New Approaches for the Development of Diagnostic Systems for Prostate Cancer

Julia Zapatero Rodríguez,  Postdoctoral Researcher, Dublin City University, Ireland, and Project Manager of AbYBiotech

Richard O’Kennedy,  Professor of Biological Sciences, Dublin City University, Ireland, and Chief Scientific Officer& Founder of AbYBiotech

Current prostate cancer diagnostic approaches fail to provide sufficient evidence for treatment decision-making. Since many prostate cancers are slow growing or non life-threatening, over treatment of indolent disease has become a major issue for doctors and prostate cancer patients. The ongoing trend towards multiplex biomarker detection holds promise for enhancing current standards of care.

Prostate Cancer (PCa) is one of the most common malignancies of men. The incidence rates are especially high in developed countries, which have the oldest population profiles and, therefore, increased risk of prostate cancer (around 6 in 10 prostate cancers cases are diagnosed in men over the age of 65).The diagnostic incidence of prostate cancer across the US, France, Germany, Italy, Spain, UK, Japan, Brazil, and Canada is expected to increase at an Annual Growth Rate (AGR) of 3.6 per cent from 2013 to 2023 (GlobalData Report, 2015).

Thus, the diagnostic and screening market for prostate cancer is estimated to reach over US$17 billion by 2017 (BCC Research, 2013). The unmet need for reliable and highly-specific diagnostic tools indicates a requirement for new diagnostic approaches aimed at overcoming the two main problems associated with current Prostate Specific  Antigen (PSA)-based testing: high over-diagnosis rates and inability to predict aggressive disease.

Current Standard for Early Detection

Prostate cancer can be detected at early stages by Digital Rectal Examination (DRE) or the PSA test. During the DRE a doctor inserts a gloved finger into the rectum to feel the prostate surface for swelling, harder areas or lumps that can indicate the presence of a tumour. Men with abnormal DRE should be referred to an urologist for further testing, regardless of PSA results. PSA, a serine protease kallikrein protein secreted by the prostate epithelial cells, is involved in seminal liquefaction. PSA testing measures the levels of this protein in blood. Normally, serum PSA levels are low in healthy men, but they increase if there is a disruption of the basement membrane of the prostate gland (e.g. during prostate cancer) and PSA is released into the peripheral circulation. PSA levels under 4 ng/mL are considered ‘normal’; levels between 4 and 10 ng/mL are considered ‘intermediate’, with cancer present in 30-35 per cent of patients; and PSA levels over 10 ng/mL are considered ‘high’, with a 67 per cent probability of advanced disease (National Comprehensive Cancer Network, 2016). DRE and PSA tests are only indicators of cancer risk and, if abnormal results are obtained, a biopsy is often recommended to examine prostate tissue samples for cancer cells. If cancer cells are present, the pathologist will assign a Gleason score on a scale of 2 to 10, based on the appearance of cancer cells compared to normal cells, to help evaluate the prognosis of patients.

The Need for Better Diagnostic Tests

Despite PSA having been considered the gold-standard biomarker for the detection of prostate cancer for almost two decades, PSA screening has led to a high rate of over-diagnosis resulting in men with indolent disease undergoing unnecessary biopsies and subsequent treatment (Etzioni et al., 2002). This is because PSA is not specific for prostate cancer, and can be elevated in other prostate conditions, such as prostatitis or Benign Prostatic Hyperplasia (BPH).

PSA Derivatives and Isoforms

The specificity of PSA for prostate cancer can be improved due to the fact that PSA can be found free in serum (free PSA, fPSA) or bound to other serum proteins (complexed PSA, cPSA) and that complexed forms (mainly bound to α1-antichymotrypsin) are more abundant in prostate cancer. Therefore, the free to total (fPSA plus cPSA) ratio could help to differentiate prostate cancer from benign prostatic diseases such as BPH. In current clinical practice, the percentage of fPSA is used to avoid unnecessary biopsies in men with normal DRE and total PSA levels between 4-10ng/mL. In this group of patients, a ‘cut-off’ point of 25 per cent fPSA can detect 95 per cent of prostate cancers while reducing unnecessary biopsies by 20 per cent (National Comprehensive Cancer Network, 2016).

Furthermore, it is now known that free PSA can be found in three different isoforms (Figure 1): pro-PSA, accounting for 33 per cent of fPSA; benign PSA (BPSA), which comprises 28 per cent fPSA; and inactive PSA (iPSA) (Özen et al., 2006). ProPSA is the precursor form of PSA and it is composed of inactive truncated forms ([–2]pPSA, [–4]pPSA and [–5]pPSA), as well as the native form, [-7]pPSA, which contains a seven amino acid pro-leader peptide that is removed after its release into the prostate lumen to be transformed to active PSA by the action ofhK2 and hK4. Some commercial tests, such as the FDA-approved 4Kscore test (OPKO Health, Inc.) have explored the potential of combining a panel of prostate-related kallikreins to improve PCa detection. The 4Kscore test combines four prostate-specific kallikrein (fPSA, iPSA, tPSA and hk2) assay results with other clinical information (age, DRE and prior biopsy results) in an algorithm that calculates the individual patient’s percent risk for aggressive prostate cancer (Gleason score ≥7) on biopsy. In a recent multi-institutional prospective study, data from 1012 men scheduled for prostate biopsy was used to confirm the utility of the 4Kscore test to detect significant prostate cancer (AUC = 0.82). Furthermore, using various 4Kscore thresholds, the number of biopsies could have been reduced by 30–58 per centwhile delaying the diagnosis on only 1.3–4.7 per cent of aggressive cancer cases (Parekh et al., 2015).

In addition to this, numerous studies suggest that [–2]pPSA is a cancer-specific form, significantly elevated in the peripheral zone of the prostate (where more cancers occur) and in the serum of prostate cancer patients (Hori et al., 2013; Filella et al., 2015). Further evidence for the association of [–2]pPSA with prostate cancer is provided by the FDA-approved Prostate Health Index (PHI), currently available from Beckman Coulter Diagnostics. This non-invasive blood test measures serum PSA, fPSA and [–2]pPSA to help physicians distinguish prostate cancer from benign conditions. With more than 80 clinical studies published, PHI provides risk stratification to aid in decision-taking management when PSA is in the 4–10 ng/mL range.

Focus on Multiplexing

As seen with previous examples, multiplexing paves the way towards the development of improved diagnostic and prognostic strategies for prostate cancer. The benefits of using multiplexed assays include improved differential diagnosis of malignant diseases, due to the use of biomarker panels, and enhanced cost-effectiveness, as a result of saving time and reducing sample volume requirements

Emerging commercial tests for prostate cancer detection and management are exploiting the advantages of multiple-marker detection approaches (Table 1). Further information on these tests can be found in our recent review (Sharma et al., 2017). Most have already been CE marked, FDA cleared and/orCLIA-waived for different clinical applications, from diagnosis and biopsy recommendations to prognosis and risk stratification following a positive biopsy result. However, the majority of them are genomic- or proteomic-only based assays. New efforts should focus on the combination of protein and nucleic acid markers to allow the investigation of tumorigenesis-associated changes that occur at both transcriptional and translational levels. Multiplexing technologies are continuously evolving and platforms like the Verigene system (Nanosphere, Luminex Corporation) ornCounter Vandage 3D assays (NanoString Technologies) enable ultra-sensitive, multiplexed detection of both protein and nucleic acids using a single platform. The gold nano particle-based Verigene system is used for ‘on-site’ diagnostics of protein (Biobarcode detection) and nucleic acid (direct genomic detection). Despite the main application of this system is in the field of pathogen detection, Verigene assays could be easily configured and implemented for a wide array of potential biomarkers (Shipp, 2006). The NanoStringn Counter Vantage 3D, recognised by The Scientist as a Top 10 Innovation of 2016, uses an optical barcoding technology for simultaneous DNA mutation detection, RNA and protein expression detection and even protein phosphorylation status. NanoString’s technology has already proved useful for medium throughput validation of prognostic markers and therapeutic targets in prostate cancer (Lee et al., 2016).

Autoantibody Signature

When cells become cancerous, they undergo a series of transformations that can result in the synthesis of tumour-associated antigens (TAA). Our body’s immune system responds by producing specific autoantibodies (AAbs) to these TAA. These autoantibodies are emerging as promising biomarker candidates due to their high specificity, their easy detection in serum and their presence during the initial, otherwise undetectable, stages of tumorigenesis. Several prostate cancer associated AAbs have been identified, includingNY-ESO-1, XAGE1b, SSX-2 and 4, AMACR, p90, LEDGF, TARDBP, TLN1, PARK7, CALD1, TTLL12, p62, Koc, Cyclin B1, PKACA, HIP1,Survivin, MUT, RAB11B, CSRP2, SPOP, RalA, ZNF671, ERB, HERV-K, PSA and HER2 (McNeel et al., 2000; Rastogi et al., 2016).To increase the sensitivity and specificity of these autoantibodies for a particular stage and type of cancer, multiplexing approaches are especially useful. An AAb panel like this has already successfully been validated for early diagnosis of bowel cancer by DCU scientists from the Applied Biochemistry Group (Dublin City University).

Targeting glycosylation in cancer

Changes in glycan (sugar chain) structures in glycoprotein biomarkers during the tumorigenesis process are also a main focus of cancer diagnostics research. Glycosylation is the most common post-translational modification in proteins and the collection of glycans present in cells at a particular time, known as the glycome, can be characteristic of various stages of tumorigenesis. Aberrant glycosylation patterns in glycoprotein prostate cancer biomarkers such as PSA, prostatic acid phosphatase (PAP), prostate stem cell antigen (PSCA), prostate specific membrane antigen (PSMA) and haptoglobin (Hp) have been reported as a result of oncogenic transformation (Belický and Tkac, 2016). Analysis of glycans has traditionally required sophisticated instrumental techniques, such as mass spectrometry (MS), capillary zone electrophoresis (CZE), high performance liquid chromatography (HPLC) or ultra performance liquid chromatography (UPLC). In recent years, lectin-based biosensors are getting attention as a feasible alternative to such systems for biomarker glycoprofiling.In contrast to more complex glycan analysis strategies like MS, lectin-based assays can discriminate different glycan structures on intact glycoproteins and even viable cells, because they do not require chemical or enzymatic separation of the glycan from the starting material. Enzyme-linked immunosorbentlectin assays (ELLA) are conceptually similar to standard enzyme-linked immunosorbent assays (ELISA), but they rely upon non-immunogenic glycan-binding proteins (lectins), instead of antibodies (immunoglobulins). Lectins can also be used for the analysis of the glycosylation state of particular proteins that had been previously captured by an antibody. Panels of proteins can be probed in parallel using antibody-lectin sandwich arrays (ALSA) to examine protein glycan alterations associated to pathological conditions (e.g. tumorigenesis),  which is especially valuable for cancer biomarker studies (Haab, 2012). However, the use of lectins may be limited due to their lower affinities, which in return leads to poor sensitivity, and their limited availability, particularly those specific to less common glycan structures. In order to overcome these limitations, companies such as GlycoSeLect Ltd. are now genetically engineering lectins with wider specificities and improved affinities. Electrochemical biosensors with a “sandwich” format in which the analyte of interest is captured by immobilised antibodies on the electrode for subsequent glycoprofiling using lectins could provide a means of highly sensitive biomarker detection coupled to glycan analysis.

Conclusion

The ambiguity associated with total PSA test-based prostate cancer diagnosis is probably the main driving force behind the search for new biomarker panels to distinguish between cancerous and non-cancerous conditions. The use of multiplexed tests based on PSA isoforms and additional cancer-specific biomarkers and glycosylation patterns may help reducing unnecessary biopsies while enhancing the capability for early detection of aggressive disease.

Acknowledgements

We would like to acknowledge support from the European Commission FP7 Programme through the Marie Curie Initial Training Network PROSENSE (grant no. 317420, 2012–2016) and the Biomedical Diagnostics Institute (BDI) though Science Foundation Ireland (SFI) under Grant No. 10/CE/BE1821.

References:

• BCC Research, 2013. Prevention and Treatment of Prostate Cancer: Technologies and Global Markets. Code - PHM113B
• Etzioni, R., Penson, D.F., Legler, J.M., Di Tommaso, D., Boer, R., Gann, P.H. and Feuer, E.J., 2002. Overdiagnosis due to prostate-specific antigen screening: lessons from US prostate cancer incidence trends. Journal of the National Cancer Institute, 94(13), pp.981-990.
• Filella, X. and Foj, L., 2015. Emerging biomarkers in the detection and prognosis of prostate cancer. Clinical Chemistry and Laboratory Medicine (CCLM), 53(7), pp.963-973.
• GlobalData Pharma Point, 2015. Prostate cancer–Global drug forecast and market analysis to 2023. Code - GDHC96PIDR.
• Haab, B.B., 2012. Using lectins in biomarker research: addressing the limitations of sensitivity and availability. PROTEOMICS-Clinical Applications, 6(7-8), pp.346-350.
• Hori, S., Blanchet, J.S. and McLoughlin, J., 2013. From prostate?specific antigen (PSA) to precursor PSA (proPSA) isoforms: a review of the emerging role of proPSAs in the detection and management of early prostate cancer. BJU International, 112(6), pp.717-728.
• Lee, J., Yan, W., Young, D., Song, Y., Chen, Y., Katta, S., Mohamed, A., Ravindranath, L., Srinivasan, A., Cullen, J. and Kagan, J., 2015. Evaluation of prostate cancer biomarkers and therapeutic targets in FFPE specimens using the NanoString platform.Proceedings: AACR 106th Annual Meeting 2015, 75(15), pp.2016.
• McNeel, D.G., Nguyen, L.D., Storer, B.E., Vessella, R., Lange, P.H. and Disis, M.L., 2000. Antibody immunity to prostate cancer associated antigens can be detected in the serum of patients with prostate cancer. The Journal of Urology, 164(5), pp.1825-1829.
• National Comprehensive Cancer Network, 2016.NCCN Guidelines version 2.2016– Prostate cancer early detection.
• Özen, H. and Sözen, S., 2006. PSA isoforms in prostate cancer detection. European Urology Supplements, 5(6), pp.495-499.
• Parekh, D.J., Punnen, S., Sjoberg, D.D., Asroff, S.W., Bailen, J.L., Cochran, J.S., Concepcion, R., David, R.D., Deck, K.B., Dumbadze, I. and Gambla, M., 2015. A multi-institutional prospective trial in the USA confirms that the 4Kscore accurately identifies men with high-grade prostate cancer. European Urology, 68(3),pp.464-470.
• Rastogi, A., Ali, A., Tan, S.H., Banerjee, S., Chen, Y., Cullen, J., Xavier, C.P., Mohamed, A.A., Ravindranath, L., Srivastav, J. and Young, D., 2016. Autoantibodies against oncogenic ERG protein in prostate cancer: potential use in diagnosis and prognosis in a panel with C-MYC, AMACR and HERV-K Gag. Genes & Cancer, 7(11-12), p.394.
• Sharma, S., Zapatero-Rodríguez, J. and O'Kennedy, R., 2017. Prostate cancer diagnostics: Clinical challenges and the ongoing need for disruptive and effective diagnostic tools. Biotechnology Advances, 35(2), p.135-149.
• Shipp, G., 2006. Ultrasensitive measurement of protein and nucleic acid biomarkers for earlier disease detection and more effective therapies.Biotechnology Healthcare, 3(2), p.35

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Author Bio

Julia Zapatero Rodríguez

Julia Zapatero Rodríguez is the Project Manager at AbYBiotech, a Dublin City University spin-out company specialising in recombinant antibody production. Before joining AbYBiotech, she was part of the prestigious Marie Curie ITN project PROSENSE, a multi-disciplinary network which aimed to develop new diagnostic tools for prostate cancer.

Richard O’Kennedy

Richard O’Kennedy is a Professor at Dublin City University (DCU), has published extensively (220 peer-reviewed papers, 50 reviews, 40 book chapters, 2 books), has many collaborations with industry, 7 patents, multiple licences and many of his innovations have been licenced. He is Founder and Chief Scientific Officer of AbYBiotech.

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