendocrine tumors. These tests have historically been conducted by radioimmunoassays, but liquid chromatography-tandem mass spectrometry (LC-MS/MS) has several advantages such as improved specificity and elimination of radioactive molecules. Here, we present our LC-MS/MS method. Initially, samples were immunopurified and subjected to LC-high resolution accurate mass tandem mass spectrometry (HRAM-MS/MS) to identify circulating forms of the peptide in human plasma. We identified 23 forms of HPP, including several glycosylated forms. The most abundant peptides then were used for targeted LC-MS/MS measurements. LC-MS/MS performance for precision, accuracy, linearity, recovery, limit of detection, and carryover met our acceptance criteria based on CLIA regulations. Additionally, we observed the expected physiological rise in HPP in response to sham feeding. Our results indicate that HPP measurement by LC-MS/MS produces clinically equivalent results to our established immunoassay when several peptides are monitored, making it a suitable replacement. The measurement of peptide fragments, including modified species, might have additional clinical value.

Discordance of insulin-like growth factor-1 results and interpretation on four different platforms.

OBJECTIVE: Inconsistent Insulin-like Growth Factor 1 (IGF-1) measurements among different platforms have been observed. In this study, we compared the IGF-1 assay on four different platforms. METHODS: A total of 110 serum specimens were analyzed in this comparison study. IGF-1 was measured on the three different chemiluminescent automated immunoassay of Siemens Immulite 2000 XPi, DiaSorin Liaison XL, IDS iSYS and LC-MS/MS method. Results were compared with Weighted Deming regression. Bias was evaluated using the Bland-Altman method. RESULTS: Weighted Deming regression analysis showed approximately 36 % negative variation on Immulite, compared to Liaison (Immulite = 0.64 * DiaSorin + 2.95, r2 = 0.95); 8 % negative variation on iSYS, compared to Liaison (iSYS = 0.92 * DiaSorin + 0.51, r2 = 0.97); 17 % negative variation on LC-MS/MS, compared to Liaison (LC-MS/MS = 0.83 * DiaSorin-11.23, r2 = 0.93); 34 % positive variation on LC-MS/MS compared to Immulite (LC-MS/MS = 1.34 * Immulite-21.97, r2 = 0.96); 81 % positive variation on IDS iSYS compared to Immulite (IDS iSYS = 1.81 * Immulite-117.65, r2 = 0.83). The Bland-Altman plot showed a significant negative variation of Immulite versus DiaSorin and positive variation of IDS iSYS versus Immulite. Overall agreement between different platforms was poor, which reflected systematic difference. The variation between platforms increased as IGF-1 values increased. CONCLUSIONS: There are wide variations between different platforms for IGF-1 measurement. The lack of standardization in IGF-1 measurement creates a challenge for clinicians to monitor IGF-1 and treat patients with pituitary disorders, when switching from one platform to another. The potential impact of the variations in IGF-1 measurement between different platforms should be taken into consideration when managing patients.

Pitfalls in Diagnosing Hypoglycemia Due to Exogenous Insulin: Validation and Utility of an Insulin Analog Assay.

OBJECTIVE: To overcome the limitations of commercially available insulin immunoassays which have variable detection of analog insulin and can lead to clinically discordant results and misdiagnosis in the workup of factitious hypoglycemia. PATIENTS AND METHODS: We performed analytical validation of a liquid chromatography high resolution accurate mass (LC-HRAM) immunoassay to detect insulin analogs. We completed clinical assessment using a large cohort of human serum samples from 78 unique individuals, and subsequently used the assay in the evaluation of eight individuals with high diagnostic suspicion for factitious hypoglycemia. RESULTS: The performance characteristics show that the LC-HRAM immunoassay can be applied to detect five commonly used synthetic insulin analogs (lispro, glulisine, aspart, glargine metabolite, and detemir) in human serum. Our clinical cases show that this assay could be used in the diagnosis of factitious hypoglycemia by identifying the analog insulin(s) in question. CONCLUSION: The LC-HRAM immunoassay reported here overcomes a gap in our diagnostic pathway for hypoglycemia. The results obtained from our studies suggest that this method is appropriate for use in clinical laboratories when factitious hypoglycemia is considered as a differential diagnosis.

Quantification of Insulin Analogs by Liquid Chromatography-High-Resolution Mass Spectrometry.

Administration of exogenous insulin or insulin analogs is a common cause of hypoglycemia. The etiology of hypoglycemic episodes can be investigated by the measurement of insulin. However, frequently used synthetic insulin analogs show variable reactivity with immunoassays designed for the quantification of human insulin and may produce misleading results. To overcome this challenge, mass spectrometric methods can be applied to differentiate and accurately quantify insulin and its analogs. Here we describe a liquid chromatography-tandem high-resolution accurate mass (LC-HRAM) for the highly specific and independent quantification of insulin and its synthetic analogs including aspart, detemir, glargine, glulisine, and lispro. This method utilizes antibody affinity extraction followed by analysis on a high-resolution accurate mass spectrometer.

Differentiation of Common IGF-1 Variants Using HRMS COM Determination with Follow-Up MS/MS Verification.

Insulin-like growth factor 1 (IGF-1), a peptide hormone regulator of growth hormone (GH), has common variants with differing functionality. These variants are a result of single amino acid changes in the peptide that can lead to significant changes in the resulting protein. The standard method of evaluating any of these variants is by using tandem mass spectrometry (MS/MS) methods. A novel method has been developed to evaluate some variants solely by high-resolution mass spectrometry (HRMS) of the intact peptide by calculating the center of mass (COM) of the [M + 7H]+7 isotopic distribution. This has allowed differentiation between the nonfunctional V44M variant and the A67T/A70T functional variants without the need for MS/MS. However, MS/MS is still needed to differentiate between the A67T and A70T variants. In this chapter we outline the LC-HRMS method for IGF-1 analysis with the inclusion of COM calculations and subsequent MS/MS differentiation.

Detection of 13C-Mannitol and Other Saccharides Using Tandem Mass Spectrometry for Evaluation of Intestinal Permeability or Leaky Gut.

Urine tests for intestinal permeability typically detect the secretion of administered saccharides with relatively different absorptions over a designated time period to determine severity of disease. Traditionally, a disaccharide/monosaccharide ratio such as lactulose/mannitol is used. Due to the potential for contamination of mannitol from different foods and commercial products causing an elevated baseline measurement, 13C mannitol can be used instead. In this chapter, a method of detecting various administered saccharides in urine for the evaluation of intestinal permeability is described. Three monosaccharides and two disaccharides are included so researchers can choose which combinations best fit their needs. Here lactulose, mannitol, 13C mannitol, rhamnose, and sucralose levels are separated and detected in urine using HPLC separation and MS/MS analysis.

Quantification of Parathyroid Hormone and its Fragments in Serum by Liquid Chromatography-High-Resolution Mass Spectrometry.

Parathyroid hormone (PTH), an 84-amino acid peptide hormone, is a major regulator of calcium homeostasis. Quantification of PTH in serum is used clinically to investigate calcium imbalances and for monitoring osteodystrophy in patients with renal failure. In addition to intact PTH, several PTH fragments are found in circulation. Recent studies have shown that accurate quantification of PTH fragments may provide valuable clinical information in certain scenarios. In this chapter, a high-resolution mass spectrometry-based method for quantification of PTH (1-84) and its fragments is described. This method involves immunoaffinity capture of intact PTH and PTH-fragments followed by liquid chromatography-high-resolution mass spectrometry (LC-HRMS).

Development of a multiomics model for identification of predictive biomarkers for COVID-19 severity: a retrospective cohort study.

BACKGROUND: COVID-19 is a multi-system disorder with high variability in clinical outcomes among patients who are admitted to hospital. Although some cytokines such as interleukin (IL)-6 are believed to be associated with severity, there are no early biomarkers that can reliably predict patients who are more likely to have adverse outcomes. Thus, it is crucial to discover predictive markers of serious complications. METHODS: In this retrospective cohort study, we analysed samples from 455 participants with COVID-19 who had had a positive SARS-CoV-2 RT-PCR result between April 14, 2020, and Dec 1, 2020 and who had visited one of three Mayo Clinic sites in the USA (Minnesota, Arizona, or Florida) in the same period. These participants were assigned to three subgroups depending on disease severity as defined by the WHO ordinal scale of clinical improvement (outpatient, severe, or critical). Our control cohort comprised of 182 anonymised age-matched and sex-matched plasma samples that were available from the Mayo Clinic Biorepository and banked before the COVID-19 pandemic. We did a deep profiling of circulatory cytokines and other proteins, lipids, and metabolites from both cohorts. Most patient samples were collected before, or around the time of, hospital admission, representing ideal samples for predictive biomarker discovery. We used proximity extension assays to quantify cytokines and circulatory proteins and tandem mass spectrometry to measure lipids and metabolites. Biomarker discovery was done by applying an AutoGluon-tabular classifier to a multiomics dataset, producing a stacked ensemble of cutting-edge machine learning algorithms. Global proteomics and glycoproteomics on a subset of patient samples with matched pre-COVID-19 plasma samples was also done. FINDINGS: We quantified 1463 cytokines and circulatory proteins, along with 902 lipids and 1018 metabolites. By developing a machine-learning-based prediction model, a set of 102 biomarkers, which predicted severe and clinical COVID-19 outcomes better than the traditional set of cytokines, were discovered. These predictive biomarkers included several novel cytokines and other proteins, lipids, and metabolites. For example, altered amounts of C-type lectin domain family 6 member A (CLEC6A), ether phosphatidylethanolamine (P-18:1/18:1), and 2-hydroxydecanoate, as reported here, have not previously been associated with severity in COVID-19. Patient samples with matched pre-COVID-19 plasma samples showed similar trends in muti-omics signatures along with differences in glycoproteomics profile. INTERPRETATION: A multiomic molecular signature in the plasma of patients with COVID-19 before being admitted to hospital can be exploited to predict a more severe course of disease. Machine learning approaches can be applied to highly complex and multidimensional profiling data to reveal novel signatures of clinical use. The absence of validation in an independent cohort remains a major limitation of the study. FUNDING: Eric and Wendy Schmidt.

Machine Learning-Based Fragment Selection Improves the Performance of Qualitative PRM Assays.

Targeted mass spectrometry-based platforms have become a valuable tool for the sensitive and specific detection of protein biomarkers in clinical and research settings. Traditionally, developing a targeted assay for peptide quantification has involved manually preselecting several fragment ions and establishing a limit of detection (LOD) and a lower limit of quantitation (LLOQ) for confident detection of the target. Established thresholds such as LOD and LLOQ, however, inherently sacrifice sensitivity to afford specificity. Here, we demonstrate that machine learning can be applied to qualitative PRM assays to discriminate positive from negative samples more effectively than a traditional approach utilizing conventional methods. To demonstrate the utility of this method, we trained an ensemble machine learning model using 282 SARS-CoV-2 positive and 994 SARS-CoV-2 negative nasopharyngeal swabs (NP swab) analyzed using a targeted PRM method. This model was then validated using an independent set of 200 positive and 150 negative samples and achieved a sensitivity of 92% relative to results obtained by RT-PCR, which was superior to a traditional approach that resulted in 86.5% sensitivity when analyzing the same data. These results demonstrate that machine learning can be applied to qualitative PRM assays and results in superior performance relative to traditional methods.

Uric Acid Expression in Carotid Atherosclerotic Plaque and Serum Uric Acid Are Associated With Cerebrovascular Events.

BACKGROUND: Uric acid (UA) concentration within carotid plaque and its association with cerebrovascular events have not been detected or quantified. Systemically, serum UA is a marker of inflammation and risk factor for atherosclerosis. However, its association with carotid plaque instability and stroke pathogenesis remains unclear. In patients undergoing carotid endarterectomy, we aimed to determine whether UA is present differentially in symptomatic versus asymptomatic carotid plaques and whether serum UA is associated with cerebrovascular symptoms (stroke, transient ischemic attack, or amaurosis fugax). METHODS: Carotid atherosclerotic plaques were collected during carotid endarterectomy. The presence of UA was assessed using Gomori methenamine silver staining as well as anti-UA immunohistochemical staining and its quantity measured using an enzymatic colorimetric assay. Clinical information was obtained through a retrospective review of data. RESULTS: UA was more commonly detected in symptomatic (n=23) compared with asymptomatic (n=9) carotid plaques by Gomori methenamine silver (20 [86.9%] versus 2 [22.2%]; P=0.001) and anti-UA immunohistochemistry (16 [69.5%] versus 1 [11.1%]; P=0.004). UA concentration was higher in symptomatic rather than asymptomatic plaques (25.1 [9.5] versus 17.9 [3.8] µg/g; P=0.021). Before carotid endarterectomy, serum UA levels were higher in symptomatic (n=341) compared with asymptomatic (n=146) patients (5.9 [interquartile range, 4.6-6.9] mg/dL versus 5.2 [interquartile range, 4.6-6.2] mg/dL; P=0.009). CONCLUSIONS: The current study supports a potential role of UA as a potential tissue participant and a systemic biomarker in the pathogenesis of carotid atherosclerosis. UA may provide a mechanistic explanation for plaque instability and subsequent ischemic cerebrovascular events.

Rhamnose Is Superior to Mannitol as a Monosaccharide in the Dual Sugar Absorption Test: A Prospective Randomized Study in Children With Treatment-Naïve Celiac Disease.

Background and Aim: We sought to correlate two different measures of gut permeability [lactulose:mannitol (L:M) and lactulose:rhamnose (L:R)] to the severity of duodenal histopathology in children with and without elevated antibodies to tissue transglutaminase (tTG). A secondary objective was to correlate gut permeability with celiac disease (CD) serology and indices of inflammation and bacterial product translocation. Methods: We prospectively randomized children undergoing endoscopy with abnormal (n = 54) and normal (n = 10) concentrations of circulating antibodies to tTG, to either L:M or L:R. Biopsies underwent modified Marsh scoring to measure mucosal injury. Circulating anticore Escherichia coli lipopolysaccharide (LPS) IgG, α-1 acid glycoprotein, LPS-binding protein, and C-reactive protein concentrations were measured by enzyme immunoassays. Results: Of the 54 cases with positive celiac serology, 31 and 69% had modified Marsh 0/1 scores or ≥3a, respectively. Circulating tTG IgA correlated with the modified Marsh score (p = 0.03). L:R, but not L:M or percent L excreted, differed according to modified Marsh scores (p = 0.01). There was no significant association between any systemic marker of inflammation or gut injury, and modified Marsh scores. Concerningly, most participants had evidence of urinary M before the challenge sugar was administered. Conclusions: L:R, but not L:M, is associated with modified Marsh scores in children undergoing small bowel biopsy for suspected CD. Despite increased intestinal permeability, we see scant evidence of systemic exposure to gut microbes in these children. Gut permeability testing with L:R may predict which patients with abnormal celiac serology will have biopsy evidence for celiac disease and reduce the proportion of such patients undergoing endoscopy whose Marsh scores are ≤1. M should not be used as a monosaccharide for permeability testing in children.

High-Resolution Mass Spectrometry for the Measurement of PTH and PTH Fragments: Insights into PTH Physiology and Bioactivity.

Full-length parathyroid hormone (PTH 1-84) is crucial for the regulation of calcium and phosphate homeostasis and bone remodeling. PTH 1-84 is metabolized into various PTH fragments, which are measured with varying levels of efficiency by PTH immunoassays. These PTH fragments, which increase in serum as CKD progresses, could potentially modulate the effects of PTH 1-84 and contribute to CKD-associated bone disorders. To obtain a true biologic representation of total PTH bioactivity, it is necessary to measure not only PTH 1-84 but also PTH fragments that are present in circulation. Traditional second-generation PTH immunoassays collectively measure PTH 1-84, PTH fragments, and post-translationally modified PTH 1-84, making it difficult to accurately predict the character of underlying renal osteodystrophy. This review highlights current advances in methods available for PTH measurement and the clinical relevance of PTH fragments in CKD. We emphasize the usefulness of mass spectrometry as a potential reference method for PTH measurement.