Circulating citrullinated proteins as candidate liquid biopsy analytes in cancer

This review outlines the functions of circulating citrullinated histone H3 (CitH3), citrullinated and matrix metalloproteinase-degraded vimentin (VICM), peptidylarginine deiminase 4 (PAD4), and related proteoforms in oncology and discusses their suitability as analytes in liquid biopsies. Evidence in the serum and plasma is different from that in the tissue, bone marrow, in vitro, or preclinical evidence. Special emphasis has been placed on the following techniques: serum and plasma-based enzyme-linked immunosorbent assay (ELISA), liquid chromatography - tandem mass spectrometry (LC-MS/MS), targeted proteomics, and biosensors. Evidence is best established for CitH3, but the biology of neutrophil extracellular traps (NETs) and their reported associations are context- and assay-dependent, with nucleosomal assays and assays of free histones/free peptides measuring distinct pools of analytes. However, for some cancers, these analytes have preliminary prognostic and treatment-response-monitoring value, but there is a lack of evidence for their clinical use in cancer diagnosis, patient stratification, or minimal residual disease (MRD) monitoring. Assay heterogeneity, matrix effects, lack of certified reference materials, limited inter-platform comparability and harmonization, lack of external quality assessment (EQA), and lack of validated clinical cut-offs hinder clinical implementation. Key preanalytical requirements include preanalytical control, harmonization of assays, assessment of the commutability of reference materials, calibrators, control materials, and EQA materials, and compliance with the International Organization for Standardization (ISO) 15,189 and the In Vitro Diagnostic Regulation (IVDR). At present, citrullinated proteins should be considered as research use/early translational analytes, and not as clinically reportable oncology biomarkers.