Analytical validation of a novel multi-target blood-based test to detect hepatocellular carcinoma.

INTRODUCTION: Surveillance is essential to diagnose and more effectively treat hepatocellular carcinoma (HCC) in at-risk patients. However, the performance of currently recommended surveillance strategies is suboptimal, particularly for early-stage detection, and patient adherence remains low. Here, we establish the analytical performance of a novel liquid biopsy test to evaluate the presence of HCC. METHODS: The multi-target HCC blood test (mt-HBT) integrates results from three DNA methylation markers (HOXA1, TSPYL5, and B3GALT6), the protein biomarker α-fetoprotein (AFP), and patient sex. The methylation markers are quantified from cell-free DNA extracted from plasma, and AFP is measured from serum. We conducted analytical validation studies on the mt-HBT, including analytical sensitivity, linearity, cross-contamination, interference, analytical accuracy, and precision. RESULTS: The mt-HBT performance met all pre-specified analytical performance criteria. The test demonstrated high reproducibility, with ≥97% concordance relative to the expected results for six categories of surrogate samples across the test's dynamic range. Of 17 candidate interfering substances, none caused significant interference to biomarker quantitation, and no occurrences of sample-to-sample cross-contamination were observed. CONCLUSION: These data demonstrate that the mt-HBT can produce consistent, reliable results for patients in the intended-use population, for whom surveillance is recommended.

TR-FRET cellular assays for interrogating posttranslational modifications of histone H3.

Posttranslational modifications such as phosphorylation, acetylation, and methylation play important roles in regulating the structures and functions of histones, which in turn regulate gene expression and DNA repair and replication. Histone-modifying enzymes, such as deacetylases, methyltransferases and demethylases, have been pursued as therapeutic targets for various diseases. However, detection of the activities of these enzymes in high-throughput cell-based formats has remained challenging. The authors have developed high-throughput LanthaScreen cellular assays for Histone H3 site-specific modifications. These assays use cells expressing green fluorescence protein-tagged Histone H3 transiently delivered via BacMam and terbium-labeled anti-Histone H3 modification-specific antibodies. Robust time-resolved Förster resonance energy transfer signals were detected for H3 lysine-9 acetylation and dimethylation (H3K9me2), serine-10 phosphorylation, K4 di- and trimethylation, and K27 trimethylation. Consistent with previous reports, hypoxic stress increased K4 methylation levels, and methyltransferase G9a inhibitor UNC-0638 decreased K9me2 levels significantly, with little effects on other modifications. To demonstrate the utility of this assay platform in screening, the K9 acetylation assay was used to profile the Enzo Epigenetics Library. Twelve known HDAC inhibitors were identified as hits and followed up in a dose-response format. In conclusion, this assay platform enables high-throughput cell-based analysis of diverse types of posttranslational modifications of Histone H3.

TR-FRET biochemical assays for detecting posttranslational modifications of p53.

The p53 tumor suppressor protein plays a pivotal role in suppressing oncogenesis by regulating a range of cellular functions, including DNA repair, cell growth, cell cycle progression, and cellular death. A network of different pathways converge upon p53, ultimately regulating the response of the tumor suppressor protein by posttranslational modifications. The authors have developed a time-resolved fluorescence resonance energy transfer (TR-FRET)-based high-throughput compatible assay to analyze the critical posttranslational modifications of p53, including phosphorylation, acetylation, and ubiquitination. By using full-length p53 protein fused with GFP (GFP-p53) as the substrate, they were able to measure all 3 different posttranslational modifications with a single substrate. In addition, with a few additional steps, the GFP-p53 substrate can also be used to assay deacetylation to aid in the discovery of inhibitors for sirtuins or other deacetylase enzymes. The flexibility of the assay to measure a diverse range of posttranslational modifications allows one to further dissect the complex regulating mechanisms of p53 and enable the discovery of specific inhibitors for these processes.

Cellular assays for high-throughput screening for modulators of Trk receptor tyrosine kinases.

Trk receptor tyrosine kinases are required for signal transduction initiated by neurotrophins leading to cell proliferation, differentiation, survival and death. Alterations in Trk kinase activity have been linked to various diseases. To address the need for cell-based assays for screening and studying the selectivity of Trk kinase modulators, we developed high-throughput cell-based assays for Trk receptor kinases using nuclear factor of activated T-cells (NFAT) beta-lactamase reporter lines stably expressing full length human Trk kinases. These assays were functionally validated with cognate neurotrophin(s), inhibitors and TRK RNAi oligos and demonstrated for their utility in identifying potent and selective modulators of Trk receptor kinases.