Utility of Commercially Available Quantitative hCG Immunoassays as Tumor Markers in Trophoblastic and Non-Trophoblastic Disease.

BACKGROUND: The use of quantitative human chorionic gonadotropin (hCG) as a tumor marker is widely accepted despite lack of FDA-approval for oncology. Differences in iso- and glycoform recognition among hCG immunoassays is well established, exhibiting wide inter-method variability. Here, we assess the utility of 5 quantitative hCG immunoassays for use as tumor markers in trophoblastic and non-trophoblastic disease. METHODS: Remnant specimens were obtained from 150 patients with gestational trophoblastic disease (GTD), germ cell tumors (GCT), or other malignancies. Specimens were identified by review of results from physician-ordered hCG and tumor marker testing. Five analyzer platforms were used for split specimen analysis of hCG: Abbott Architect Total, Roche cobas STAT, Roche cobas Total, Siemens Dimension Vista Total, and Beckman Access Total. RESULTS: Frequency of elevated hCG concentrations (above reference cutoffs) was highest in GTD (100%), followed by GCT (55% to 57%), and other malignancies (8% to 23%). Overall, the Roche cobas Total detected elevated hCG in the greatest number of specimens (63/150). Detection of elevated hCG in trophoblastic disease was nearly equivalent among all immunoassays (range, 41 to 42/60). CONCLUSIONS: While no immunoassay is likely to be perfect in all clinical situations, results for the 5 hCG immunoassays evaluated suggest that all are adequate for use of hCG as a tumor marker in gestational trophoblastic disease and select germ cell tumors. Further harmonization of hCG methods is needed as serial testing for biochemical tumor monitoring must still be performed using a single method. Additional studies are needed to assess the utility of quantitative hCG as a tumor marker in other malignant disease.

Reprogramming fatty acyl specificity of lipid kinases via C1 domain engineering.

C1 domains are lipid-binding modules that regulate membrane activation of kinases, nucleotide exchange factors and other C1-containing proteins to trigger signal transduction. Despite annotation of typical C1 domains as diacylglycerol (DAG) and phorbol ester sensors, the function of atypical counterparts remains ill-defined. Here, we assign a key role for atypical C1 domains in mediating DAG fatty acyl specificity of diacylglycerol kinases (DGKs) in live cells. Activity-based proteomics mapped C1 probe binding as a principal differentiator of type 1 DGK active sites that combined with global metabolomics revealed a role for C1s in lipid substrate recognition. Protein engineering by C1 domain swapping demonstrated that exchange of typical and atypical C1s is functionally tolerated and can directly program DAG fatty acyl specificity of type 1 DGKs. Collectively, we describe a protein engineering strategy for studying metabolic specificity of lipid kinases to assign a role for atypical C1 domains in cell metabolism.

Association between SARS-CoV-2 Neutralizing Antibodies and Commercial Serological Assays.

BACKGROUND: Commercially available SARS-CoV-2 serological assays based on different viral antigens have been approved for the qualitative determination of anti-SARS-CoV-2 antibodies. However, there are limited published data associating the results from commercial assays with neutralizing antibodies. METHODS: Sixty-six specimens from 48 patients with PCR-confirmed COVID-19 and a positive result by the Roche Elecsys Anti-SARS-CoV-2, Abbott SARS-CoV-2 IgG, or EUROIMMUN SARS-CoV-2 IgG assays and 5 control specimens were analyzed for the presence of neutralizing antibodies to SARS-CoV-2. Correlation, concordance, positive percent agreement (PPA), and negative percent agreement (NPA) were calculated at several cutoffs. Results were compared in patients categorized by clinical outcomes. RESULTS: The correlation between SARS-CoV-2 neutralizing titer (EC50) and the Roche, Abbott, and EUROIMMUN assays was 0.29, 0.47, and 0.46, respectively. At an EC50 of 1:32, the concordance kappa with Roche was 0.49 (95% CI; 0.23-0.75), with Abbott was 0.52 (0.28-0.77), and with EUROIMMUN was 0.61 (0.4-0.82). At the same neutralizing titer, the PPA and NPA for the Roche was 100% (94-100) and 56% (30-80); Abbott was 96% (88-99) and 69% (44-86); and EUROIMMUN was 91% (80-96) and 81% (57-93) for distinguishing neutralizing antibodies. Patients who were intubated, had cardiac injury, or acute kidney injury from COVID-19 infection had higher neutralizing titers relative to those with mild symptoms. CONCLUSIONS: COVID-19 patients generate an antibody response to multiple viral proteins such that the calibrator ratios on the Roche, Abbott, and EUROIMMUN assays are all associated with SARS-CoV-2 neutralization. Nevertheless, commercial serological assays have poor NPA for SARS-CoV-2 neutralization, making them imperfect proxies for neutralization.

Deconstructing Lipid Kinase Inhibitors by Chemical Proteomics.

Diacylglycerol kinases (DGKs) regulate lipid metabolism and cell signaling through ATP-dependent phosphorylation of diacylglycerol to biosynthesize phosphatidic acid. Selective chemical probes for studying DGKs are currently lacking and are needed to annotate isoform-specific functions of these elusive lipid kinases. Previously, we explored fragment-based approaches to discover a core fragment of DGK-α (DGKα) inhibitors responsible for selective binding to the DGKα active site. Here, we utilize quantitative chemical proteomics to deconstruct widely used DGKα inhibitors to identify structural regions mediating off-target activity. We tested the activity of a fragment (RLM001) derived from a nucleotide-like region found in the DGKα inhibitors R59022 and ritanserin and discovered that RLM001 mimics ATP in its ability to broadly compete at ATP-binding sites of DGKα as well as >60 native ATP-binding proteins (kinases and ATPases) detected in cell proteomes. Equipotent inhibition of activity-based probe labeling by RLM001 supports a contiguous ligand-binding site composed of C1, DAGKc, and DAGKa domains in the DGKα active site. Given the lack of available crystal structures of DGKs, our studies highlight the utility of chemical proteomics in revealing active-site features of lipid kinases to enable development of inhibitors with enhanced selectivity against the human proteome.

Chemoproteomic Discovery of a Ritanserin-Targeted Kinase Network Mediating Apoptotic Cell Death of Lung Tumor Cells.

Ritanserin was tested in the clinic as a serotonin receptor inverse agonist but recently emerged as a novel kinase inhibitor with potential applications in cancer. Here, we discovered that ritanserin induced apoptotic cell death of non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) cells via a serotonin-independent mechanism. We used quantitative chemical proteomics to reveal a ritanserin-dependent kinase network that includes key mediators of lipid [diacylglycerol kinase α, phosphatidylinositol 4-kinase ß] and protein [feline encephalitis virus-related kinase, rapidly accelerated fibrosarcoma (RAF)] signaling, metabolism [eukaryotic elongation factor 2 kinase, eukaryotic translation initiation factor 2-α kinase 4], and DNA damage response [tousled-like kinase 2] to broadly kill lung tumor cell types. Whereas ritanserin exhibited polypharmacology in NSCLC proteomes, this compound showed unexpected specificity for c-RAF in the SCLC subtype, with negligible activity against other kinases mediating mitogen-activated protein kinase signaling. Here we show that ritanserin blocks c-RAF but not B-RAF activation of established oncogenic signaling pathways in live cells, providing evidence in support of c-RAF as a key target mediating its anticancer activity. Given the role of c-RAF activation in RAS-mutated cancers resistant to clinical B-RAF inhibitors, our findings may have implications in overcoming resistance mechanisms associated with c-RAF biology. The unique target landscape combined with acceptable safety profiles in humans provides new opportunities for repositioning ritanserin in cancer.

Increased specimen minimum volume reduces turnaround time and hemolysis.

Quantity not sufficient (QNS) specimens with minimal blood volume for testing are common in clinical laboratories. However, there is no universal definition of minimum volume for a QNS specimen and little data is available addressing the impact of QNS / low volume specimens on turnaround time (TAT) and sample hemolysis. We compared the TAT and hemolysis index from samples ≤1.0 mL to all specimens received and quantified the number of specimens with reduced blood volume. A new QNS policy requiring ≥1.5 mL of sample in a blood tube for laboratory analysis was implemented and the results were assessed by sample hemolysis and TAT. The median laboratory TAT for samples with ≤1.0 mL of blood was 61 min (Interquartile Range, IQR: 50-82), in contrast to 28 min (26-34) for all samples. The hemolysis index for samples ≤1.0 mL was 112 (65-253) and 15 (8-29) for all samples. Requirement of a minimum volume of 1.5 mL of blood resulted in the proportion of samples with TAT ≥ 60 min to decrease from 10.4% to 4.24% in the ED, and for specimens cancelled due to hemolysis to decrease from 4.24% to 3.38%. This policy was introduced hospital wide with similar effects. Together, we correlate limited specimen volume with an increase in laboratory TAT and hemolysis. Implementation of a QNS policy of ≥1.5 mL and provider education provided a significant and durable reduction in TAT and specimen hemolysis.

Comparison between BNP and NT-proBNP in pediatric populations.

BACKGROUND: B-type natriuretic peptide (BNP) and N-terminal pro-B-type natriuretic peptide (NT-proBNP) are essential biomarkers for the evaluation of cardiac pathologies. However, pediatric reference intervals for BNP and NT-proBNP are not well defined and concordance between them in the evaluation of pediatric patients has been poorly described. METHODS: Paired BNP and NT-proBNP testing was performed on 311 specimens representing 175 pediatric patients. Pediatric BNP and NT-proBNP reference intervals derived from the literature were used to evaluate concordance of results based on age group and cardiac pathology. RESULTS: Deming regression analysis of BNP and NT-proBNP results revealed a slope of 13.63 (95% CI, 10.35-16.92) and y-intercept of -977.8 (-2063-107.2) with a positive Spearman correlation (r = 0.91). By age group, concordance kappa between BNP and NT-proBNP was 1.0 for 0-10 days, 0.23 (0-0.62) for 11-30 days, 0.82 (0.67-0.97) for 31 days-1 year, 0.81 (0.57-1.0) for 1-2 years and 0.73 (0.64-0.86) for 2-18 years. The ratio of NT-proBNP to BNP was lowest in heart transplant patients (ratio, 6.5 [95% CI, 5.1-8.1]) relative to those with heart disease (10.5 [8.8-13.7]) and pulmonary hypertension (14.2 [11.3-16.0]) but no differences in concordance were observed. For serial specimens, 21% displayed inverse, discordant changes in BNP and NT-proBNP results. Review of discordant serial results revealed that kinetics of changes was comparable and unlikely to be clinically significant. CONCLUSIONS: There is positive correlation and moderate concordance between BNP and NT-proBNP in the pediatric population studied.

On the Basis of Race: The Utility of a Race Factor in Estimating Glomerular Filtration.

BACKGROUND: Glomerular filtration rate (GFR) is a measure of the combined rate of filtration of all functional nephrons in the kidney. Measurement of GFR is used in the clinic to detect, stratify, and monitor progression of kidney dysfunction, and also serves as a prognostic tool for staging chronic kidney disease (CKD). The gold standard method for measuring GFR is by plasma or urine clearance of exogenous filtration markers, but this is not feasible in routine clinical practice. The most commonly used method to assess GFR is using equations for estimated GFR (eGFR). CONTENT: Addition of a race factor to eGFR equations has been recommended to optimize performance for Black individuals. Here, we review the basis of the race-based equation and assess its utility and widespread applicability. SUMMARY: Although evidence supporting the performance of a race factor exists in the unique populations in which these estimation equations were derived, more studies are needed to assess the need, or lack thereof, for race factors for all ethnicities. Furthermore, ethnicity is complex and likely cannot be qualified with a 2-level descriptor.

Detecting Fentanyl Analogs in Urine Using Precursor Ion Scan Mode.

The opioid crisis has led many providers to inquire about the capabilities of urine drug testing to detect contemporary compounds such as fentanyl and fentanyl analogs. However, current methods for clinical urine drug testing, including immunoassays and targeted liquid chromatography tandem mass spectrometry, are not designed to broadly screen for the variety of fentanyl analogs that may be encountered. In this proof-of-principle study we developed a precursor ion scan method to enable semi-targeted data acquisition for structurally related fentanyl analogs. Based on the knowledge that many analogs fragment to m/z=188 and m/z=105, data was acquired on all precursor ions 250-400 Da that fragmented to these product ions. Using a tandem mass spectrometer we analyzed 102 residual urine specimens, in which we identified fentanyl, acetylfentanyl and acrylfentanyl. In 30 contrived urine samples, the precursor ion scan was also able to identify furanylfentanyl, butryrlfentanyl, 4-fluroisobutrylfentanyl, and despropionylfentanyl with accuracy ranging from 83-100%.

A Comparison of Four 3-Axis-Accelerometers for Monitoring Hospital Pneumatic Tube Systems.

BACKGROUND: Validation of hospital pneumatic tube systems (PTS) is recommended to predict and prevent errors caused by sample hemolysis. 3-Axis accelerometer dataloggers have been successfully implemented as tools for PTS validation, but the most suitable device for such validation has not been investigated. The aim of this study was to evaluate the performance of four commercially available 3-axis accelerometers for PTS validation. METHODS: PCE-VD3 (PCE), CEM DT-178A (CEM), Extech VB300 (EXT), and MSR 145 (MSR) dataloggers were placed into a single PTS carrier and repeatedly transported through one of three PTS routes. The number and magnitude of accelerations within each PTS route was collected by each device. Deming regression analysis was used to compare device performance. RESULTS: The MSR datalogger captured the greatest number of g-forces >3 g, 5 g, 10 g, and 15 g, and the greatest magnitude of g-force (26.7 g) relative to the other devices (CEM: 23.0 g, EXT: 23.3 g, PCE: 23.7 g). As a result of increased sampling frequency, the MSR recorded the lowest AUC and the greatest number of g-forces exceeding 3 g relative to the other devices. Subjectively, the data were difficult to extract from 4 tested devices. CONCLUSIONS: Commercially available dataloggers differ in their ability to detect the number and magnitude of g-forces within PTSs. We recommend that one device be used to perform all PTS evaluations, with baseline evaluations for tolerable AUC, number, and magnitude of g-forces established internally. Lack of harmonization, cumbersome data processing, and time-consuming data analysis are substantial barriers to universal implementation of dataloggers for PTS validation and monitoring.

Comparison of Two Commercially Available Fentanyl Screening Immunoassays for Clinical Use.

BACKGROUND: Fentanyl is a synthetic opioid associated with illicit drug use and overdose deaths. The SEFRIA Immunalysis (IAL) and ARK fentanyl assays are both FDA-cleared, open channel immunoassays for fentanyl detection in urine. However, limited data are available in the literature comparing these assays. The objective of this study was to perform a direct comparison of these two fentanyl immunoassays. METHODS: IAL and ARK fentanyl immunoassays were performed on a Roche Cobas e602 automated chemistry analyzer. Repeatability and total imprecision were compared by diluting fentanyl into urine at concentrations above, below, and at the manufacturers' cutoffs of 1.0 ng/mL. Cross-reactivity was assessed for norfentanyl and the fentanyl analogs acetylfentanyl, acrylfentanyl, and furanylfentanyl. Concordance was assessed in 90 patient samples using liquid chromatography-tandem mass spectrometry (LC-MS/MS) as the gold standard. RESULTS: Repeatability varied from 11.4%-17.8% on the IAL assay and 2.8%-5.5% on the ARK assay. Total imprecision was 18.9%-40.7% on the IAL assay and 2.9%-6.4% on the ARK assay. Both assays cross-reacted with acetylfentanyl (∼100%), acrylfentanyl (∼100%), and furanylfentanyl (∼20%), but only the ARK assay cross-reacted with norfentanyl (∼3%). An admixture of 0.5 ng/mL fentanyl and 6 ng/mL norfentanyl produced a positive result on the ARK assay. Total concordance between IAL and ARK for 90 tested patient samples was 93% (kappa = 0.85). Relative to LC-MS/MS, the IAL assay had a concordance of 90% (kappa = 0.79) and the ARK assay had a concordance of 94% (kappa = 0.88). Including norfentanyl in the LC-MS/MS confirmation increased the concordance of the ARK to 96% (kappa = 0.90). CONCLUSIONS: The ARK assay recognized the metabolite norfentanyl, demonstrated superior precision, and had better concordance with LC-MS/MS compared to the IAL assay.

Activity-Based Kinome Profiling Using Chemical Proteomics and ATP Acyl Phosphates.

Human kinases are a large family of proteins (500+) that catalyze ATP-dependent phosphorylation of protein and metabolite substrates to regulate diverse facets of cell biology. Dysregulation and mutations of protein kinases are linked to human disease, providing opportunities for developing pharmacological agents as potential therapy. Assessing the selectivity of pharmacological compounds targeting this enzyme class is critical given that off-target activity of kinase inhibitor drugs may result in toxicity. This set of protocols outlines use of ATP acyl phosphate activity-based probes to evaluate the potency and selectivity of kinase inhibitors via fluorescent gel- and mass spectrometry-based detection methods. These competitive chemical proteomic assays can evaluate engagement of >200 native kinase targets directly in complex proteomes. © 2019 by John Wiley & Sons, Inc.

Isoform-selective activity-based profiling of ERK signaling.

Extracellular signal-regulated kinases (ERKs) mediate downstream signaling of RAS-RAF-MEK as key regulators of the mitogen-activated protein kinase (MAPK) pathway. Activation of ERK signaling is a hallmark of cancer and upstream MAPK proteins have been extensively pursued as drug targets for cancer therapies. However, the rapid rise of resistance to clinical RAF and MEK inhibitors has prompted interest in targeting ERK (ERK1 and ERK2 isoforms) directly for cancer therapy. Current methods for evaluating activity of inhibitors against ERK isoforms are based primarily on analysis of recombinant proteins. Strategies to directly and independently profile native ERK1 and ERK2 activity would greatly complement current cell biological tools used to probe and target ERK function. Here, we present a quantitative chemoproteomic strategy that utilizes active-site directed probes to directly quantify native ERK activity in an isoform-specific fashion. We exploit a single isoleucine/leucine difference in ERK substrate binding sites to enable activity-based profiling of ERK1 versus ERK2 across a variety of cell types, tissues, and species. We used our chemoproteomic strategy to determine potency and selectivity of academic (VX-11e) and clinical (Ulixertinib) ERK inhibitors. Correlation of potency estimates by chemoproteomics with anti-proliferative activity of VX-11e and Ulixertinib revealed that >90% inactivation of both native ERK1 and ERK2 is needed to mediate cellular activity of inhibitors. Our findings introduce one of the first assays capable of independent evaluation of native ERK1 and ERK2 activity to advance drug discovery of oncogenic MAPK pathways.

The Ligand Binding Landscape of Diacylglycerol Kinases.

Diacylglycerol kinases (DGKs) are integral components of signal transduction cascades that regulate cell biology through ATP-dependent phosphorylation of the lipid messenger diacylglycerol. Methods for direct evaluation of DGK activity in native biological systems are lacking and needed to study isoform-specific functions of these multidomain lipid kinases. Here, we utilize ATP acyl phosphate activity-based probes and quantitative mass spectrometry to define, for the first time, ATP and small-molecule binding motifs of representative members from all five DGK subtypes. We use chemical proteomics to discover an unusual binding mode for the DGKα inhibitor, ritanserin, including interactions at the atypical C1 domain distinct from the ATP binding region. Unexpectedly, deconstruction of ritanserin yielded a fragment compound that blocks DGKα activity through a conserved binding mode and enhanced selectivity against the kinome. Collectively, our studies illustrate the power of chemical proteomics to profile protein-small molecule interactions of lipid kinases for fragment-based lead discovery.