Prostate-specific Antigen Glycosylation Research Articles

Liquid-Biopsy Glycan Score Biomarker Accurately Indicates and Stratifies Primary and Metastatic Prostate Cancers.

Prostate cancer (PCa) is the most commonly diagnosed cancer in males. Early PCa usually shows no clinical symptoms and its primary diagnosis is currently guided by liquid-biopsy testing of serum prostate-specific antigen (PSA). This testing suffers from high false-positive and false-negative rates. Identifying new biomarkers for precise liquid-biopsy detection of PCa is, thus, an acute clinical request. Here, by using an advanced dual-functional aptamer assay, we quantified the extent of glycosylation of PSA circulating in cancer patients' serum, linked it to cancer-related breakage of PSA complexes with serum-circulating proteins, and proved its facility for stratification of primary and metastatic PCa. PSA's "Glycan Score" 100% accurately informed about PCa status in a 30-patient cohort, while serum PSA's concentration correctly classified only 53% of PCa patients and did not inform about their PCa status. The Glycan Score liquid-biopsy test thus has a huge potential for accurate diagnosis and staging of PCa, enabling mass-screening program progress and advanced PCa treatment monitoring.

Characterisation of the main PSA glycoforms in aggressive prostate cancer

Serum levels of prostate specific antigen (PSA) are commonly used for prostate cancer (PCa) detection. However, their lack of specificity to distinguish benign prostate pathologies from PCa, or indolent from aggressive PCa have prompted the study of new non-invasive PCa biomarkers. Aberrant glycosylation is involved in neoplastic progression and specific changes in PSA glycosylation pattern, as the reduction in the percentage of α2,6-sialic acid (SA) are associated with PCa aggressiveness. In this study, we have characterised the main sialylated PSA glycoforms from blood serum of aggressive PCa patients and have compared with those of standard PSA from healthy individuals’ seminal plasma. PSA was immunoprecipitated and α2,6-SA were separated from α2,3-SA glycoforms using SNA affinity chromatography. PSA N-glycans were released, labelled and analysed by hydrophilic interaction liquid chromatography combined with exoglycosidase digestions. The results showed that blood serum PSA sialylated glycoforms containing GalNAc residues were largely increased in aggressive PCa patients, whereas the disialylated core fucosylated biantennary structures with α2,6-SA, which are the major PSA glycoforms in standard PSA from healthy individuals, were markedly reduced in aggressive PCa. The identification of these main PSA glycoforms altered in aggressive PCa opens the way to design specific strategies to target them, which will be useful to improve PCa risk stratification.

High-throughput glycopeptide profiling of prostate-specific antigen from seminal plasma by MALDI-MS

An altered total seminal plasma glycosylation has been associated with male infertility, and the highly abundant seminal plasma glycoprotein prostate-specific antigen (PSA) plays an important role in fertilization. However, the exact role of PSA glycosylation in male fertility is not clear. To understand the involvement of PSA glycosylation in the fertilization process, analytical methods are required to study the glycosylation of PSA from seminal plasma with a high glycoform resolution and in a protein-specific manner. In this study, we developed a novel, high-throughput PSA glycopeptide workflow, based on matrix-assisted laser desorption/ionization-mass spectrometry, allowing the discrimination of sialic acid linkage isomers via the derivatization of glycopeptides. The method was successfully applied on a cohort consisting of seminal plasma from infertile and fertile men (N = 102). Forty-four glycopeptides were quantified in all samples, showing mainly complex-type glycans with high levels of fucosylation and sialylation. In addition, N,N-diacetyllactosamine (LacdiNAc) motives were found as well as hybrid-type and high mannose-type structures. Our method showed a high intra- and interday repeatability and revealed no difference in PSA glycosylation between fertile and infertile men. Next to seminal plasma, the method is also expected to be of use for studying PSA glycopeptides derived from other biofluids and/or in other disease contexts.

HILIC-MRM-MS for Linkage-Specific Separation of Sialylated Glycopeptides to Quantify Prostate-Specific Antigen Proteoforms.

Elevated serum prostate-specific antigen (PSA) levels in body fluids may indicate prostate cancer (PCa), but it is noted that the clinical performance is rather poor. Specificity and sensitivity values of 20 and 94% at a cutoff value of 4.1 ng/mL, respectively, result in overdiagnosis and unnecessary interventions. Previous exploratory studies have indicated that the glycosylation of PSA potentially leads to improved PCa diagnosis based on qualitative analyses. However, the applied methods are not suited for a quantitative evaluation or implementation in a medical laboratory. Therefore, in this proof-of-principle study, we have evaluated the use of hydrophilic interaction liquid chromatography (HILIC) in combination with targeted quantitative mass spectrometry for the sialic acid linkage-specific analysis of PSA glyco-proteoforms based on either trypsin or ArgC peptides. The efficiency of PSA proteolysis was optimized as well as the glycopeptide separation conditions (buffer type, strength, and pH). The HILIC-based analysis of PSA glyco-proteoforms presented here has the potential for the clinical validation of patient cohorts. The method shows the feasibility of the use of a HILIC stationary phase for the separation of isomeric glycopeptides to detect specific glyco-proteoforms. This is the first step toward the development and evaluation of PSA glyco-proteoforms for use in a clinical chemistry setting aiming for improved PCa diagnosis or screening.

Novel Diagnostic Biomarkers of Prostate Cancer: An Update.

In recent years, several biomarkers alternative to standard prostate specific antigen (PSA) for prostate cancer (PCa) diagnosis have become available. The aim of this systematic review is to assess the current knowledge about alternative serum and urinary biomarkers for the diagnosis of PCa. A research was conducted in Medline, restricted to English language articles published between December 2014 and June 2018 with the aim to update previously published series on PCa biomarkers. The preferred reporting items for systematic reviews and meta-analyses (PRISMA) criteria were used for selecting studies with the lowest risk of bias. Emerging role and actual controversies on serum and urine alternative biomarkers to standard PSA for PCa diagnosis, staging and prognosis assessment, such as prostate health index (PHI), PCA3, ConfirmMDx, Aberrant PSA glycosylation, MiPS, miRNAs are critically presented in the current review. Although the use of several biomarkers has been recommended or questioned by different international guidelines, larger prospective randomized studies are still necessary to validate their efficacy in PCa detection, discrimination, prognosis and treatment effectiveness. To date, only PHI and 4Kscore have shown clinical relevance for discriminating more aggressive PCa. Furthermore, a new grading classification based on molecular features relevant for PCa risk-stratification and tailoring treatment is still needed.

Glycans as Biomarkers in Prostate Cancer.

Prostate cancer is the most commonly diagnosed malignancy in men, claiming over 350,000 lives worldwide annually. Current diagnosis relies on prostate-specific antigen (PSA) testing, but this misses some aggressive tumours, and leads to the overtreatment of non-harmful disease. Hence, there is an urgent unmet clinical need to identify new diagnostic and prognostic biomarkers. As prostate cancer is a heterogeneous and multifocal disease, it is likely that multiple biomarkers will be needed to guide clinical decisions. Fluid-based biomarkers would be ideal, and attention is now turning to minimally invasive liquid biopsies, which enable the analysis of tumour components in patient blood or urine. Effective diagnostics using liquid biopsies will require a multifaceted approach, and a recent high-profile review discussed combining multiple analytes, including changes to the tumour transcriptome, epigenome, proteome, and metabolome. However, the concentration on genomics-based paramaters for analysing liquid biopsies is potentially missing a goldmine. Glycans have shown huge promise as disease biomarkers, and data suggests that integrating biomarkers across multi-omic platforms (including changes to the glycome) can improve the stratification of patients with prostate cancer. A wide range of alterations to glycans have been observed in prostate cancer, including changes to PSA glycosylation, increased sialylation and core fucosylation, increased O-GlcNacylation, the emergence of cryptic and branched N-glyans, and changes to galectins and proteoglycans. In this review, we discuss the huge potential to exploit glycans as diagnostic and prognostic biomarkers for prostate cancer, and argue that the inclusion of glycans in a multi-analyte liquid biopsy test for prostate cancer will help maximise clinical utility.

An In-Depth Glycosylation Assay for Urinary Prostate-Specific Antigen.

The concentration of prostate-specific antigen (PSA) in serum is used as an early detection method of prostate cancer (PCa); however, it shows low sensitivity, specificity, and a poor predictive value. Initial studies suggested the glycosylation of PSA to be a promising marker for a more specific yet noninvasive PCa diagnosis. Recent studies on the molecular features of PSA glycosylation (such as antenna modification and core fucosylation) were not successful in demonstrating its potential for an improved PCa diagnosis, probably due to the lack of analytical sensitivity and specificity of the applied assays. In this study, we established for the first time a high-performance PSA Glycomics Assay (PGA), allowing differentiation of α2,6- and α2,3-sialylated isomers, the latter one being suggested to be a hallmark of aggressive types of cancer. After affinity purification from urine and tryptic digestion, PSA samples were analyzed by CE-ESI-MS (capillary electrophoresis–electrospray ionization coupled to mass spectrometry). Based on positive controls, an average interday relative standard deviation of 14% for 41 N-glycopeptides was found. The assay was further verified by analyzing PSA captured from patients’ urine samples. A total of 67 N-glycopeptides were identified from the PSA pooled from the patients. In summary, the first PGA successfully established in this study allows an in-depth relative quantitation of PSA glycoforms from urine. The PGA is a promising tool for the determination of potential glycomic biomarkers for the differentiation between aggressive PCa, indolent PCa, and benign prostate hyperplasia in larger cohort studies.

Analysis of urinary PSA glycosylation is not indicative of high-risk prostate cancer

The levels of core fucosylation and α2,3-linked sialic acid in serum Prostate Specific Antigen (PSA), using the lectins Pholiota squarrosa lectin (PhoSL) and Sambucus nigra agglutinin (SNA), can discriminate between Benign Prostatic Hyperplasia (BPH) and indolent prostate cancer (PCa) from aggressive PCa. In the present work we evaluated whether these glycosylation determinants could also be altered in urinary PSA obtained after digital rectal examination (DRE) and could also be useful for diagnosis determinations. For this purpose, α2,6-sialic acid and α1,6-fucose levels of urinary PSA from 53 patients, 18 biopsy-negative and 35 PCa patients of different aggressiveness degree, were analyzed by sandwich ELLA (Enzyme Linked Lectin Assay) using PhoSL and SNA. Changes in the levels of specific glycosylation determinants, that in serum PSA samples were indicative of PCa aggressiveness, were not found in PSA from DRE urine samples. Although urine is a simpler matrix for analyzing PSA glycosylation compared to serum, an immunopurification step was necessary to specifically detect the glycans on the PSA molecule. Those specific glycosylation determinants on urinary PSA were however not useful to improve PCa diagnosis. This could be probably due to the low proportion of PSA from the tumor in urine samples, which precludes the identification of aberrantly glycosylated PSA.

Sample preparation of serum to allow capillary electrophoresis analysis of prostate specific antigen isoforms

Prostate cancer is the second most frequently diagnosed cancer in men worldwide. Currently prostate specific antigen (PSA) serum concentration is the most used prostate cancer marker, but it only shows limited specificity. Because PSA glycosylation is altered by prostate cancer, detecting glycosylation changes could increase PSA specificity as a prostate cancer marker. Changes in PSA glycosylation can modify its electrophoretic- behavior and techniques such as capillary zone electrophoresis (CZE) and two-dimensional electrophoresis (2-DE) could be applied to detect changes in PSA glycosylation.Most serum PSA is complexed with alpha-1 antichymotrypsin (ACT). To have access to most of the PSA, the complexed PSA has to be released as free PSA (fPSA); in addition, this total fPSA must be purified from the serum matrix so that it can be analyzed using CZE.In this work a methodology for isolating PSA from serum for its CZE analysis was established. By using PSA standard, the effect of this methodology, which combines conditions for dissociating complexed PSA and immunoaffinity chromatographic purification, was studied. It was seen that this highly repeatable sample treatment did not noticeably alter the circular dichroism (CD) spectrum or the CZE pattern of PSA standard. Therefore, as a proof-of-concept, the developed sample treatment was applied to serum from a cancer patient with a high PSA content. The following observations can be made from these experiments: first of all, the 2-DE pattern of serum PSA remained unchanged after sample treatment; second, as hypothesized, the established sample preparation methodology made it possible to obtain the CZE pattern of PSA from serum; and third, the CZE pattern of serum PSA and of PSA standard from seminal plasma of healthy individuals, both submitted to the sample treatment method, showed some differences regarding the proportion of CZE peaks of the glycoprotein. These differences could be related to possible changes in the linkages of peptide backbone, in glycosylation or in other post-translational modifications between samples from both origins.

Aberrant sialylation of a prostate-specific antigen: Electrochemical label-free glycoprofiling in prostate cancer serum samples

Electrochemical detection method allowing to detect prostate-specific antigen (PSA), a biomarker of prostate cancer (PCa), with PSA glycoprofiling was applied in an analysis of PCa serum samples for the first time. Electrochemical impedance spectroscopy (EIS) as a label-free method with immobilized anti-PSA was applied for PSA detection and lectins to glycoprofile captured PSA on the same surface. A proper choice of blocking agent providing high selectivity of biosensor detection with the immobilized anti-PSA antibody was done. The biosensor could detect PSA down to 100 ag/mL with a linear concentration working range from 100 ag/mL up to 1 μg/mL, i.e. 10 orders of concentration magnitude and the sensitivity of (5.5 ± 0.2)%/decade. The results showed that a commercial carbo-free blocking solution was the best one, reducing non-specific binding 55-fold when compared to the immunosensor surface without any blocking agent applied, while allowing to detect PSA. The biosensor response obtained after addition of lectin (i.e. proportional to the amount of a particular glycan on PSA) divided by the biosensor response obtained after incubation with a sample (i.e. proportional to the PSA level in the sample) was applied to distinguish serum samples of PCa patients from those of healthy individuals. The results showed that Maackia amurensis agglutinin (MAA) recognizing α-2,3-terminal sialic acid can be applied to distinguish between these two sets of samples since the MAA/PSA response obtained from the analysis of the PCa samples was significantly higher (5.3-fold) compared to the MAA/PSA response obtained by the analysis of samples from healthy individuals. Thus, combined analysis of serological PSA levels together with PSA glycoprofiling of aberrant glycosylation of PSA (i.e. increase in the level of α-2,3-terminal sialic acid) has a potential to improve detection of PCa.

Altered glycosylation in prostate cancer.

Prostate cancer is annually the most common newly diagnosed cancer in men. The prostate functions as a major secretory gland for the production of glycoproteins critical to sperm activation and reproduction. Prostate-specific antigen (PSA), produced by the prostate, is one of the most commonly assayed glycoproteins in blood, serving as a biomarker for early detection and progression of prostate cancer. The single site of N-glycosylation on PSA has been the target of multiple glycan characterization studies. In this review, the extensive number of studies that have characterized the changes in O-linked and N-linked glycosylations associated with prostate cancer development and progression will be summarized. This includes analysis of the glycosylation of PSA, and other prostate glycoproteins, in tissues, clinical biofluids, and cell line models. Other studies are summarized in the context of understanding the complexities of these glycan changes in order to address the many confounding questions associated with prostate cancer, as well as efforts to improve prostate cancer biomarker assays using targeted glycomic-based strategies.

Glycoproteomics: identifying the glycosylation of prostate specific antigen at normal and high isoelectric points by LC-MS/MS.

Prostate specific antigen (PSA) is currently used as a biomarker to diagnose prostate cancer. PSA testing has been widely used to detect and screen prostate cancer. However, in the diagnostic gray zone, the PSA test does not clearly distinguish between benign prostate hypertrophy and prostate cancer due to their overlap. To develop more specific and sensitive candidate biomarkers for prostate cancer, an in-depth understanding of the biochemical characteristics of PSA (such as glycosylation) is needed. PSA has a single glycosylation site at Asn69, with glycans constituting approximately 8% of the protein by weight. Here, we report the comprehensive identification and quantitation of N-glycans from two PSA isoforms using LC–MS/MS. There were 56 N-glycans associated with PSA, whereas 57 N-glycans were observed in the case of the PSA-high isoelectric point (pI) isoform (PSAH). Three sulfated/phosphorylated glycopeptides were detected, the identification of which was supported by tandem MS data. One of these sulfated/phosphorylated N-glycans, HexNAc5Hex4dHex1s/p1 was identified in both PSA and PSAH at relative intensities of 0.52 and 0.28%, respectively. Quantitatively, the variations were monitored between these two isoforms. Because we were one of the laboratories participating in the 2012 ABRF Glycoprotein Research Group (gPRG) study, those results were compared to that presented in this study. Our qualitative and quantitative results summarized here were comparable to those that were summarized in the interlaboratory study.

Increased expression of GCNT1 is associated with altered O-glycosylation of PSA, PAP, and MUC1 in human prostate cancers.

Protein glycosylation is a common posttranslational modification and glycan structural changes have been observed in several malignancies including prostate cancer. We hypothesized that altered glycosylation could be related to differences in gene expression levels of glycoprotein synthetic enzymes between normal and malignant prostate tissues. We interrogated prostate cancer gene expression data for reproducible changes in expression of glycoprotein synthetic enzymes. Over-expression of GCNT1 was validated in prostate samples using RT-PCR. ELISA was used to measure core 2 O-linked glycan sialyl Lewis X (sLe(x) ) of prostate specific antigen (PSA), Mucin1 (MUC1), and prostatic acidic phosphatase (PAP) proteins. A key glycosyltransferase, GCNT1, was consistently over-expressed in several prostate cancer gene expression datasets. RT-PCR confirmed increased transcript levels in cancer samples compared to normal prostate tissue in fresh-frozen prostate tissue samples. ELISA using PSA, PAP, and MUC1 capture antibodies and a specific core 2 O-linked sLe(x) detection antibody demonstrated elevation of this glycan structure in cancer compared to normal tissues for MUC1 (P = 0.01), PSA (P = 0.03) and near significant differences in PAP sLe(x) levels (P = 0.06). MUC1, PSA and PAP protein levels alone were not significantly different between paired normal and malignant prostate samples. GCNT1 is over-expressed in prostate cancer and is associated with higher levels of core 2 O-sLe(x) in PSA, PAP and MUC1 proteins. Alterations of O-linked glycosylation could be important in prostate cancer biology and could provide a new avenue for development of prostate cancer specific glycoprotein biomarkers.

Measurement of aberrant glycosylation of prostate specific antigen can improve specificity in early detection of prostate cancer

IntroductionWe previously identified prostate cancer (PCa)-associated aberrant glycosylation of PSA, where α2,3-linked sialylation is an additional terminal N-glycan on free PSA (S2,3PSA). We then developed a new assay system measuring S2,3PSA using a magnetic microbead-based immunoassay. We compared the diagnostic accuracy of conventional PSA and percent-free PSA (%fPSA) tests. MethodsWe used MagPlex beads to measure serum S2,3PSA levels using anti-human fPSA monoclonal antibody (8A6) for capture and anti-α2,3-linked sialic acid monoclonal antibody (HYB4) for detection. We determined the cutoff values in a training test and measured serum S2,3PSA levels in 314 patients who underwent biopsy, including 138 PCa and 176 non-PCa patients with PSA of <10.0ng/ml. Serum S2,3PSA levels were presented as mean fluorescence intensity (MFI). Receiver operating characteristic curves were used to evaluate the diagnostic accuracy of total PSA, %fPSA, and S2,3PSA. ResultsWe determined an MFI cutoff value of 1130 with a sensitivity of 95.0% and specificity of 72.0% for the diagnosis of PCa in the training test. In the validation study, the area under the curve for the detection of PCa with S2,3PSA was 0.84, which was significantly higher than that with PSA or %fPSA. ConclusionsAlthough the present study is small and preliminary, these results suggest that the measurement of serum S2,3PSA using a magnetic microbead-based immunoassay may improve the accuracy of early detection of PCa and reduce unnecessary prostate biopsy.

112 Measurement of prostate cancer-associated aberrant glycosylation of prostate specific antigen can improve diagnostic accuracy

112 Measurement of prostate cancer-associated aberrant glycosylation of prostate specific antigen can improve diagnostic accuracy

Immunoaffinity chromatographic isolation of prostate-specific antigen from seminal plasma for capillary electrophoresis analysis of its isoforms

Prostate-specific antigen (PSA) concentration in serum has been the biomarker employed for prostate cancer diagnosis in the last two decades. However, new more specific biomarkers allowing a better differentiation of cancer from non-malignant prostate diseases are necessary. Glycosylation of PSA gives rise to different forms of the protein which can be separated into several isoforms by analytical techniques, such as CE. Because PSA glycosylation is influenced by pathological conditions, the CE pattern of PSA isoforms could be different in prostate cancer than in non-malignant prostate diseases. To study this CE pattern of PSA, prior purification of the protein from the biological fluid is mandatory. In this study an immunoaffinity chromatography method which allows PSA purification without altering the CE pattern is developed. An in-house prepared column produced with commercial anti-PSA antibodies is employed. The use of 1M propionic acid as elution agent provides higher than 40% recovery of high purity PSA. CE analysis of PSA immunopurified from seminal plasma of a healthy individual shows the same 8 peaks as the commercially available PSA standard. Sample preparation only requires dilution with phosphate buffered saline prior to immunoaffinity purification. High repeatability for the sample preparation step was achieved (RSD% for percentage of corrected peak area in the range 0.6–5.3 for CE analysis of three independently purified seminal plasma aliquots compared to range 0.8–4.9 for a given aliquot analyzed three times by CE). IAC of five microliters seminal plasma provided enough PSA to achieve signal/noise ratio larger than 5 for the smallest CE isoforms.

CE methods for analysis of isoforms of prostate‐specific antigen compatible with online derivatization for LIF detection

Prostate-specific antigen (PSA) is the usual biomarker for prostate cancer (PCa). However, its lack of selectivity has lead to the search for new biomarkers. PSA glycosylation seems to depend on the pathophysiological conditions of the individual. Thus, methods to separate PSA isoforms (peaks) to study their role as PCa markers are needed. In this work, CE methods for PSA isoforms separation, based on the use of different dynamic coatings, are developed using UV detection. Three complementary CE methods allowing the separation of 8 or 9 PSA isoforms are selected. The longest method takes only 17 min, while the shortest one separates 9 isoforms in < 8 min. Depending on the isoforms of interest for their use as PCa biomarker, the CE method to be used can be chosen or various of them can be combined. A remarkable aspect of these methods is that the BGEs employed are devoid of compounds with primary amino groups, making the CE methods compatible with fluorescent on-column derivatization through amino residues. As a proof-of-concept, a preliminary result shows that LIF detection of labeled PSA analyzed by one of the three developed methods permits detection of glycoprotein isoforms.

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

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.

Glycoproteomics for Prostate Cancer Detection: Changes in Serum PSA Glycosylation Patterns

Currently, serum prostate-specific antigen (PSA) is used for the early detection of prostate cancer despite its low specificity in the range of 4-10 ng/mL. Because aberrant glycosylation is a fundamental characteristic of tumor genesis, the objective of this study was to investigate whether changes in PSA glycosylation may be used to improve the cancer specificity of PSA. We developed five lectin immunosorbant assays to analyze the glycosylation patterns of PSA in serum. Each assay sandwiches serum PSA between a PSA monoclonal antibody and a biotinylated lectin and then tags the biotin complex using a streptavidin SULFO TAG for electrochemiluminescence detection. Low limits of detection (0.04-1.35 ng/mL), good reproducibility (%CVs < 10%), and direct analysis of PSA glycosylation in sera suggest these assays may have a potential role in improving PSA's cancer specificity. Clinical performance was evaluated in 52 human subjects (26 cancer and 26 noncancer). ROC analysis showed that the total SNA assay (AUC = 0.71) appeared to perform better than percent free PSA (AUC = 0.54) in its diagnostic gray zone between 10 and 20% in a subset of 21 subjects. A separate study of 16 additional subjects showed similar findings.

Different glycan structures in prostate-specific antigen from prostate cancer sera in relation to seminal plasma PSA

Prostate-specific antigen (PSA), the tumor marker currently used for prostate cancer (PCa), is not specific enough to distinguish between PCa and benign prostate hyperplasia (BPH). Glycan processing is normally perturbed in tumors, therefore we investigated whether changes in glycosylation of PSA could be useful diagnostic indicators. Previously we determined that the glycosylation of PSA secreted by the tumor prostate cell line LNCaP differs significantly from that of PSA from seminal plasma (normal control). We therefore undertook a detailed glycan analysis of PSA derived from sera from PCa patients and, importantly, established that the glycosylation of the PCa serum PSA was significantly different from the PSA from the LNCaP cell line. In comparison with seminal plasma PSA, the fucose content of PSA from the PCa patient serum was significantly lower and there was a decrease in alpha2,3-linked sialic acid. Differences in the glycosylation of PSA derived from PCa patients' sera, seminal plasma, and LNCaP cells were further established by lectin detection, glycosylation immunosorbent assay, and two-dimensional electrophoresis. We also investigated whether the impact of glycosylation changes initiated by the tumor was reflected in the serum glycome. By comparing the glycans released from the total glycoproteins in PCa patient serum with those of normal serum we found an increase in the proportion of sialyl-Lewis x structures. Further analysis of the glycosylation of PSA from PCa and BPH sera will be required in order to determine the utility of these glycan differences to discriminate specifically between benign and malignant prostate states.