An Automated Micro-Total Immunoassay System for Measuring Cancer-Associated α2,3-linked Sialyl N-Glycan-Carrying Prostate-Specific Antigen May Improve the Accuracy of Prostate Cancer Diagnosis

Abstract

Abstract:The low specificity of the prostate-specific antigen (PSA) for early detection of prostate cancer (PCa) is a major issue worldwide. The aim of this study to examine whether the serum PCa-associated α2,3-linked sialyl N-glycan-carrying PSA (S2,3PSA) ratio measured by automated micro-total immunoassay systems (µTAS system) can be applied as a diagnostic marker of PCa. The µTAS system can utilize affinity-based separation involving noncovalent interaction between the immunocomplex of S2,3PSA and Maackia amurensis lectin to simultaneously determine concentrations of free PSA and S2,3PSA. To validate quantitative performance, both recombinant S2,3PSA and benign-associated α2,6-linked sialyl N-glycan-carrying PSA (S2,6PSA) purified from culture supernatant of PSA cDNA transiently-transfected Chinese hamster ovary (CHO)-K1 cells were used as standard protein. Between 2007 and 2016, fifty patients with biopsy-proven PCa were pair-matched for age and PSA levels, with the same number of benign prostatic hyperplasia (BPH) patients used to validate the diagnostic performance of serum S2,3PSA ratio. A recombinant S2,3PSA-and S2,6PSA-spiked sample was clearly discriminated by µTAS system. Limit of detection of S2,3PSA was 0.05 ng/mL and coefficient variation was less than 3.1%. The area under the curve (AUC) for detection of PCa for the S2,3PSA ratio (%S2,3PSA) with cutoff value 43.85% (AUC; 0.8340) was much superior to total PSA (AUC; 0.5062) using validation sample set. Although the present results are preliminary, the newly developed µTAS platform for measuring %S2,3PSA can achieve the required assay performance specifications for use in the practical and clinical setting and may improve the accuracy of PCa diagnosis. Additional validation studies are warranted.

Urinary biomarkers of prostate cancer

“…Serum PSA with aberrant glycosylation was found to be superior to serum PSA itself. 19 High levels of PSA were secreted into the prostatic fluids, and a change in glycosylated PSA was also found in urine. Fucosylated PSA levels in urine after massage were measured by lectin-antibody enzyme-linked immunosorbent assay in 69 men (20 men with negative biopsy and 49 men with prostate cancer) and were significantly decreased in the men with prostate cancer compared with the men whose biopsies were negative for cancer.…”

“…PSA is also a glycoprotein with one N‐glycosylation site (Asn). Serum PSA with aberrant glycosylation was found to be superior to serum PSA itself . High levels of PSA were secreted into the prostatic fluids, and a change in glycosylated PSA was also found in urine.…”

Comparative Study of Blood-Based Biomarkers, α2,3-Sialic Acid PSA and PHI, for High-Risk Prostate Cancer Detection

“…To improve the specificity of the PSA as a biomarker, different strategies using several PSA isoforms (ratio free PSA/total PSA, PSA density and velocity, proPSA forms, 4K score, and Prostate Health Index (PHI)) have been developed [7], recently including, PSA glycoforms [8,9,10]. …”

Activation and activity of glycosylated KLKs 3, 4 and 11

“…Unfortunately, peptides of KLK3 S/T from both tryptic and chymotryptic digest were not resolved in these measurements, most likely due to the presence of inhomogeneous N-glycosylation at Asn61. This single glycosylation site may carry mannose-rich hybrid glycans and a mixture of bi-and triantennary N-glycans with varying numbers of fucose and terminal sialic acids that were observed for KLK3, similar to mammalian KLK4 ( Figure 1C) (Sarrats et al, 2010;Stura et al, 2011;Yamakoshi et al, 2011;Ishikawa et al, 2017). Nevertheless, the high molecular weight on SDS gels and downshifts upon enzymatic deglycosylation confirm the N-glycosylation of KLK3 S/T .…”