The glycoprofiling of two proteins, the free form of the prostate-specific antigen (fPSA) and zinc-α-2-glycoprotein (ZA2G), was assessed to determine their suitability as prostate cancer (PCa) biomarkers. The glycoprofiling of proteins was performed by analysing changes in the glycan composition on fPSA and ZA2G using lectins (proteins that recognise glycans, i.e. complex carbohydrates). The specific glycoprofiling of the proteins was performed using magnetic beads (MBs) modified with horseradish peroxidase (HRP) and antibodies that selectively enriched fPSA or ZA2G from human serum samples. Subsequently, the antibody-captured glycoproteins were incubated on lectin-coated ELISA plates. In addition, a novel glycoprotein standard (GPS) was used to normalise the assay. The glycoprofiling of fPSA and ZA2G was performed in human serum samples obtained from men undergoing a prostate biopsy after an elevated serum PSA, and prostate cancer patients with or without prior therapy. The results are presented in the form of an ROC (Receiver Operating Curve). A DCA (Decision Curve Analysis) to evaluate the clinical performance and net benefit of fPSA glycan-based biomarkers was also performed. While the glycoprofiling of ZA2G showed little promise as a potential PCa biomarker, the glycoprofiling of fPSA would appear to have significant clinical potential. Hence, the GIA (Glycobiopsy ImmunoAssay) test integrates the glycoprofiling of fPSA (i.e. two glycan forms of fPSA). The GIA test could be used for early diagnoses of PCa (AUC = 0.83; n = 559 samples) with a potential for use in therapy-monitoring (AUC = 0.90; n = 176 samples). Moreover, the analysis of a subset of serum samples (n = 215) revealed that the GIA test (AUC = 0.81) outperformed the PHI (Prostate Health Index) test (AUC = 0.69) in discriminating between men with prostate cancer and those with benign serum PSA elevation.

Department of Immunochemistry Diagnostics University Hospital in Pilsen Pilsen Czech Republic

Department of Pharmacology and Toxicology Faculty of Medicine in Pilsen Charles University Pilsen Czech Republic

Division of Experimental Urology Department of Urology Medical University Innsbruck Innsbruck Austria

Glycanostics Ltd Bratislava Slovak Republic

Institute of Chemistry Bratislava Slovak Republic

Klinikum Lippe Clinic for Urology Detmold Germany

Private Urological Ambulance Trencin Slovak Republic

University Hospital Bratislava Bratislava Slovakia

Zobrazit více v PubMed

Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, et al.. Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA: Cancer J Clin. 2021;71(3):209–49. doi: 10.3322/caac.21660 PubMed DOI

Wright P, Wilding S, Watson E, Downing A, Selby P, Hounsome L, et al.. Key factors associated with social distress after prostate cancer: Results from the United Kingdom Life after Prostate Cancer diagnosis study. Cancer Epidemiol. 2019;60:201–7. doi: 10.1016/j.canep.2019.04.006 PubMed DOI

Houédé N, Rébillard X, Bouvet S, Kabani S, Fabbro-Peray P, Trétarre B, et al.. Impact on quality of life 3 years after diagnosis of prostate cancer patients below 75 at diagnosis: an observational case-control study. BMC Cancer. 2020;20(1):1–12. PubMed PMC

Trujillo B, Wu A, Wetterskog D, Attard G. Blood-based liquid biopsies for prostate cancer: clinical opportunities and challenges. Br J Cancer. 2022;127(8):1394–402. doi: 10.1038/s41416-022-01881-9 PubMed DOI PMC

Heijnsdijk EA, de Carvalho TM, Auvinen A, Zappa M, Nelen V, Kwiatkowski M, et al.. Cost-effectiveness of prostate cancer screening: a simulation study based on ERSPC data. J Natl Cancer Inst. 2015;107(1):366. doi: 10.1093/jnci/dju366 PubMed DOI PMC

Vickers AJ. Redesigning prostate cancer screening strategies to reduce overdiagnosis. Clin Chem. 2019;65(1):39–41. doi: 10.1373/clinchem.2018.287094 PubMed DOI PMC

Campos-Fernández E, Barcelos LS, de Souza AG, Goulart LR, Alonso-Goulart V. Research landscape of liquid biopsies in prostate cancer. Am J Cancer Res. 2019;9(7):1309. PubMed PMC

Bai Y, Zhao H. Liquid biopsy in tumors: opportunities and challenges. Annals Translat Med. 2018;6(Suppl 1):S89. PubMed PMC

Bertok T, Bertokova A, Hroncekova S, Chocholova E, Svecova N, Lorencova L, et al.. Novel Prostate Cancer Biomarkers: Aetiology, Clinical Performance and Sensing Applications. Chemosensors. 2021;9(8):205.

Bertokova A, Svecova N, Kozics K, Gabelova A, Vikartovska A, Jane E, et al.. Exosomes from prostate cancer cell lines: Isolation optimisation and characterisation. Biomed Pharmacother. 2022;151:113093. doi: 10.1016/j.biopha.2022.113093 PubMed DOI

Tkac J, Bertok T, Hires M, Jane E, Lorencova L, Kasak P. Glycomics of prostate cancer: Updates. Exp Rev Proteomics. 2019;16(1):65–76. PubMed PMC

Tkac J, Gajdosova V, Hroncekova S, Bertok T, Hires M, Jane E, et al.. Prostate-specific antigen glycoprofiling as diagnostic and prognostic biomarker of prostate cancer. Interface Focus. 2019;9(2):20180077. doi: 10.1098/rsfs.2018.0077 PubMed DOI PMC

Petrosyan A. Onco-Golgi: is fragmentation a gate to cancer progression? Biochem Mol Biol J. 2015;1(1):16. doi: 10.21767/2471-8084.100006 PubMed DOI PMC

Bui S, Mejia I, Díaz B, Wang Y. Adaptation of the Golgi apparatus in cancer cell invasion and metastasis. Front Cell Develop Biol. 2021;9:806482. doi: 10.3389/fcell.2021.806482 PubMed DOI PMC

Zhang X. Alterations of golgi structural proteins and glycosylation defects in cancer. Front Cell Develop Biol. 2021;9:665289. doi: 10.3389/fcell.2021.665289 PubMed DOI PMC

Liu L, Doray B, Kornfeld S. Recycling of Golgi glycosyltransferases requires direct binding to coatomer. Proc Natl Acad Sci USA. 2018;115(36):8984–9. doi: 10.1073/pnas.1810291115 PubMed DOI PMC

Tu L, Banfield DK. Localization of Golgi-resident glycosyltransferases. Cell Mol Life Sci. 2010;67:29–41. doi: 10.1007/s00018-009-0126-z PubMed DOI PMC

Bertok T, Jane E, Bertokova A, Lorencova L, Zvara P, Smolkova B, et al.. Validating fPSA Glycoprofile as a Prostate Cancer Biomarker to Avoid Unnecessary Biopsies and Re-Biopsies. Cancers. 2020;12(10):2988. doi: 10.3390/cancers12102988 PubMed DOI PMC

Bertokova A, Bertok T, Jane E, Hires M, Ďubjaková P, Novotná O, et al.. Detection of N, N-diacetyllactosamine (LacdiNAc) containing free prostate-specific antigen for early stage prostate cancer diagnostics and for identification of castration-resistant prostate cancer patients. Biorg Med Chem. 2021;39:116156. PubMed

Bertok T, Tkac J. Means and methods for glycoprofiling of a protein. US Patent App. 17/041,816; 2021.

Peracaula R, Tabarés G, Royle L, Harvey DJ, Dwek RA, Rudd PM, et al.. Altered glycosylation pattern allows the distinction between prostate-specific antigen (PSA) from normal and tumor origins. Glycobiology. 2003;13(6):457–70. doi: 10.1093/glycob/cwg041 PubMed DOI

Pihikova D, Pakanova Z, Nemcovic M, Barath P, Belicky S, Bertok T, et al.. Sweet characterisation of prostate specific antigen using electrochemical lectin-based immunosensor assay and MALDI TOF/TOF analysis: Focus on sialic acid. Proteomics. 2016;16(24):3085–95. doi: 10.1002/pmic.201500463 PubMed DOI PMC

Pihíková D, Belicky Š, Kasák P, Bertok T, Tkac J. Sensitive detection and glycoprofiling of a prostate specific antigen using impedimetric assays. Analyst. 2016;141(3):1044–51. doi: 10.1039/c5an02322j PubMed DOI PMC

Tkac J, Bertok T, inventors; PCT/EP2022/072138, https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2023012352&_cid=P22-LDXXGC-67374-1, assignee. Standard for glycoprofiling of proteins. 2023.

Bertok T, Bertokova A, Jane E, Hires M, Aguedo J, Potocarova M, et al.. Identification of whole-serum glycobiomarkers for colorectal carcinoma using reverse-phase lectin microarray. Front Oncol. 2021;11:735338. doi: 10.3389/fonc.2021.735338 PubMed DOI PMC

Kuhn M. Building Predictive Models in R Using the caret Package. Journal of Statistical Software. 2008;28(5):1–26. PubMed

R Core Team: _R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, 2023. https://www.R-project.org/.

Robin X, Turck N, Hainard A, Tiberti N, Lisacek F, Sanchez J-C, et al.. pROC: an open-source package for R and S+ to analyze and compare ROC curves. BMC Bioinformatics. 2011;12(1):1–8. doi: 10.1186/1471-2105-12-77 PubMed DOI PMC

Zeileis A, Hothorn T. Diagnostic checking in regression relationships. R News 2002, 2(3), 7–10. https://CRAN.R-project.org/doc/Rnews/2002.

Nyalwidhe JO, Betesh LR, Powers TW, Jones EE, White KY, Burch TC, et al.. Increased bisecting N‐acetylglucosamine and decreased branched chain glycans of N‐linked glycoproteins in expressed prostatic secretions associated with prostate cancer progression. Proteom Clin Appl. 2013;7(9–10):677–89. doi: 10.1002/prca.201200134 PubMed DOI PMC

Kohler RS, Anugraham M, López MN, Xiao C, Schoetzau A, Hettich T, et al.. Epigenetic activation of MGAT3 and corresponding bisecting GlcNAc shortens the survival of cancer patients. Oncotarget. 2016;7(32):51674–86. doi: 10.18632/oncotarget.10543 PubMed DOI PMC

Chen Q, Tan Z, Guan F, Ren Y. The essential functions and detection of bisecting GlcNAc in cell biology. Front Chem. 2020;8:511. doi: 10.3389/fchem.2020.00511 PubMed DOI PMC

Hassan MI, Waheed A, Yadav S, Singh TP, Ahmad F. Zinc α2-glycoprotein: a multidisciplinary protein. Mol Cancer Res. 2008;6(6):892–906. PubMed

Butler W, Huang J. Glycosylation Changes in Prostate Cancer Progression. Front Oncol. 2021;11:809170. doi: 10.3389/fonc.2021.809170 PubMed DOI PMC

Bajaj R, Warner AN, Fradette JF, Gibbons DL. Dance of The Golgi: Understanding Golgi Dynamics in Cancer Metastasis. Cells. 2022;11(9):1484. doi: 10.3390/cells11091484 PubMed DOI PMC

Narimatsu Y, Joshi HJ, Nason R, Van Coillie J, Karlsson R, Sun L, et al.. An Atlas of Human Glycosylation Pathways Enables Display of the Human Glycome by Gene Engineered Cells. Molecular Cell. 2019;75(2):394–407.e5. doi: 10.1016/j.molcel.2019.05.017 PubMed DOI PMC

Narimatsu Y, Büll C, Chen Y-H, Wandall HH, Yang Z, Clausen H. Genetic glycoengineering in mammalian cells. J Biol Chem. 2021;296:100448. doi: 10.1016/j.jbc.2021.100448 PubMed DOI PMC

Mathew S, Rapsey CM, Wibowo E. Psychosocial Barriers and Enablers for Prostate Cancer Patients in Starting a Relationship. J Sex Marital Ther. 2020;46(8):736–46. doi: 10.1080/0092623X.2020.1808549 PubMed DOI

Klotz LH. PSAdynia and other PSA-related syndromes: a new epidemic—a case history and taxonomy. Urology. 1997;50(6):831–2. Epub 1998/01/14. doi: 10.1016/S0090-4295(97)00490-1 PubMed DOI

Pihikova D, Kasak P, Kubanikova P, Sokol R, Tkac J. Aberrant sialylation of a prostate-specific antigen: Electrochemical label-free glycoprofiling in prostate cancer serum samples. Anal Chim Acta. 2016;934(-):72–9. doi: 10.1016/j.aca.2016.06.043 PubMed DOI PMC

Paleček E, Tkáč J, Bartosik M, Bertók Ts, Ostatná V, Paleček J. Electrochemistry of nonconjugated proteins and glycoproteins. Toward sensors for biomedicine and glycomics. Chem Rev. 2015;115(5):2045–108. doi: 10.1021/cr500279h PubMed DOI PMC

Serum levels of prostate specific antigen, free PSA, [-2]proPSA, fPSA/tPSA ratio, Prostate Health Index, and glycosylation patterns of free PSA in patients with benign prostatic hyperplasia pharmacotherapy