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Inclusive Quantification Assay of Serum Des-γ-Carboxyprothrombin Proteoforms for Hepatocellular Carcinoma Surveillance by Targeted Mass Spectrometry.

ABSTRACT: Hepatocellular carcinoma (HCC) is a malignant cancer with one of the highest mortality rates. Des-γ-carboxyprothrombin (DCP) is an HCC serologic surveillance marker that can complement the low sensitivity of alpha-fetoprotein (AFP). DCP exists in the blood as a mixture of proteoforms from an impaired carboxylation process at glutamic acid (Glu) residues within the N-terminal domain. The heterogeneity of DCP may affect the accuracy of measurements because DCP levels are commonly determined using an immunoassay that relies on antibody reactivity to an epitope in the DCP molecule. In this study, we aimed to improve the DCP measurement assay by applying a mass spectrometry (MS)-based approach for a more inclusive quantification of various DCP proteoforms. We developed a multiple-reaction monitoring-MS (MRM-MS) assay to quantify multiple noncarboxylated peptides included in the various des-carboxylation states of DCP. We performed the MRM-MS assay in 300 patients and constructed a robust diagnostic model that simultaneously monitored three noncarboxylated peptides. The MS-based quantitative assay for DCP had reliable surveillance power, which was evident from the area under the receiver operating characteristic curve (AUROC) values of 0.874 and 0.844 for the training and test sets, respectively. It was equivalent to conventional antibody-based quantification, which had AUROC values at the optimal cutoff (40 mAU/mL) of 0.743 and 0.704 for the training and test sets, respectively. The surveillance performance of the MS-based DCP assay was validated using an independent validation set consisting of 318 patients from an external cohort, resulting in an AUROC value of 0.793. Conclusion: Due to cost effectiveness and high reproducibility, the quantitative DCP assay using the MRM-MS method is superior to antibody-based quantification and has equivalent performance.

SUBMITTER: Lee J

PROVIDER: S-EPMC8485883 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Patients with hepatocellular carcinoma (HCC) who respond to sorafenib have been reported to exhibit an increase in the level of des-γ-carboxyprothrombin (DCP) in the blood, subsequent to the initiation of sorafenib treatment. In the present study, the levels of secretion of DCP and DCP with more γ-carboxyglutamic residues (NX-DCP) and the effects of hypoxic conditions were examined in 13 liver cancer cell lines, and the presence of vitamin K and sorafenib, in the KYN-2 cell line, which resulted in confirmed DCP and NX-DCP secretion. DCP, NX-DCP and prothrombin secretion were confirmed in 2/13 cell lines, KYN-2 and KIM-1. The level of secretions increased under hypoxic conditions. The addition of vitamin K suppressed cell proliferation, and DCP expression decreased to below detectable levels, however the level of prothrombin expression increased. Sorafenib treatment increased the level of apoptosis and suppressed cell proliferation, and decreased DCP and NX-DCP. In contrast, levels of prothrombin and vascular endothelial growth factor (VEGF) expression exhibited a slight increase. When the same experiment was conducted under hypoxic conditions, DCP secretion significantly decreased in the presence of sorafenib. The level of DCP secretion increased by several fold in the sorafenib-treated and non-treated cells compared with the normoxic conditions. Prothrombin and VEGF values with normoxic conditions remained almost similar with hypoxic conditions. Under hypoxic conditions, NX-DCP significantly decreased below the control values for the first 48 h subsequent to sorafenib treatment, but significantly increased at 72 h. In vivo experiments demonstrated that sorafenib inhibited angiogenesis and tumor proliferation, but the levels of DCP and NX-DCP did not differ significantly from the controls. These findings indicate that the suppression of neovascularization by sorafenib promotes blood vessel ischemia, producing hypoxic conditions whereby vitamin K uptake and utilization efficiency is reduced.

Project description:Exposure to environmental ionizing radiation (IR) is ubiquitous, and large-dose exposure to IR is known to cause DNA damage and genotoxicity which is associated with an increased risk of cancer. Whether such detrimental effects are caused by exposure to low-dose IR is still debated. Therefore, rapid and early estimation of absorbed doses of IR in individuals, especially at low levels, using radiation response markers is a pivotal step for early triage during radiological incidents to provide adequate and timely clinical interventions. However, there is currently a crucial shortage of methods capable of determining the extent of low-dose IR exposure to human beings. The phosphorylation of histone H2AX on serine 139 (designated γ-H2AX), a classic biological dosimeter, can be used to evaluate the DNA damage response. We have developed an estimation assay for low-level exposure to IR based on the mass spectrometry quantification of γ-H2AX in blood. Human peripheral blood lymphocytes sensitive to low-dose IR, maintaining low temperature (4°C) and adding enzyme inhibitor are proven to be key steps, possibly insuring that a stable and marked γ-H2AX signal in blood cells exposed to low-dose IR could be detected. For the first time, DNA damage at low dose exposures to IR as low as 0.01 Gy were observed using the sensitive variation of γ-H2AX with high throughput mass spectrometry quantification in human peripheral blood, which is more accurate than the previously reported methods by virtue of isotope-dilution mass spectrometry, and can observe the time effect of DNA damage. These in vitro cellular dynamic monitoring experiments show that DNA damage occurred rapidly and then was repaired slowly over the passage of post-irradiation time even after exposure to very low IR doses. This assay was also used to assess different radiation exposures at the in vitro cellular level. These results demonstrate the potential utility of this assay in radiation biodosimetry and environmental risk assessment.

Dataset Information

Inclusive Quantification Assay of Serum Des-γ-Carboxyprothrombin Proteoforms for Hepatocellular Carcinoma Surveillance by Targeted Mass Spectrometry.

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