Pre-analytical considerations in the development of a prototype SARS-CoV-2 antigen ARCHITECT automated immunoassay.

OBJECTIVES: To evaluate pre-analytical challenges related to high-volume central laboratory SARS-CoV-2 antigen testing with a prototype qualitative SARS-CoV-2 antigen immunoassay run on the automated Abbott ARCHITECT instrument. METHODS: Contrived positive and negative specimens and de-identified nasal and nasopharyngeal specimens in transport media were used to evaluate specimen and reagent on-board stability, assay analytical performance and interference, and clinical performance. RESULTS: TCID50/mL values were similar for specimens in various transport media. Inactivated positive clinical specimens and viral lysate (USA-WA1/2020) were positive on the prototype immunoassay. Within-laboratory imprecision was ≤0.10 SD (<1.00 S/C) with a ≤10% CV (≥1.00 S/C). Assay reagents were stable on board the instrument for 14 days. No high-dose hook effect was observed with a SARS-CoV-2 stock of Ct 13.0 (RLU>1.0 × 106). No interference was observed from mucin, whole blood, 12 drugs, and more than 20 cross-reactants. While specimen stability was limited at room temperature for specimens with or without viral inactivation, a single freeze/thaw cycle or long-term storage (>30 days) at -20 °C did not adversely impact specimen stability or assay performance. Specificity of the prototype SARS-CoV-2 antigen immunoassay was ≥98.5% and sensitivity was ≥89.5% across two ARCHITECT instruments. Assay sensitivity was inversely correlated with Ct and was similar to that reported for the Roche Elecsys® SARS-CoV-2 Ag immunoassay. CONCLUSIONS: The prototype SARS-CoV-2 antigen ARCHITECT immunoassay is sensitive and specific for detection of SARS-CoV-2 in nasal and nasopharyngeal specimens. Endogenous proteases in mucus may degrade the target antigen, which limits specimen storage and transport times and complicates assay workflow.

Development of the Abbott RealTime ZIKA assay for the qualitative detection of Zika virus RNA from serum, plasma, urine, and whole blood specimens using the m2000 system.

Zika virus is an arthropod-borne flavivirus that has rapidly developed into a world-wide concern. Discovered in 1947, the virus was relatively obscure until an outbreak occurred in 2007 in the Yap islands and spread eventually to the Americas in 2015. Only 20% of patients infected with Zika virus develop symptoms. However, there can be serious consequences of infection including birth defects in developing fetuses and links to Guillain-Barré syndrome. The swift rise in infections has necessitated the development of diagnostic tests for both the detection of viral RNA and the presence of virus-specific antibodies. Abbott has developed a dual target RT-PCR assay for the detection of Zika virus RNA within serum, plasma, whole blood, and urine using the automated m2000 system for sample extraction to result reporting. The Abbott RealTime ZIKA assay has a limit of detection of 30 copies per mL in serum, 40 copies per mL in plasma and urine, and 120 copies per mL in whole blood and demonstrates high specificity against challenges from closely related infectious agents.

PKCα Attenuates Jagged-1-Mediated Notch Signaling in ErbB-2-Positive Breast Cancer to Reverse Trastuzumab Resistance.

PURPOSE: Breast cancer is the second leading cause of cancer mortality among women worldwide. The major problem with current treatments is tumor resistance, recurrence, and disease progression. ErbB-2-positive breast tumors are aggressive and frequently become resistant to trastuzumab or lapatinib. We showed previously that Notch-1 is required for trastuzumab resistance in ErbB-2-positive breast cancer. EXPERIMENTAL DESIGN: Here, we sought to elucidate mechanisms by which ErbB-2 attenuates Notch signaling and how this is reversed by trastuzumab or lapatinib. RESULTS: The current study elucidates a novel Notch inhibitory mechanism by which PKCα downstream of ErbB-2 (i) restricts the availability of Jagged-1 at the cell surface to transactivate Notch, (ii) restricts the critical interaction between Jagged-1 and Mindbomb-1, an E3 ligase that is required for Jagged-1 ubiquitinylation and subsequent Notch activation, (iii) reverses trastuzumab resistance in vivo, and (iv) predicts better outcome in women with ErbB-2-positive breast cancer. CONCLUSIONS: The clinical impact of these studies is PKCα is potentially a good prognostic marker for low Notch activity and increased trastuzumab sensitivity in ErbB-2-positive breast cancer. Moreover, women with ErbB-2-positive breast tumors expressing high Notch activation and low PKCα expression could be the best candidates for anti-Notch therapy.

Clinical characteristics associated with ALK rearrangements in never-smokers with pulmonary adenocarcinoma.

OBJECTIVES: Anaplastic lymphoma kinase (ALK) rearrangement is a validated predictive marker to define patients with non-small cell lung cancer (NSCLC) who can benefit from selective ALK inhibitors. Therefore, accurate assessment of its prevalence and clinical characteristics is increasingly important in the treatment of NSCLC. Also, this ALK rearrangement was previously reported to be more common in patients with no smoking history or those with adenocarcinoma. PATIENTS AND METHODS: Never-smokers with completely resected pulmonary adenocarcinoma were screened for ALK rearrangements using Nanostring's gene expression platform. Clinicopathologic data, such as information about epidermal growth factor receptor (EGFR) and KRAS mutation status were retrospectively reviewed. RESULTS: Of 231 tumors screened, 20 (9%) had an ALK rearrangement and all were confirmed to be positive with immunohistochemical and fluorescent in situ hybridization analysis. Of the tumors with available data on the EGFR/KRAS mutation status, EGFR and KRAS mutation rates were 64% (69/108) and 5% (5/102), respectively. Amongst the tumors that were free of EGFR and KRAS mutations, the proportion of ALK rearrangements reached up to 33%. At the time of data cut-off, total of 68 tumors were recurred. Although the recurrence rate was similar between the ALK-positive and negative groups (30% vs. 29%), there was a tendency for ALK-positive tumors to recur more frequently in the pleural space (15% vs. 5%). The five-year disease-free survival (61%) and overall survival rates (79%) in the ALK-positive group were similar to those in the ALK-negative group (51% and 83%, respectively). Even after excluding two patients treated with crizotinib after disease recurrence, overall survival was similar between the two groups. CONCLUSION: In an NSCLC subpopulation based on smoking history, histology, and EGFR and KRAS mutation status, the prevalence of ALK rearrangements is considerably high. However, ALK rearrangement status itself has no prognostic relevance in patients with completely resected NSCLC.

Laboratory automation of high-quality and efficient ligand-binding assays for biotherapeutic drug development.

BACKGROUND: Regulated bioanalytical laboratories that run ligand-binding assays in support of biotherapeutics development face ever-increasing demand to support more projects with increased efficiency. Laboratory automation is a tool that has the potential to improve both quality and efficiency in a bioanalytical laboratory. The success of laboratory automation requires thoughtful evaluation of program needs and fit-for-purpose strategies, followed by pragmatic implementation plans and continuous user support. RESULTS: In this article, we present the development of fit-for-purpose automation of total walk-away and flexible modular modes. We shared the sustaining experience of vendor collaboration and team work to educate, promote and track the use of automation. CONCLUSION: The implementation of laboratory automation improves assay performance, data quality, process efficiency and method transfer to CRO in a regulated bioanalytical laboratory environment.

Specific method validation and sample analysis approaches for biocomparability studies of denosumab addressing method and manufacture site changes.

Manufacturing changes during a biological drug product life cycle occur often; one common change is that of the manufacturing site. Comparability studies may be required to ensure that the changes will not affect the pharmacokinetic properties of the drug. In addition, the bioanalytical method for sample analysis may evolve during the course of drug development. This paper illustrates the scenario of both manufacturing and bioanalytical method changes encountered during the development of denosumab, a fully human monoclonal antibody which inhibits bone resorption by targeting RANK Ligand. Here, we present a rational approach to address the bioanalytical method changes and provide considerations for method validation and sample analysis in support of biocomparability studies. An updated and improved ELISA method was validated, and its performance was compared to the existing method. The analytical performances, i.e., the accuracy and precision of standards and validation samples prepared from both manufacturing formulation lots, were evaluated and found to be equivalent. One of the lots was used as the reference standard for sample analysis of the biocomparability study. This study was sufficiently powered using a parallel design. The bioequivalence acceptance criteria for small molecule drugs were adopted. The pharmacokinetic parameters of the subjects dosed with both formulation lots were found to be comparable.

NOTCH-1 and NOTCH-4 are novel gene targets of PEA3 in breast cancer: novel therapeutic implications.

INTRODUCTION: Women with triple-negative breast cancer have the worst prognosis, frequently present with metastatic tumors and have few targeted therapy options. Notch-1 and Notch-4 are potent breast oncogenes that are overexpressed in triple-negative and other subtypes of breast cancer. PEA3, an ETS transcription factor, is also overexpressed in triple-negative and other breast cancer subtypes. We investigated whether PEA3 could be the critical transcriptional activator of Notch receptors in MDA-MB-231 and other breast cancer cells. METHODS: Real-time PCR and Western blot analysis were performed to detect Notch-1, Notch-2, Notch-3 and Notch-4 receptor expression in breast cancer cells when PEA3 was knocked down by siRNA. Chromatin immunoprecipitation was performed to identify promoter regions for Notch genes that recruited PEA3. TAM-67 and c-Jun siRNA were used to identify that c-Jun was necessary for PEA3 enrichment on the Notch-4 promoter. A Notch-4 luciferase reporter was used to confirm that endogenous PEA3 or AP-1 activated the Notch-4 promoter region. Cell cycle analysis, trypan blue exclusion, annexin V flow cytometry, colony formation assay and an in vivo xenograft study were performed to determine the biological significance of targeting PEA3 via siRNA, Notch signaling via a γ-secretase inhibitor, or both. RESULTS: Herein we provide new evidence for transcriptional regulation of Notch by PEA3 in breast cancer. PEA3 activates Notch-1 transcription in MCF-7, MDA-MB-231 and SKBr3 breast cancer cells. PEA3 activates Notch-4 transcription in MDA-MB-231 cells where PEA3 levels are endogenously high. In SKBr3 and BT474 breast cancer cells where PEA3 levels are low, overexpression of PEA3 increases Notch-4 transcripts. Chromatin immunoprecipitation confirmed the enrichment of PEA3 on Notch-1 and Notch-4 promoters in MDA-MB-231 cells. PEA3 recruitment to Notch-1 was AP-1-independent, whereas PEA3 recruitment to Notch-4 was c-JUN-dependent. Importantly, the combined inhibition of Notch signaling via a γ-secretase inhibitor (MRK-003 GSI) and knockdown of PEA3 arrested growth in the G1 phase, decreased both anchorage-dependent and anchorage-independent growth and significantly increased apoptotic cells in vitro. Moreover, either PEA3 knockdown or MRK-003 GSI treatment significantly reduced tumor growth of MDA-MB-231 xenografts in vivo. CONCLUSIONS: Taken together, the results from this study demonstrate for the first time that Notch-1 and Notch-4 are novel transcriptional targets of PEA3 in breast cancer cells. Targeting of PEA3 and/or Notch pathways might provide a new therapeutic strategy for triple-negative and possibly other breast cancer subtypes.

Strategies to minimize variability and bias associated with manual pipetting in ligand binding assays to assure data quality of protein therapeutic quantification.

Bioanalytical laboratories require accurate and precise pipetting to assure reproducible and accurate results for reliable data. Two areas where pipetting differences among analysts lead to poor reproducibility are long term stability testing and sample dilution. The purpose of this paper is to illustrate the problems with manual pipetting, describe an automation strategy to mitigate risks associated with manual pipetting, and provide recommendations on a control strategy that properly monitors samples requiring dilutions. We determined differences among various manual pipetting techniques by analysts within a laboratory. To reduce variability in pipetting, a flexible modular liquid handling script was created on the Hamilton Microlab Star (HMS) to perform sample dilution, pre-treatment and plate loading. The script is capable of handling variable dilution factors. Additionally, two dilution controls were prepared and tested at concentrations of high and mid quality controls (QC). These same dilution controls were incorporated into both pre-study validation and in-study QCs to monitor dilution processing and assay performance. Variability of manual pipetting among 11 analysts was more negatively biased with increasing dilution. Forward and reverse pipetting delivering different volumes contributed to the discordance. The dilutional bias with manual pipetting was eliminated using the liquid handler. Total error of dilution controls was less than 20%. The in-study pass rate was 100%. Application of liquid handlers minimizes the variability and bias due to manual pipetting differences among analysts. The incorporation of dilution QCs serves a dual purpose to monitor the dilution process of the samples as well as the binding assay performance.

Protection of early phase hepatic ischemia-reperfusion injury by cholinergic agonists.

BACKGROUND: Cytokine production is critical in ischemia/reperfusion (IR) injury. Acetylcholine binds to macrophages and inhibits cytokine synthesis, through the cholinergic anti-inflammatory pathway. This study examined the role of the cholinergic pathway in cytokine production and hepatic IR- injury. METHODS: Adult male mice underwent 90-min of partial liver ischemia followed by reperfusion. The AChR agonists (1,1-dimethyl-4-phenyl-L-pioperazinium-iodide [DMPP], and nicotine) or saline-vehicle were administered i.p. before ischemia. Plasma cytokine tumor necrosis factor (TNF)-alpha, macrophage inflammatory protein-2, and Interleukin-6 were measured. Liver injury was assessed by plasma alanine transaminase (ALT) and liver histopathology. RESULTS: A reperfusion time-dependent hepatocellular injury occurred as was indicated by increased plasma-ALT and histopathology. The injury was associated with marked elevation of plasma cytokines/chemokines. Pre-ischemic treatment of mice with DMPP or nicotine significantly decreased plasma-ALT and cytokines after 3 h of reperfusion. After 6 h of reperfusion, the protective effect of DMPP decreased and reached a negligible level by 24 h of reperfusion, despite significantly low levels of plasma cytokines. Histopathology showed markedly diminished hepatocellular injury in DMPP- and nicotine-pretreated mice during the early-phase of hepatic-IR, which reached a level comparable to saline-treated mice at late-phase of IR. CONCLUSION: Pharmacological modulation of the cholinergic pathway provides a means to modulate cytokine production and to delay IR-induced heaptocellular injury.