Elsevier

Clinica Chimica Acta

Volume 411, Issues 23–24, 14 December 2010, Pages 1935-1939
Clinica Chimica Acta

A sensitive assay to measure biomarker glycosylation demonstrates increased fucosylation of prostate specific antigen (PSA) in patients with prostate cancer compared with benign prostatic hyperplasia

https://doi.org/10.1016/j.cca.2010.08.009Get rights and content

Abstract

Background

Prostate specific antigen (PSA) measurement is used for the diagnosis of prostate cancer (PCa) but the test lacks specificity due to the number of false positive readings. The glycosylation of PSA is altered in PCa but studies in this area have been limited to few clinical samples and/or require advanced laboratory facilities. An assay to assess PSA glycosylation was established using equipment available in most routine biomedical testing laboratories.

Methods

Serum samples from patients with PCa or benign prostatic hyperplasia (BPH) were used. PSA (range 4–10 ng/ml) was affinity purified, separated and probed with the lectin Ulex europaeus (UEA-1; specific for α1,2 linked fucose). An enzyme-linked immunosorbent lectin assay (ELLA) with colorimetric detection was devised and PSA fucosylation assessed in a further independent set of 26 samples.

Results

Free PSA (fPSA) from PCa patients showed a significant increase in fucosylation compared with fPSA from patients with BPH. The ELLA was 92% specific and 69% sensitive for PCa over BPH. In comparison, fPSA measurement was 70% specific and 56% sensitive (threshold set to 25% tPSA) for PCa over BPH.

Conclusions

Changes in glycosylation of PSA were identified using 50 μl of serum with PSA in the range of 4–10 ng/ml, this represents a more specific and sensitive test for PCa based on fucosylation changes of fPSA.

Introduction

Prostate specific antigen (PSA) has been used for the diagnosis of prostate cancer (PCa) and monitoring patient response to treatment for approximately 20 years. PSA is a serine protease belonging to the kallikrein family, produced in the epithelium of the periurethral glands and functions to liquefy seminal gel [1]. The enzyme is a single chain glycoprotein with one N-linked glycosylation site at Asn-45; the molecular mass is 26,079 Da and the carbohydrate moiety adds approximately 2300 Da further to the overall weight, therefore, free PSA (fPSA) is approximately 28,400 Da [2].

One of the inherent problems of PSA use in the diagnostic setting is the poor specificity for PCa as patients with benign prostatic hyperplasia (BPH) may exhibit serum levels of PSA of 4 ng/ml or higher. Various approaches have been used to improve the selectivity and sensitivity of PSA for PCa, for example, PSA density [3] and PSA velocity [4]. PSA is found in two predominant forms in the serum either as fPSA or complexed with α1-chymotrypsin and α2-macroglobulin [5]. PCa patients have less fPSA than patients with BPH [6] and the ratio of fPSA to total PSA (tPSA) is used for the diagnosis of PCa but is not without problems, as the levels of fPSA vary with age [7].

The modification of proteins by glycosylation occurs in approximately half of all serum proteins and changes in glycosylation have been found to accompany both tumour formation and aggressive clinical behaviour [8]. Aberrant glycosylation of proteins in PCa tissue has been observed compared with BPH and normal tissue [9], [10], [11], [12]. Similarly, changes in the glycans on PSA have been observed in PCa compared with BPH but studies in this area have either used very few samples with extremely high PSA levels or technology only available in specialised glycobiology/glycoproteomic laboratories [13], [14], [15], [16].

We hypothesised that alterations in the tissue fucosylation of PCa compared with BPH [12] would be reflected in changes in the glycosylation of PSA and that this might add an element of specificity to the PSA test. The lectin from Ulex europaeus (UEA-1; specific for α1,2 linked fucose) was used and showed an elevation in the fucosylation of immuno-purified PSA from PCa serum samples compared with BPH. The observations were validated using a separate set of 26 serum samples in a simple enzyme-linked immunosorbent lectin assay (ELLA) developed in-house. The ELLA system – when used to study fPSA fucosylation – showed 92% specificity and 69% sensitivity for PCa over BPH, this compared with 70% specificity and 56% sensitivity for fPSA alone, when the threshold value was taken of 25% of the tPSA levels.

Section snippets

Subjects and sample collection

All patients had pathologically confirmed BPH or PCa and gifted a blood sample to the study, the patient details are given in Table 1. The study was approved by the Local Ethics Committee at the University College London Hospital Trust. 10 ml of blood was collected pre-operatively from each patient after informed consent. A sample of the blood was sent for routine PSA testing and the remainder was allowed to clot; centrifuged at 1500 g for 5 min, the supernatant was transferred to a new tube and

UEA-1 binding to immuno-purified PSA

Serum PSA from patients with PCa and BPH was separated by SDS-PAGE and probed with UEA-1, or anti-PSA antibody, Fig. 1. Several bands of MW lower than fPSA were observed; these most probably represent clipped forms of PSA. The higher MW bands (between 50 kDa and 100 kDa) are likely to be PSA complexed with serum proteins. Fucosylation of affinity purified PSA from serum of patients with PCa and BPH was compared by assessing the ratio of UEA-1 to PSA binding (Fig. 1) and was observed to be

Discussion

In this study an ELLA was developed in which the lectin UEA-1 was used to assess the glycosylation of the protein biomarker PSA. Only 50 μl of serum sample from patients with PSA in the “grey zone” range of 4–10 ng/ml was used in the assay.

The results showed an increase in the fucosylation of serum PSA in the PCa patient samples. An important next development for this field will be the availability of a recombinant form of PSA with “normal” and “cancer” glycoforms, these will find use as

Acknowledgements

This study was supported by a grant from Against Breast Cancer (registered charity 1121258) for AJ. Conflicts of interest: MD and AL are inventors on a British Patent (number 2379444) that is based on the observations described in this manuscript.

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