Wisteria floribunda Agglutinin and Its Reactive-Glycan-Carrying Prostate-Specific Antigen as a Novel Diagnostic and Prognostic Marker of Prostate Cancer

Abstract

Wisteria floribunda agglutinin (WFA) preferably binds to LacdiNAc glycans, and its reactivity is associated with tumor progression. The aim of this study to examine whether the serum LacdiNAc carrying prostate-specific antigen-glycosylation isomer (PSA-Gi) and WFA-reactivity of tumor tissue can be applied as a diagnostic and prognostic marker of prostate cancer (PCa). Between 2007 and 2016, serum PSA-Gi levels before prostate biopsy (Pbx) were measured in 184 biopsy-proven benign prostatic hyperplasia patients and 244 PCa patients using an automated lectin-antibody immunoassay. WFA-reactivity on tumor was analyzed in 260 radical prostatectomy (RP) patients. Diagnostic and prognostic performance of serum PSA-Gi was evaluated using area under the receiver-operator characteristic curve (AUC). Prognostic performance of WFA-reactivity on tumor was evaluated via Cox proportional hazards regression analysis and nomogram. The AUC of serum PSA-Gi detecting PCa and predicting Pbx Grade Group (GG) 3 and GG ≥ 3 after RP was much higher than those of conventional PSA. Multivariate analysis showed that WFA-reactivity on prostate tumor was an independent risk factor of PSA recurrence. The nomogram was a strong model for predicting PSA-free survival provability with a c-index ≥0.7. Serum PSA-Gi levels and WFA-reactivity on prostate tumor may be a novel diagnostic and pre- and post-operative prognostic biomarkers of PCa, respectively.

Keywords: LacdiNAc; N-glycan; Wisteria floribunda agglutinin (WFA) lectin; biomarker; prostate-specific antigen.

Figure A1

Representative hematoxylin-eosin (HE) staining and WFA reactive-glycan expression of tumors of each Gleason grade among RP specimens. WFA staining intensity was classified into three groups: weakly positive, moderately positive, and strongly positive at a tumor site, respectively. Scale bar indicated 500 μm.

Figure 1

Prostate cancer (PCa)-associated aberrant N-glycosylation of prostate-specific antigen (PSA). PSA derived from PCa serum and culture supernatant of LNCaP carries Wisteria floribunda agglutinin (WFA)-reactive LacdiNAc glycans; this is not the case for PSA derived from benign prostatic hyperplasia (BPH) serum. PCa-associated aberrant LacdiNAc carrying PSA glycosylation isomer designated as PSA–glycosylation isomer (PSA-Gi) [21]. Carbon linkage positions are denoted by the bond position drawn on each monosachharide. IRNK indicate N-glycosylation site of PSA.

Figure 2

The schematic representation of serum PSA-Gi detection using a two-step surface plasmon field-enhanced fluorescence spectrometry (SPFS)-based WFA lectin-anti-PSA antibody immunoassay. Gray line arrows indicated that reagent dispense from reagent container to mixing reactor using pump. Gray dotted line arrows indicated mixing the content of mixing reactor by pump.

Figure 3

Serum levels of the PSA-Gi at Pbx in the patients who diagnosed as BPH or PCa by an SPFS-based lectin-antibody immunoassay. (a) PSA-Gi and (b) total PSA levels in patients with a diagnosis of BPH or PCa at a total PSA ≤ 20 ng/mL; (c) receiver-operator characteristic (ROC) curve analysis of total PSA, PSA-Gi, and PSA-Gi/total PSA in patients who had a diagnosis of BPH or PCa at a total PSA ≤ 20 ng/mL. The areas under the ROC curve (AUCs) for the prediction of PCa of PSA-Gi, total PSA, and PSA-Gi/total PSA were 0.795, 0.638, and 0.734, respectively; (d) PSA-Gi and (e) total PSA levels in patients with BPH or PCa at total PSA ≤ 10 ng/mL; (f) ROC curve analysis of total PSA, PSA-Gi, and PSA-Gi/total PSA in patients with BPH or PCa at a total PSA ≤ 10 ng/mL. The AUCs for the prediction of PCa by means of PSA-Gi, total PSA, and PSA-Gi/total PSA were 0.752, 0.550, and 0.718, respectively; (g) correlation between PSA-Gi and total PSA. Correlation coefficient was analyzed by non-parametric Spearman’s r-test. (a–g) The cutoff level at 90% sensitivity of PSA-Gi and/or total PSA is presented as a blue dotted line.

Figure 4

The serum PSA-Gi levels at Pbx in PCa patients who underwent radical prostatectomy (RP). (a) PSA-Gi levels before Pbx among PCa patients classified by the Pbx grade group (Pbx GG); (b) total PSA level before Pbx of PCa patients classified by the Pbx GG. Cutoff levels at 57.1% sensitivity of PSA-Gi and/or total PSA is presented as a blue dotted line; (c) ROC curve analysis of total PSA and PSA-Gi in PCa patients with Pbx GG 2 and Pbx GG 3. The AUCs for the prediction of patients with Pbx GG 3 of PSA-Gi and total PSA were 0.649 and 0.520, respectively.

Figure 5

The serum PSA-Gi levels at Pbx in PCa patients who underwent RP. (a) PSA-Gi levels before Pbx among PCa patients classified by the grade group after RP (ope GG); (b) total PSA level before Pbx of PCa patients classified by the ope GG; (c,d) PSA-Gi and total PSA levels before Pbx between patients with ope GG ≤ 2 and ope GG ≥ 3. Cutoff levels at 60% sensitivity of PSA-Gi and/or total PSA is presented as a blue dotted line; (e) ROC curve analysis of total PSA and PSA-Gi in PCa patients with ope GG ≤ 2 and ope GG ≥ 3. The AUCs for the prediction of patients with ope GG ≥ 3 of PSA-Gi and total PSA were 0.724 and 0.618, respectively; (f,g) PSA-Gi and total PSA levels in patients with a GG upgrade from 2 at Pbx to ope GG ≥ 3 and a GG downgrade from ≥3 at Pbx to ope GG ≤ 2.

Figure 6

Immunohistochemical analysis of RP specimens using WFA lectin and post-operative nomogram predicting PSA-free survival probability. (a) Representative hematoxylin-eosin (HE) staining and WFA reactive-glycan expression of tumors of RP specimens. WFA-reactivity was classified into three groups: weakly positive, moderately positive, and strongly positive at a tumor site, respectively. Scale bar indicated 500 μm; (b) association between with WFA-reactive glycan expression and ope GG (ope GS), pathological stage, and perineural invasion-status; (c) PSA-free survival was evaluated using Kaplan–Meier curves and differences between three groups were assessed using the log-rank test. Patients with tumors strongly or moderately positive for WFA had a much shorter period of PSA recurrence after RP than did patients with tumors weakly positive for WFA; (d) Cox hazard regression analysis-based post-operative nomogram predicting PSA-free survival probability after RP. The c-index (0.754, 95% CI, 0.697–0.812), which is similar to the area under a receiver operating characteristic curve, was used to estimate the discrimination ability of the nomogram [25].