Tesis sobre el tema "Biomarker of prostate cancer"

Successful treatment of prostate cancer (PCa) depends on early diagnosis and screening, which currently relies on the measurement of serum prostate specific antigen (PSA) levels. The overarching aim of the project was to generate molecularly imprinted polymers for PCa biomarkers, with subsequent integration with a sensing platform to allow for rapid, point of care detection and monitoring. The initial work involved the use of simple PSA epitopes for epitope imprinting using conventional imprinting techniques. A four amino acid sequence from the Cterminus of PSA was imprinted with MAA, Aam and Urea monomers to obtain bulk imprinted polymers. Apparent Kd of 102 μM, 154 μM, 194 μM was obtained for MAA, AAm, Urea based bulk mini-MIPs respectively. Epitope imprinting was further developed using a surface imprinting approach, via electropolymersiation of dopamine to detect an epitopic sequence from pro-PSA. An improvement in Kd from bulk-imprinted polymers, with an apparent Kd of 2.9 μM was obtained with the surface electrochemical MIP sensor. However, both epitope imprinting technique lacked sensitivity to measure clinical relevant concentrations of PSA (nM range). As a consequence, a more sophisticated technique called hybrid imprinting was developed to build an electrochemical MIP sensor. Hybrid MIP imprinting utilised an aptamer with established affinity towards PSA to trap the aptamer-PSA complex into a surface grown electropolymer (polydopamine). The resulting aptamer lined polymer pockets exhibited high selectivity and affinity towards PSA (apparent Kd 0.3 nM). The apta-MIP sensor was also able to discriminate from a homologous protein (human Kallikrein 2) and was resilient to fouling from serum proteins. The apta-MIP sensor was further translated to a MOSFET device whereby successful detection of PSA at clinically relevant concentration was obtained in human plasma. Although good sensitivity and selectivity was obtained with the hybrid-MIP sensors, further research is required to understand the binding mechanism of the template to the MIP.

Prostate cancer is a leading cause of cancer-related death in men, and while many men diagnosed with the disease will have an indolent clinical course, 20-25% of men will experience disease recurrence which is invariably lethal. There is an urgent need for prognostic biomarkers that will predict disease recurrence and risk-stratify patients upon diagnosis, allowing for personalised therapies. This thesis attempts to identify new prognostic biomarkers for prostate cancer and investigates their patterns of protein expression in human primary prostate tumour tissue. Cancer stem cells are cancer cells thought to be uniquely capable of self-renewal and tumorigenicity, and may have a role in tumour recurrence. Using a literature searching approach, potential biomarkers related to stem cells, cancer stem cells or recurrence in prostate cancer were identified, and ALDH7A1, BMI1, SDC1, MUC1-C, Nestin and ZSCAN4 were chosen for investigation. An in silico approach was also used for biomarker identification, with RS1 and SLC31A1 selected as their mRNA was found to be upregulated in recurrent tumours. The expression patterns of all 7 potential biomarkers were examined by immunohistochemistry on prostate tumour tissue and benign tissue from prostate biopsies and prostatectomies. BMI1, ALDH7A1, MUC1-C and Nestin showed no relationship to recurrence or other clinical features. RS1 protein levels increased in patients with recurrence within 5 years, negatively correlated with AR expression, and a meta-analysis showed that the RS1 gene was amplified in up to 32% of castration-resistant prostate tumours. ZSCAN4 was heterogeneously expressed in a subset of 26% of prostate tumours with unclear characteristics and was not expressed in benign tissue, but was not associated with recurrence. Finally, SDC1 expression was lost in tumour epithelium, but a population of unidentified SDC1-expressing cells were found in the stroma of a third of tumours, and an increased burden of these cells was associated with primary Gleason pattern 5 tumours. These cells do not overlap with common epithelial, mesenchymal or stromal lineages, but may be migratory. In summary, the data presented in this thesis identifies 3 potential new biomarkers for prostate cancer, and provides the basis for future characterisation of their wider roles in the disease.

Abstract   The prostate gland, a male organ, situated right under the urine bladder, is involved in male reproduction. It can also be the place for more or less serious diseases such as inflammation, abnormal growth and cancer. Especially prostate cancer is very common in the Western world. Today PSA is the most widely used marker for detection of prostate cancer. Unfortunately, this method is not specific enough. Therefore, there is a need for a better marker for screening of malignant prostate cancer. The marker should be specific both for the organ prostate and for the cancer disease. One promising marker is the prostasome, a small vesicle emanating from epithelial cells in the ejaculatory ducts in the prostate. The aim of this project was to set up an ELISA and test a number of antibodies for their ability to work as suitable capture or detection antibodies. As blocking agent different concentrations of BSA were tested. Biotin-Streptavidin conjugate was used in the detection step. Two surface proteins, PSCA and PSMA were used as capture antigens; they are specific for prostasomes. Clusterin, a prostasomal surface-bound protein, was used as antigen for the secondary antibody in the assay. With this experimental setup the detection limit was 2500ng/mL, which is probably not enough to detect prostasomes in cancer. The development of the ELISA did not reach its final stage, a ready-to-use assay, during this project. We have not yet the knowledge of optimal antibody concentrations and the other test parameters are also at experimental state.

In metastatic castration-resistant prostate cancer (mCRPC), elevated plasma sphingolipids and a 3-lipid signature (3LS) are associated with worse clinical outcomes. The thesis aims to: assess the association between 3LS, somatic genetic aberrations and clinical outcomes in mCRPC; determine whether simvastatin therapy modulates the circulating lipidomic profile in men with mCRPC; and characterise the effect of extracellular sphingolipids on prostate cancer cells in vitro. Elevated circulating sphingolipids were found to be associated with AR, TP53, RB1 and PI3K aberrations in mCRPC, which are somatic aberrations linked to poor outcomes. Notably, the combination of lipid and genetic abnormalities conferred a worse prognosis. This suggests that certain genotypes in mCRPC may benefit from metabolic therapies. A proof-of-concept study of simvastatin in addition to standard therapy for mCRPC showed that simvastatin can modulate the circulating lipid profile by reducing plasma sphingolipids. Simvastatin eliminated the 3LS in almost half the men with this poor prognostic biomarker, thus demonstrating its potential as a metabolic targeting strategy. Treatment of prostate cancer cells with plasma from men with mCRPC and the 3LS increased cell viability in vitro. This phenotype was associated with gene expression and lipidomic changes that likely involve the NF-κB, sphingosine-1-phsosphate and sphingomyelin pathways. Therefore, plasma lipids may have a direct effect on prostate cancer growth and be a potential therapeutic target. In conclusion, elevated plasma sphingolipids are associated with worse clinical outcomes in mCRPC, providing prognostic information in addition to established genomic biomarkers. Extracellular sphingolipids increase prostate cancer cell viability, potentially through activation of ceramide metabolism towards pro-survival sphingolipids. This can be therapeutically targeted with simvastatin, which can eliminate the poor prognostic 3LS in men with mCRPC.

Prostate cancer (PCa) is the most commonly diagnosed cancer amongst Western males. PCa progression is strongly linked to steroid receptor signalling, however the modulation of steroid receptor expression in PCa is incompletely understood. Lipocalin 2 (LCN2) is a secreted protein which binds to Fe3+-containing siderophores and was originally identified as part of the innate immune response. LCN2 has been proposed as a potential biomarker for a range of cancers. However, LCN2 effects appear to be tissue specific. LCN2 expression is associated with poor prognosis in breast cancer, but with good prognosis in pancreatic cancer where it has been used therapeutically. The role of LCN2 in prostate cancer is poorly understood, in particular its effects on steroid receptor regulation. To elucidate the role of LCN2 in prostate cancer, the LCN2 gene was ectopically expressed in LNCaP cells to generate the LNCaP-LCN2 cell line. LNCaP-LCN2 cells had elevated androgen receptor expression which was linked to increased levels of KLK3 (PSA). LNCaP-LCN2 cells also had reduced levels of Estrogen receptor α (ERα), but increased expression of ERβ. This was combined with higher levels of E-cadherin, but not to changes in other EMT markers. Reciprocally, LCN2 was suppressed using RNAi in the PC3 cell line to generate PC3-shLCN2 cells. PC3-shLCN2 displayed a distinct change in morphology, with increased cell size and a sub-population of multi-nucleated and highly enlarged cells. PC3-shLCN2 cells had reduced proliferation, and lost the ability to form colonies in a 3D substrate. With regards to steroid receptors, PC3-shLCN2 cells had increased ERα expression, but reduced ERβ expression. This was also combined with a loss of E-cadherin and EGFR. Microarray analysis of PC3-shLCN2 cells identified changes to expression of a wide range of genes including VEGF-R, SPARC and KLK6. Functional grouping of differentially expressed genes suggests that LCN2 in involved in a range of cellular processes including hormone receptor response, Wnt signalling and cell cytoskeletal integrity. Many, but not all genes identified by microarray were responsive to recombinant LCN2 protein indicating a paracrine function for the protein. Treatment of PC3 cells with the iron chelator Deferoxamine resulted in phenotypic changes similar to those found in PC3-shLCN2 cells which suggest that LCN2 functions in part due to intracellular iron regulation. In summary, the data presented in this thesis suggests that LCN2 has both pro- and anti- tumourigenic properties in prostate cancer and that the protein is involved in a much wider range of functions than previously described.

>Magister Scientiae - MSc
Prostate cancer is a deadly disease that occurs in the male’s prostate gland. A prostate gland is a walnut structure that forms part of the male’s reproductive system. Prostate cancer is caused by high level than normal of PSA (Gleason score > 4 ng ml-1) in human blood. Some symptoms associated with high levels of PSA include blood in urine, pain when urinating, difficulty in getting and keeping an erection, blood in semen and pain in upper thigh. An immunosensor is a type of biosensor that has an antigen or antibody fragment as its biological recognition component. The specificity of the molecular recognition of antigen by antibodies to form a stable complex is the basis of immunosensor technology. In this work, overoxidized polypyrrole (OvoxPpy) was electrosynthesized as a novel sensor platform on glassy carbon electrode (GCE). The OvoxPpy was then doped with osmium telluride quantum dots(OsTe2QDs) by drop-coating method to form OsTe2QDs|OvoxPpy|GCE system. The morphology and the size of OsTe2QDs|OvoxPpy|GCE nanocomposite were determined using scanning electron microscopy. The size of thioglycolic acid capped osmium telluride quantum dots (TGA-OsTe2QDs) used as support material for the biosensor was about 2.289 nm. These quantum dots showed an excellent photo-absorption properties with an ultraviolet- visible (UV-Vis) photo absortion band occurring at 406nm associated with high band energy of 3.05 eV. The electrochemical immunosensor for PSA was prepared by immobilizing anti- PSA-antibody onto the OsTe2QDs|OvoxPpy|GCE by drop-coating and allowing it to dry for 2h. The nanocomposite sensor platform and the immunosensor were electrochemically characterised by voltammetric and impedimetric techniques. The phase shift in Bode diagram at maximum frequency was indicative of kinetic changes. Charge transfer resistance, Rct, was used as the analytical parameter for measuring the interfacial kinetics which occurred as a result of the bio-recognition event between anti-PSA-antibody and PSA. The impedance of the quantum dot electrode (TGA-OsTe2QDs-Nafion|GCE) was lower (1.490 x 104 kΩ) than the impedance of the immunosensor platform (BSA-Anti-PSA-antibody|TGA-OsTe2 QDs|OvoxPpy|GCE), 2.754 x 104. The Rct of the immunosensor was found to increase with increasing concentration of PSA. The linearity of the immunosensor at the very low concentration range (1.266 - 4.207 fg ml-1) tested, confirms its high sensitivity for PSA.

Prostate cancer (PC) is the most commonly diagnosed non-cutaneous cancer in men in developed countries. Although most prostate cancers behave in an indolent fashion, approximately 20% are more aggressive, a situation which is reflected in the large gap between the incidence and mortality figures. The 42 publications included in this thesis revolve around a key issue in the management of prostate cancer: how to identify those cancers which are more likely to manifest adverse behaviour so that men with such tumours can be offered appropriate therapeutic interventions, while those patients with more indolent cancers can be managed with surveillance and avoid the risk of toxicities associated with more intensive treatments. The first section includes studies of morphological parameters, including tumour grade, local spread and histological type, as well as proposing the novel prognostic factor of Gleason pattern at a positive surgical margin. The latter has now been incorporated into routine clinical practice and is included in the 2016 WHO Classification and College of American Pathologists reporting protocol for prostate cancer. Other articles in this section analyse structured reporting protocols for prostate cancer, analyse deficiencies and discrepancies, and propose evidence-based international standards. In section 2 the ability of molecular biomarkers to supplement or replace traditional morphological parameters in predicting biological behaviour is investigated. Several of these publications focus on AZGP1, which was found to be a robust independent predictor of prostate cancer recurrence in both retrospective and multicentre prospective studies. The final section includes investigations of the molecular mechanisms underpinning the progression of prostate cancer, since an understanding of these mechanisms facilitates the identification of potential biomarkers that stratify patients into different risk categories, enabling better targeting of therapy.

Serum prostate-specific antigen and Gleason grade are parameters routinely used for risk stratification in prostate cancer (PCa), but they present some limitations in the prediction of disease progression and in their use to guide clinical decision making. Prostate cancer stem cells (PCSCs) are widely considered to be responsible for tumorigenesis, disease progression and therapy failure. Their identification and characterization are mandatory for understanding the intricate intratumoral heterogeneity (spatial and molecular) of PCa and for developing relevant clinical tools to effectively manage patients and tailor therapy. Here, we proposed the surface glycoprotein neural cell adhesion molecule (NCAM1/CD56), a known marker of neuroendocrine (NE) cells, as a novel PCSC marker that provides prognostic information and molecular insights into the process of tumorigenesis. NCAM1 defines clusters of cells enriched in proliferative inflammatory atrophy (PIA) regions, without NE traits (hereafter referred as NCAM1+). In a retrospective cohort of 406 PCa patients treated with radical prostatectomy (RP), we uncovered that NCAM1 is an independent prognostic marker for predicting distant metastasis and biochemical recurrence and its expression in radical prostatectomy biopsies concurred with diagnostic biopsies (concordance 87.6%). Using the human cell lines, LNCaP (androgen-sensitive) and DU145 (androgen-insensitive), we found that NCAM1, but not other candidate PCSC markers, allowed FACS-based prospective purification of PCa cells displaying i) unique self-renewal ability in vitro, in a serial 3D-Matrigel organoid propagation assay, ii) tumorigenic potential upon limiting dilution transplantation in vivo. Relevant to real-life human PCa, we found that the ability to generate primary-derived organoids (PDOs) from dissociated high Gleason PCa biopsies exclusively resided in the purified NCAM1+ cell fraction, a property that was efficiently inhibited by treatment with an anti-NCAM1 blocking monoclonal antibody. We also found that the progressive development of adenocarcinoma in transgenic TRAMP mice crossed with NCAM1-/- mice was blocked at very early stages of tumorigenesis, indicating that genetic NCAM1 ablation prevents premalignant lesions to expand and progress to advanced stages. PCa is a paradigm tumor model for clinical, spatial and molecular heterogeneity and this heterogeneity is reflected in the NCAM1+ cell population. Single cell-RNA sequencing (sc-RNASeq) of purified NCAM1+ cells (cell lines and primary human PCa biopsies) uncovered heterogenous cellular states reflected in several distinct clusters. Phylogenetic tree reconstruction of the evolutionary relationship among the different clusters along with single cell trajectory analysis revealed the existence of a cell fraction with basal traits (p63+/AR-/CD117+ cells) and a quiescent phenotype, sitting at the apex of the hierarchical structure of the NCAM1+ population. These cells were functionally characterized by Hedgehog signaling which drives NCAM1+/CD117+ -PCSC self-renewal ability. Moreover, they were molecularly characterized by a transcriptional signature called “Stem Score” which could have potential as a prognostic tool for identifying patients at risk of biochemical recurrence (BCR) and distant metastasis. Androgen deprivation therapy (ADT) is the standard management for advanced PCa. Despite its initial effectiveness, the majority of patients relapse and develop castration resistant prostate cancer (CRPC), which is thought to be mediated by resistant PCSCs. ADT-treated NCAM1+ cells isolated from both dissociated human PCa biopsies and the LNCaP cell line enter into a quiescent state and retained the ability to generate organoids in vitro and tumors in vivo, escaping the ADT-induced senescence observed in NCAM1- cells. By global transcriptional profiling RNASeq analysis of NCAM1-overexpressing LNCaP cells, followed up by high-resolution studies, we uncovered an NCAM1-FGFR2-FGF18 molecular circuitry and an NCAM1-DNER-Notch1 molecular circuitry that mediate resistance to ADT-induced senescence. Thus, targeting these pathways with FGFR or Notch pathway inhibitors could represent a promising strategy to eradicate ADT-resistant NCAM1+ cells and prevent CRPC. Taken together, these data highlight NCAM1 as a novel predictive-prognostic biomarker in PCa, which could significantly improve the clinical management of PCa patients and pointed out druggable molecular pathways that could be targeted to eradicate “true” NCAM1+-PCSCs (self-renewal) and stem-like NCAM1+ progenitors (ADT-resistance).

Introduzione: Il tumore alla prostata (PCa) è una delle neoplasie più comuni tra gli uomini nel mondo occidentale e, globalmente, rappresenta la sesta causa di morte dovuta a cancro. L'approccio diagnostico attuale si basa sulla misurazione dei livelli sierici dell’antigene prostatico PSA (prostate specific antigen), nonostante recenti studi clinici abbiano dimostrato che questo marcatore non riduce significativamente la mortalità associata a PCa. In questo scenario abbiamo ipotizzato che i microRNA possano essere nuovi potenziali biomarcatori di PCa. Scopo del lavoro: L'identificazione di microRNA, coinvolti nella progressione neoplastica del tumore prostatico come nuovi biomarcatori diagnostici aggiuntivi al PSA, prognostici e predittivi di aggressività tumorale. La nostra strategia sperimentale ha previsto l’uso di linee cellulari di prostata non tumorigeniche e a diverso grado di malignità, modelli murini di carcinoma prostatico e casistiche di pazienti affetti da PCa. Materiali e metodi: Linee cellulari commerciali: analisi dell'espressione globale dei miRNA tramite piattaforma low-density array nelle linee di PCa (LNCap, PC3, DU145), normali o iperplastiche (RWPE-1 e BPH-1). Casistiche cliniche: analisi di miRNA selezionati, in un set di pazienti (n=58) nei tessuti di parenchima normale, pre-neoplastico (prostatic intraepithelial neoplasia, PIN) e tumorale. I risultati ottenuti sono stati correlati a parametri clinicopatologici dei pazienti. I potenziali target proteici dei miRNA selezionati sono stati valutati in una casistica più ampia mediante tissue micro-array (TMA). Modello murino transgenico TRAMP: analisi globale di espressione dei miRNA in ghiandole di PIN e tumorali, e nello stroma associato. Linee cellulari primarie: ottenimento di linee di fibroblasti derivate da resezioni chirurgiche di PCa (n=10). Risultati: Dallo screening nelle linee cellulari abbiamo selezionato 23 miRNA poi valutati nei 58 pazienti. Tredici miRNA hanno mostrato differenze di espressione significative (p<0.05). In particolare, nove miRNA (miR-135b,-193a-5p,-205,-224,-22,-34b,-34c-5p,-452,miR-886-3p) sono risultati progressivamente diminuiti nella progressione neoplastica (N-PIN-PCa). Viceversa, i miR-130a, -218, -532, -542-5p, -489 e let-7c hanno mostrato una diminuzione di espressione nel PIN rispetto al tessuto normale. È noto che i miR-205, miR-218, miR-224 abbiano come bersaglio proteico RUNX2 (Runt-related transcription factor 2), un fattore di trascrizione coinvolto nel tropismo osseo di cellule metastatiche. La valutazione di RUNX2 nei TMA di prostata ha rivelato che la positività nucleare è specifica delle cellule tumorali (p<0.0001) e correla con l’estensione del tumore e con l'invasione capsulare. Inoltre il confronto tra l’espressione dei miRNA e RUNX2 ha mostrato correlazione inversa significativa. Dall’analisi del modello TRAMP abbiamo identificato un pannello di miRNA differenzialmente espressi tra le componenti epiteliali e stromali associate a PIN o PCa (n=52). Conclusioni: I nostri risultati mostrano una sinergica perdita di miRNA con funzione oncosoppressiva e contemporaneo aumento di RUNX2 nei tessuti di PCa. Questo dato ha importanti implicazioni a livello di progressione di malattia che sarà valutata in una successiva fase del progetto. Le nostre analisi hanno indicato il miR-205 come potenziale marcatore di aggressività di malattia. Hanno inoltre identificato 9 miRNA precocemente persi nelle lesioni precancerose (PIN) rispetto al parenchima sano. Studi in casistiche indipendenti potranno confermare queste molecole come nuovi biomarcatori di neoplasia da affiancare alla valutazione del PSA. Inoltre lo studio dei miRNA “stromali” ha evidenziato una profonda deregolazione di queste molecole nel microambiente tumorale rispetto a quello non neoplastico. Questo risultato sottolinea dal punto di vista molecolare l’importanza dello stroma nel sostenere la sopravvivenza e la crescita tumorale e fornisce una possibile strategia terapeutica alternativa, mirata alle cellule stromali anziché epiteliali per indurre regressione di malattia. Saranno necessari ulteriori studi per valutare il ruolo dei miRNA nell’interazione tra epitelio tumorale e stroma circostante.
Introduction: Prostate cancer (PCa) is one of the most common cancers among men and the sixth cause of cancer-related death in men worldwide. The current diagnostic approach is based on serum measurement of prostate specific antigen (PSA) levels, despite recent clinical studies showed that did not considerably reduced mortality incidence in prostate cancer patients. In this scenario, we hypothesized that microRNAs (miRNAs) could be novel biomarkers for PCa disease. Aim of the study: We propose to identify miRNA signatures associated to PCa progression that could represent a novel generation of diagnostic biomarkers adjunctive to PSA, prognostic and predictive of cancer progression. Our experimental strategy included the use of normal or tumorigenic prostate cell lines, mouse model of PCa and patients’ series. Methods: Prostate cell lines: global miRNA expression analysis using a low-density array platform in PCa (LNCap, PC3, DU145) or non-tumorigenic cells (RWPE-1, BPH-1). Clinical series. Analysis of selected miRNAs in 58 PCa patients for which normal parenchyma and prostatic intraepithelial neoplasia (PIN) was available. Correlation of molecular profiles to clinicopathological characteristics. Potential miRNA targets were investigated using a larger series of PCa patients arranged in tissue micro-arrays (TMAs). TRAMP mouse: global miRNA profiles were obtained from epithelial and stromal compartments of PIN or tumoral lesions. Primary cell lines: fibroblasts were obtained from prostate resection of PCa patients (n=10). Results: miRNA screening in cell lines provided a panel of 23 miRNAs that were then investigated in the 58 PCa patients. Thirteen miRNA displayed significant deregulation (p<0.05) in disease tissues. Specifically nine miRNAs (miR-135b,-193a-5p,-205,-224,-22,-34b,-34c-5p,-452, miR-886-3p) were progressively down-regulated during neoplastic progression (N-PIN-PCa). Conversely, miR-130a, -218, -532, -542-5p, -489 and let-7c displayed lower levels in PIN compared to normal prostate. A recognized target of miR-205, miR-218 and miR-224 is the Runt-related transcription factor 2 (RUNX2), a protein involved in metastatic dissemination to the bone. In our patients’ TMA, RUNX2 was overexpressed in tumoral cell nuclei (p<0.0001) and it was related to tumor size and capsular invasion. Moreover RUNX2 was inversely related to miRNA levels. TRAMP mice analysis has provided a signature of miRNAs (n=52) differentially expressed in epithelial and stromal compartments of PIN or PCa cells. Conclusions: Our results show a simultaneous loss of oncosuppressive miRNAs and increased RUNX2 expression in PCa tissues. This data is particularly relevant in disease progression monitoring, an aspect that will be studied in future project’s phases. Our analysis showed that miR-205 loss is a potential biomarker of aggressive disease. Furthermore, we identified nine miRNAs which expression is decreased from early stage of disease (PIN). Validation of this result in independent patients’ series could provide novel biomarkers of PCa useful as adjunts to PSA monitoring. Lastly, profound stromal miRNAs deregulation underlines the importance of tumour microenvironment in sustaining cancer cell survival and growth. Moreover this result suggest that targeting tumour stroma could represent an alternative strategy for anti-cancer therapies. Future studies are needed to shed light on this aspect.

The introduction of prostate-specific antigen (PSA) testing about 3 decades ago led to the possibility of early detection of prostate cancer (PCa). Although PSA testing reduced the mortality rate, it is also associated with high risk of over diagnosis in patients with and without PCa. Despite the current drawbacks, it would be a challenge to replace PSA testing entirely. Instead, there is a need to develop parallel testing of other potential biomarkers that can complement the results from PSA tests. To address alternative biomarker sensing, this thesis highlights on the development of oligonucleotide-based biosensors for the detection of different biomarkers of PCa. Using PSA as a gold standard, the first study of this dissertation investigates the use of DNA aptamers to detect PSA using electrochemical impedance spectroscopy (EIS). The study compares 6-mercapto 1-hexanol chemistry with sulfo-betaine chemistry for the development of PSA aptasensor in terms of performance and selectivity. The second study focuses on glycoprofiling in order to complement PSA quantification as an additional information for reliable PCa diagnosis. This strategy was developed in a microfluidic channel with an optical read out using chemiluminescence. This study addresses one of the major problems of cross-reactivity with lectins in glycoprofiling, which can be solved using DNA aptamers. A third study concentrates on the development of an aptasensor for Alpha-Methylacyl-CoA Racemase (AMACR). AMACR has been reported for its high specificity and sensitivity to PCa. For the fabrication of the biosensor, a new strategy using polyethylene glycol was developed by electrochemical grafting it to a polypyrrole film. Since PCa diagnosis can be improved by looking at different biomarkers, an electrochemical platform for miRNA/DNA detection using a gold nanoparticle amplification strategy was also investigated. The sensor was fabricated using peptide nucleic acids (PNA) probes on gold electrodes. The study presents non-Faradaic EIS and amperometric techniques in order to exploit the inherent charges of nucleic acids. In conclusion, this thesis wants to serve as a potential orientation for overcoming the shortcomings of the current PCa testing and contribute towards the development of oligonucleotide-based biosensors for PCa biomarker detection and hopefully enhance the diagnosis and prognosis of PCa.

Il carcinoma della prostata è uno dei tumori più frequenti e rappresenta quasi il 30% di tutti i tumori di nuova diagnosi nel sesso maschile. La concomitanza di fattori quali l'elevata mortalità, la tardività della diagnosi clinica abituale ed i benefici della diagnosi precoce hanno negli ultimi tempi suscitato un notevole interesse per iniziative sistematiche di diagnosi precoce e di screening. La ricerca di una o più sostanze potenzialmente utili per identificare i pazienti con un cancro della prostata o per stabilire la prognosi della neoplasia non ha messo in evidenza fin’ora un marker completamente soddisfacente. Circa il 20-30% dei tumori della prostata non sono associati ad elevati livelli di PSA. Il PSA, infatti, si innalza anche in caso di infiammazione della prostata o di ipertrofia prostatica benigna, e in caso di massaggio o manipolazione prostatica. Lo studio del profilo genico del tessuto prostatico permette di identificare biomarcatori specifici utili nella diagnosi. Il tessuto prostatico è stato ottenuto da 30 pazienti sottoposti a biopsia prostatica. Il livello sierico di PSA è compreso tra 2,68 ng/ml e 100ng/ml. Tramite Real Time PCR, abbiamo determinato l’espressione dei geni ODC1, DPP4, IMPDH1 e 2, ZIP1, ZIP2, ZIP3, ZIP4. La valutazione quantitativa è stata ottenuta tramite Light Cycler 1.5. Dal nostro studio è emerso chiaramente che in caso di PCa è presente una down-regulation dell’espressione dei geni ZIP, trasportatori dello zinco. Questo dato potrebbe risultare utile nella diagnosi e prognosi del tumore della prostata ed i geni ZIP potrebbero essere utili in qualità di biomarcatori tumorali.
Prostate cancer is the most common cancer among men and it is a significant cause of morbidity and mortality worldwide. Screening for prostate specific antigen has led to earlier detection of prostate cancer. However PSA is neither tissue specific. Thus the serum PSA screening is characterized by poor specificity as well as poor sensitivity. This low specificity of PSA is a reason of marker improvement. Therefore it is of prime interest to develop clinical markers with a superior specificity for prostate cancer lesions for use in the initial diagnosis. Characterization of gene expression profiles that molecularly distinguish prostatic neoplasms may identify genes involved in prostate carcinogenesis. For this study, we determined the expression level of ODC1, DPP4, IMPDH1, IMPDH2, ZIP1, ZIP2, ZIP3 & ZIP4 by means of Real-Time PCR (qPCR). Quantitative detection of human genes was performed with a Light-Cycler 1.5 Instrument. Prostate tissue specimens were obtained from 30 patients undergoing prostate needle biopsy. These included 14 patients who were diagnosed for Adenocarcinoma, 14 who had a diagnosis of benign prostate hyperplasia (BPH), and 2 of prostatic intraepithelial neoplasia (PIN). The serum PSA levels of these patients were determined and all patients had a range between 2,68ng/ml and 100ng/ml (mean PSA value=13,95ng/ml). The mean age of the selected patients was between 43 and 80 years (mean age= 65,3years) and Gleason score between 0 and 8 ( mean score =3,1). Our results clearly establish that Zip1, Zip2, and Zip3 mRNA are down regulated in malignant prostate glands and up regulated in BPH. This is the first report that identifies the expression of Zip1, Zip2, Zip3 and Zip4 in human prostate needle biopsy. The down regulation of these transporters in the malignant cells is essential for the cellular depletion of zinc to prevent the anti tumor effects of zinc. These findings are consistent with the concept that Zip1, Zip2 and Zip3 are tumor-suppressor genes in prostate cancer. The identification of new prostate cancer specific genes such as ZIP genes would represent a considerable advance in the improvement of diagnostics tests for prostate cancer.

>Magister Scientiae - MSc
Prostate Cancer is the leading cause of cancer-related death in males in the Western world. It is a common biological disease originating from the reproductive system of the male namely, the prostate gland, usually in older patients (over the age of 50) and with a family history of this disease. The disease shows clinical aggressiveness due to genetic alterations of gene expression in prostate epithelial cells. Prostate cancer is currently diagnosed by biopsy and prostate cancer screening via the Prostate-Specific Antigen (PSA) blood test. Early detection is critical and although PSA was discovered to aid in the diagnoses of this cancer at its early stages, it has a disadvantage due to its low specificity thus causing unnecessary biopsies of healthy individuals and overtreatment of patients. Although various studies and efforts have been made to identify the ideal biomarker for prostate cancer and many even being applied to clinical use, it is still challenging and has not replaced the best-known biomarker PSA. PSA test has minimal invasive characteristics, at relatively low cost together with high sensitivity but low specificity. Biomarker discovery is a challenging process and a good biomarker has to be sensitive, specific and its test highly standardized and reproducible as well as identify risk for or diagnose a disease, assess disease severity or progression, predict prognosis or guide treatment. Computational biology plays a significant role in the discovery of new biomarkers, the analyses of disease states and the validation of potential biomarkers. Bioinformatic approaches are effective for the detection of potential micro ribonucleic acid (miRNA) in cancer. Altered miRNA expression may serve as a biomarker for cancer diagnosis and treatment. Small non-protein coding RNA, miRNA are small regulatory RNA molecules that modulate the expression of their target genes. miRNAs influence numerous cancer-relevant processes such as proliferation, cell cycle control, apoptosis, differentiation, migration and metabolism. Discovery and existence of extracellular miRNAs that circulate in the blood of cancer patients has raised the possibility that miRNAs may serve as novel diagnostic markers. Since a single miRNA is said to be able to target several mRNAs, aberrant miRNA expression is capable of disrupting the expression of several mRNAs and proteins. Biomarker discovery for prostate cancer of mRNA and miRNA expression are strongly needed to enable more accurate detection of prostate cancer, improve prediction of tumour aggressiveness and facilitate diagnosis. The aim of this project was to focus on functional analyses of genes and their protein products regulated by previously identified miRNA in prostate cancer using bioinformatics as a tool. Most proteins function in collaboration with other proteins and therefore this study further aims to identify these protein-protein interactions and the biological relevance of these interactions as it relates to Prostate cancer. Various computational databases were used such as STRING, DAVID and GeneHub-GEPIS for functional analyses of these miRNA regulated genes. The main focus was on the 21 genes regulated by several miRNAs identified in a previous study. Results from this study identified six genes; ERP44, GP1BA, IFNG, SEPT2, TNFRSF13C and TNFSF4, as possible diagnostic biomarkers for prostate cancer. These results are promising, since the targeted biomarkers would be easily detectable in bodily fluids with the Gene Ontology (GO) analysis of these gene products showing enrichment for cell surface expression. The six genes identified in silico were associated to transcription factors (TFs) to confirm regulatory control of these TFs in cancer promoting processes and more specifically prostate cancer. The CREB, E2F, Nkx3-1 and p53 TFs were discovered to be linked to the genes IFNG, GP1BA, SEPT2 and TNFRSF13C respectively. The expression of these TFs show strong association with cancer and cancer related pathways specifically prostate cancer and thus demonstrates that these genes can be assessed as possible biomarkers for prostate cancer. The prognostic and predictive values of the candidate genes were evaluated to assess their relationship to prognosis of this disease by means of several in silico prognostic databases. The results revealed expression differences for the majority of the candidate genes were not significantly sufficient to be distinguished as strong prognostic biomarkers in several prostate cancer populations. Although one marker, GP1BA was supported as having prognostic value for prostate cancer based on it's statistical pvalue in one of the prostate cancer patient datasets used. Another candidate gene SEPT2 showed promise as it has some prognostic value in the early stages of the disease. Although the results yielded, based on the in silico analysis, were not the discovery of an ideal diagnostic marker based on the set criteria in this study, further analysis using a molecular approach qRT-PCR can be considered for a detailed followup study on selected candidate genes to evaluate their roles in disease initiation and progression of prostate cancer using cell lines as well as patient samples.
CSIR

Philosophiae Doctor - PhD
There are over 100 different types of cancer, and each of these cancers are classified by the type of cell that it initially affects. For the purpose of this research we will be focussing on prostate cancer (PC). Prostate cancer is the second most common form of cancer in men around the world and annually approximately 4500 men in South Africa are diagnosed making PC a global epidemic. Prostate cancer is a type of cancer which starts in the prostate it is normally a walnut-sized gland found right below the bladder. PC follows a natural course, starting as a tiny group of cancer cells that can grow into a tumour. In some men if PC is not treated it may spread to surrounding tissue by a process called direct invasion/ spread and could lead to death. Current diagnostic tests for prostate cancer have low specificity and poor sensitivity. Although many PC's are slow growing there is currently no test to distinguish between these and cancers that will become aggressive and life threatening. Therefore the need for a less invasive early detection method with the ability to overcome the lack of specificity and sensitivity of current available diagnostic test is required. Biomarkers have recently been identified as a viable option for early detection of disease for example biological indicators ie. DNA, RNA, proteins and microRNAs (miRNAs). Since first described in the 1990s, circulating miRNAs have provided an active and rapidly evolving area of research that has the potential to transform cancer diagnostics and prognostics. In particular, miRNAs could provide potentially new biomarkers for PC as diagnostic molecules. Circulating miRNAs are highly stable and are both detectable and quantifiable in a range of accessible bio-fluids, having the potential to be useful as diagnostic, prognostic and predictive biomarkers. In this study we aimed to identify miRNAs as potential biomarkers to detect and distinguish between various types of PC in its earliest stage. The major objectives of the study were to identify miRNAs and their gene targets that play a critical role in disease onset and progression to further understand their mechanism of action in PC using several in silico methods, and to validate the potential diagnostic miRNAs using qRT-PCR in several cell lines. The identification of specific miRNAs and their targets was done using an "in-house" designed pipeline. Bioinformatic analyses was done using a number of databases including STRING, DAVID, DIANA and mFold database, and these combined with programming and statistical analyses was used for the identification of potential miRNAs specific to PC. Our study identified 40 miRNAs associated with PC using our "in-house" parameters in comparison to the 20-30 miRNAs known to be involved in PC found in public databases e.g. miRBase. A comparison between our parameters and those used in public databases showed a higher degree of specificity for the identification PC-associated miRNAs. These selected miRNAs were analysed using different bioinformatics tools, and were confirmed to be novel miRNAs associated with PC. The identified miRNAs were experimentally validated using qRT-PCR to generate expression profiles for PC as well as various other cancers. Prostate lines utilised in this study included PNT2C2 (normal) which was compared to BPH1 (Benign) and LNCaP (Metastatic). In the study the expression profiles of eight potential miRNA biomarkers for the detection of PC was determined using qRT-PCR, and to distinguish PC from other cancers. QRT-PCR data showed that miRNA-3 and -5 were up-regulated in the BPH1 and LNCaP when compared to PNT2C2. In addition miRNA-8 was also shown to be up-regulated in LNCaP. Based on these results it was shown that a miRNA profile could be established to distinguish between BPH1 and the LNCaP prostate cell lines. The results suggest that one miRNA as a diagnostic marker may be sufficient to differentiate between different cancer cell lines. Furthermore by creating a unique profile for each cancer cell line by using a combination of miRNAs could be a suitable approach as well. Finally, it was shown that through the use of a single or combination of all eight miRNAs a unique profile for all the cancer cell lines tested in this study can be created. This is an important finding which could have potential diagnostic or prognostic implications in clinical practice.

Prostate cancer is one of the most challenging global medical issues today. In 2011, prostate cancer was the most diagnosed malignancy in the United States, making up 29% of new cancer cases. In that year it was the second leading cause of cancer related deaths among men in the USA and the second most common cause of cancer related death overall from the EU. The prostate remains, however, an under studied organ, making insights into the anatomy and biology of prostate cancer difficult to achieve. After 30 years, PSA screening of men of the appropriate age is still the first step in prostate cancer diagnosis, usually followed by a manual prostate exam which may lead to a transrectal biopsy. This study makes use of Next Generation Sequencing to successfully identify a superior miRNA based urinary assay for the detection of prostate cancer. A receiver operating curve AUC of 0.90 was achieved for patients vs. non-patients using an additive risk model defined by empirically derived critical threshold values of eight urinary miRNAs identified with this method. This is superior to the PSA blood test’s AUC of 0.66 which illustrates that a miRNA profile such as this has the potential to surpass protein biomarkers such as PSA in terms of specificity and sensitivity. It was also demonstrated that a geometric mean of three urinary miRNAs were useful for endogenous normalization. One significant risk factor for prostate cancer is being African American. Again using Next Generation Sequencing technology, we have established a miRNA expression profile for the stages of a prostate cancer cell line progression model derived from the normal prostate epithelium of an African American man. Normal prostate epithelium was immortalized only with SV40 large T antigen (P69) and passaged three times in nude mice, producing the highly aggressive and metastatic M12 cell line. The M2182 cell line is an intermediate between the P69s and M12s having only been passaged twice and not yet having acquired metastatic potential. The F6 cell line was derived by reintroducing a copy of chromosome 19 missing from the M12 cell line via microcell mediated chromosome transfer. These profiles show a large downregulation of miRNAs early in tumorigenesis (from P69 toM2182) affecting the DLK1-DIO3 megacluster and the miR-200 family. The later acquisition of metastatic potential (from M2182 to M12) is concomitant with the upregulation of specific miRNAs including the HOX gene miRNAs miR-10a and miR-196 and miR-9. Thus, the analysis of this progression model has uncovered relevant miRs and genes the dysregulation of which contribute to prostate tumorigenesis.

In practice, despite using existing clinicopathological biomarkers to predict outcomes, many localised prostate cancers are still over-treated. Hence improved markers in localised prostate cancer (PC) are urgently required. The aim of this thesis was to validate potential prognostic markers of localised PC, with an emphasis on zinc-alpha 2-glycoprotein (AZGP1) as a lipid-based biomarker. The thesis also explores the contributions of lipids in PC progression using various pre-clinical platforms. AZGP1 is putative biomarker in PC. Data from our prospective phase III validation study demonstrates that AZGP1 status at radical prostatectomy (RP) is predictive of outcomes. Samples from 347 patients undergoing RP were analysed for AZGP1 expression. Absent/low AZGP1 expression was an independent predictor of shorter metastasis-free survival. AZGP1 also improved the discriminatory value of existing prognostic risk models. Over decades, our research group has also contributed to other studies of molecular biomarkers in PC. These candidate biomarkers have been re-analysed in discovery and validation cohorts to examine for more clinically meaningful endpoints of metastasis and PC death. Out of 12 evaluable markers, AZGP1 and Ki67 were the key predictors of these clinically relevant outcomes. Importantly, this thesis demonstrates that PSA relapse is a poor surrogate for metastasis and death. AZGP1 is a known lipid-mobilising factor. Using an in vitro spheroid model and a novel ex vivo platform, we investigated the relationship between lipids and PC cells. With fatty acid co-culture, we demonstrated that there is gross uptake of fatty acids from the tumour microenvironment. We identified the role of external fatty acid uptake as the dominant component of energy transfer over de novo lipid synthesis from glucose and glutamine utilisation in PC. The resultant lipid uptake led to increased PC growth and proliferation.

BACKGROUND: Prostate cancer (PCa) is the most commonly diagnosed solid tumor and the second leading cancer death in the United States, and also one of the major cancer-related deaths in Chinese. Androgen deprivation therapy (ADT) is the first line treatment for metastatic PCa. PCa ultimately relapses with subsequent ADT treatment failure and becomes castrate-resistant (CR). It is important to develop effective therapies with a surrogate marker towards CR PCa. METHOD: Histone deacetylase (HDAC) inhibitors were examined to determine their effects in androgen receptor (AR)/ cellular prostatic acid phosphatase (cPAcP)-positive PCa cells, including LNCaP C-33, C-81, C4-2 and C4-2B and MDA PCa2b androgen-sensitive and androgen-independent cells, and AR/cPAcP-negative PCa cells, including PC-3 and DU 145 cells. Cell growth was determined by cell number counting. Western blot analyses were carried out to determine AR, cPAcP and PSA protein levels. RESULTS: cPAcP protein level was increased by HDAC inhibitor treatment. Valproic acid, a HDAC inhibitor, suppressed the growth of AR/cPAcP-positive PCa cells by over 50% in steroid-reduced conditions, higher than on AR/cPAcP-negative PCa cells. Further, HDAC inhibitor pretreatments increased androgen responsiveness as demonstrated by PSA protein level quantitation. CONCLUSION: Our results clearly demonstrate that HDAC inhibitors can induce cPAcP protein level, increase androgen responsiveness, and exhibit higher inhibitory activities on AR/cPAcP-positive PCa cells than on AR/cPAcP-negative PCa cells. Upon HDAC inhibitor pretreatment, PSA level was greatly elevated by androgens. This data indicates the potential clinical importance of cPAcP serving as a useful biomarker in the identification of PCa patient sub-population suitable for HDAC inhibitor treatment.

Aims: The purpose of the research was to investigate the benefits of a hereditary colorectal cancer registry in the management of patients and families with Lynch syndrome. In study one, a systematic review was performed to quantify the impact of registration and screening on colorectal cancer (CRC) incidence and mortality, with comparison between familial adenomatous polyposis (FAP) and Lynch syndrome (LS). In study two, a regional Lynch syndrome registry was utilised to evaluate the uptake of predictive testing and colorectal screening among first-degree relatives (FDRs) and investigate novel methods for engaging at-risk relatives, including an enhanced role for the general practitioner (GP). In study three, the registry was used to investigate proposed associations between Lynch syndrome and prostate and bladder cancer. In study four, mismatch repair-deficient (dMMR) CRCs from Lynch syndrome patients and randomised-controlled trials (RCTs) were used to evaluate a novel prognostic biomarker, beta-2 microglobulin (B2M). Methods: An electronic database search was conducted to identify studies describing CRC incidence and/or mortality in FAP or LS, with comparison of either: 1) screened and unscreened patients or 2) patients ‘before and after’ establishment of the registry. Using the Manchester regional Lynch syndrome registry database, the uptake of predictive testing and colorectal screening among FDRs was assessed with Kaplan-Meier analysis. Novel strategies for improving engagement were explored via a patient advisory group discussion and a regional primary care questionnaire. Cases of prostate and bladder cancer in male mutation carriers and their male FDRs were identified, and cumulative and relative risks were calculated, using expected rates from cancer registry data. DNA from 350 dMMR CRC specimens from Lynch syndrome patients and RCTs were tested for B2M mutations using Sanger sequencing, and correlated with clinical outcome. Results: 43 studies were included in the systematic review (33 FAP; 10 Lynch). Registry-based screening was associated with a significant reduction in CRC incidence and in Lynch syndrome, CRC-related mortality was negligible in those undergoing surveillance. 242 Lynch syndrome families were recorded on the Manchester Lynch syndrome registry. 329 of 591 (55.7%) eligible FDRs had undergone predictive testing. Uptake was significantly lower in males and younger age groups (<25 yrs). Compliance with colorectal screening was excellent following a mutation positive predictive test but poor in untested individuals (97.3% vs 35.0%). Eight prostate cancers were identified in 821 male LS mutation carriers and male FDRs. MSH2 mutation carriers had a ten-fold increased risk of prostate cancer (RR 10.41; 95%CI 2.80, 26.65) but no association with bladder cancer was identified. 69/286 (24.1%) of dMMR CRCs contained significant B2M mutations. B2M mutations were associated with complete absence of recurrence (0/39) during follow-up in the QUASAR trial (stage II), compared with 14/77 (18.2%) in wild-type B2M (p=0.005). Conclusion: Studies consistently report that registration and screening result in a reduction of CRC incidence and mortality in FAP and LS (Level 2a evidence, Grade B recommendation). Funding and managerial support for registries should be made available. Uptake of predictive testing and colorectal screening in Lynch syndrome could be substantially improved, particularly among males and younger age groups, but this requires advances in communication with at-risk relatives. It is unlikely that GPs will actively participate without considerable support from genetics services. A trial of PSA screening in MSH2 mutation carriers from 50 years would be appropriate. B2M mutation status has potential clinical utility as a prognostic biomarker in stage II dMMR CRC.

Traf2- and Nck-interacting kinase (TNIK) is a serine/threonine kinase upregulated and amplified in pancreatic and gastric cancer respectively. TNIK has also been identified as a potential therapeutic target of colorectal cancer. However, the role of TNIK in prostate cancer (PCa) has not been investigated. Interrogating public human PCa patient data, we found that TNIK expression is associated with an aggressive form of PCa termed neuroendocrine prostate cancer (NEPC). Treatment-induced NEPC can arise as a consequence of strong selective pressure from androgen receptor (AR) pathway inhibition. Clinically, TNIK expression is positively correlated with neuroendocrine (NE) markers and inversely correlated with androgen regulated genes. In agreement, our in vitro studies reveal that TNIK expression is increased under AR pathway inhibition. We found that TNIK is transcriptionally repressed by androgen via direct binding of the AR at the TNIK locus. Through gain of function studies, we demonstrated that TNIK is not required for NE differentiation. Likewise, loss of function studies using siRNA or small molecule inhibitors targeting TNIK did not have significant effect on the growth of Enzalutamide-resistant cells with NE phenotype in vitro. Overall, our results indicate that TNIK may serve as a possible biomarker for NEPC.
Medicine, Faculty of
Experimental Medicine, Division of
Medicine, Department of
Graduate

BORIS, a paralogue of the transcription factor CTCF, is a member of the cancer-testis antigen family. BORIS is normally present only in the testis, however, it is aberrantly expressed in various tumours and, as recently reported, in leukocytes from breast cancer patients. The main aim of this study was to investigate BORIS expression in tissues and leukocytes from prostate cancer patients, and to correlate BORlS levels with clinical and pathological variables. To achieve this, BORIS immunoexpression was evaluated in human prostate tissues, cancer and benign prostate hyperplasia (BPH), and also in leukocytes from patients diagnosed with prostate cancer and BPH, and healthy donors. The staining was quantified using the immunoreactive score (IRS). BORIS, whilst absent in BPH tissues, was observed at varying levels in all prostate tumours analyzed, with the mean IRS= 6.78±0.89. Increased levels of BORIS protein positively correlated with Gleason score, T -stage and Androgen Receptor (AR) protein levels in prostate tumours. BORIS localization in the nucleus plus cytoplasm was also associated with higher BORIS levels and Gleason score. BORIS was not detected in leukocytes from healthy donors and patients with BPH, whereas 89% leukocyte specimens from prostate cancer patients were BORIS positive; the IRS values ranged between 1 and 8, with the average IRS= 2.51 ± 0.18. Positive relationship was observed between BORIS IRS, tumour stage and Gleason score however these results were not statistic all y significant. Detection of BORIS in prostate tumours suggests potential applications of BORIS as a biomarker for prostate cancer diagnosis, as an immunotherapy target and, potentially, a prognostic marker of more aggressive prostate cancer. The association of BORIS with AR indicates BORIS involvement in the growth and development of prostate tumours. Although BORIS has been shown to have properties as a blood biomarker, further validation will be required due to the lack of statistical significance.

Prostate cancer is a common cancer, particularly in Western countries. Increasing body mass index (BMI) has been associated with a modest increased risk of advanced and fatal prostate cancer (12-15% per 5 kg/m2 increase in BMI). Adipokines (adipose derived hormones) have been proposed as mediators of this association, but the results of several (mostly small) observational studies have been conflicting. The thesis describes investigations conducted into associations of adiposity, adipokines and lifestyle factors with prostate cancer; firstly, a systematic review and dose-response meta-analysis of the association of the adipokines adiponectin and leptin with prostate cancer incidence and progression. Secondly, a nested case-control study in men aged 50-69 with PSA-detected prostate cancer recruited during the case-finding stage of ProtecT (a population-based randomised trial of treatments for localised prostate cancer) compared BMI, WHR, adiponectin, leptin and leptin:adiponectin (L:A) ratio between 311 men with advanced (the cases) and 413 men with localised (the controls) prostate cancer. Finally, an exploration of associations of BMI, lifestyle factors and adipokines with age-related PSA change (,PSA growth') in men with localised prostate cancer undergoing active monitoring in ProtecT. The meta-analysis found adiponectin levels to be associated with a small reduced risk of total (OR 0.96, 95%CI 0.94-0.98, per 2.5~g/m1) and aggressive (OR 0.89, 95%CI 0.83-0.95) prostate cancer. The ProtecT case-control study indicated an inverse association of adiponectin with risk of advanced stage prostate cancer in overweight and obese men (p for interaction by BMI - 0.006; OR 0.62, 95% Cl 0.42- 0.90 per log(~g/ ml) in men ~25kg/m2}. The meta-analysis did not find a clear association between leptin and total (OR 1.01, 95% Cl 0.98-1.03 per 2.5 ng/ml) or aggressive (OR 1.01, 95%CT 0.99-1.06) prostate cancer and the ProtecT case-control study found little evidence of an association of leptin with prostate cancer stage or grade. There was weak evidence that L:A ratio was inversely associated with stage in normal weight men (OR 0.69, 95%CI 0.45-1.04) and positively associated with stage in overweight men (OR 1.22, 95%CI 0.97-1 .54) (p for interaction = 0.009). BMI was not associated with risk of advanced stage or high grade prostate cancer, or PSA growth, in ProtecT. In men undergoing active monitoring in ProtecT, exercise was inversely, and smoking was positively, associated with PSA at age 50 and yearly PSA growth; both of these factors have been linked to risk of aggressive or fatal prostate cancer. In conclusion, adiponectin appears protective against aggressive prostate cancer, particularly in overweight and obese men. Leptin and adiponectin (or L:A ratio) alone are unlikely to be major risk factors for overall prostate cancer and therefore their measurement at diagnosis would not aid prognostication. However, modification of adipokine levels through diet, exercise and weight loss is not likely to be harmful and may reduce the risk of other obesity-related cancers and diseases (e.g. cardiovascular disease). It is also unlikely that leptin is mediating the association of BMI with advanced or aggressive prostate cancer.

Evidence from international comparisons and migrant studies suggest that environmental factors, such as a Western diet, may be important in prostate cancer development, possibly through effects on hormone and growth factor secretion and metabolism. However, despite considerable research, convincing associations between diet and risk for prostate cancer have not been established. Random and systematic measurement error in dietary assessment using traditional survey methods may contribute to inconsistent findings, particularly as they may not capture adequately specific nutritional constituents of the diet that may be associated with risk, such as fatty acids or vitamins. Validated biomarkers of nutritional factors and hormonal activity, as used in this thesis, provide more precise, objective and integrated measures of exposure, with the capacity to clarify potential mechanisms in the causal pathway of prostate cancer development. Nutritional and hormonal biomarkers investigated for their potential role in the development of prostate cancer include: folate and vitamin B₁₂, which are essential for DNA methylation, repair and synthesis; phytanic acid, obtained predominantly from ruminant fat intake and associated with an enzyme (α-Methylacyl-Coenzyme A Racemase (AMACR)) that is consistently over-expressed in prostate cancer tissue; and insulin-like growth factor (IGF-I), a growth factor influenced by diet and involved in the regulation of cell proliferation, differentiation, and apoptosis. All work presented in this thesis is from the European Prospective Investigation into Cancer and Nutrition (EPIC) study of 500,000 European men and women, using prospectively collected diet and lifestyle data and biological samples. The large number of prostate cancer cases diagnosed during long-term follow-up of EPIC participants enabled investigation of heterogeneity in risk for prostate cancer by time from recruitment to diagnosis (of particular importance for a disease with a long pre-clinical phase) and cancer characteristics such as disease grade and stage. Plasma phytanic acid concentration was highly correlated with dietary intake of fat from dairy products (r = 0.46) and beef (r = 0.30); capturing differences between countries in consumption of fat from these foods. Although phytanic acid is a useful biomarker of ruminant fat consumption, there was little evidence to support the hypothesis that the association between dairy products and prostate cancer risk (as suggested by previous work in EPIC and other studies) is mediated by phytanic acid (OR for doubling in concentration 1.05; 95%CI 0.91 – 1.21; P _trend = 0.53). There was strong evidence for an association between higher circulating IGF-I concentration and risk for prostate cancer (OR for highest versus lowest fourth 1.69; 95% CI: 1.35, 2.13; P _trend = 0.0002). Furthermore, the positive association observed among men diagnosed with advanced stage disease and among men diagnosed more than seven years after blood collection, supports the hypothesis that high IGF-I concentration is associated with clinically significant prostate cancer many years before diagnosis. There was no evidence of an association between prostate cancer risk and dietary folate or vitamin B₁₂ intake, or between circulating levels of folate (OR for doubling in concentration 1.05; 95%CI 0.95 – 1.15; P _trend = 0.33) or vitamin B₁₂ (1.05; 95%CI 0.92 – 1.21; P _trend = 0.47) and only limited evidence for an increased risk associated with elevated vitamin B₁₂ in a meta-analysis of six prospective studies, that included the present study. All of these analyses were based on a blood sample taken at one point in time, with the assumption that this reflects the ‘true’ underlying concentration over the long-term. The poor to modest reliability estimates (intra-class correlation coefficients ranging from 0.18 to 0.48) for circulating concentrations of folate, IGF-I, phytanic acid and vitamin B₁₂ taken in samples approximately six years apart in a sub-sample of participants from EPIC Oxford, show that estimates of usual concentrations based on a single blood measurement weaken the ability to detect associations with disease risk. Where small effect sizes are anticipated, this may bias associations toward the null. In conclusion, there is convincing evidence that IGF-I is an important and potentially modifiable risk factor for prostate cancer many years before diagnosis. However, there is little evidence for an association between biomarkers of folate, vitamin B₁₂ and phytanic acid concentrations and risk for prostate cancer. Future studies should, where possible, incorporate multiple blood samples taken several years apart to better characterise long term relationships between biomarkers of nutritional and hormonal exposure and disease risk and pool individual participant data from multiple prospective studies to strengthen the power to detect modest associations.

>Magister Scientiae - MSc
Prostate cancer, commonly referred to as adenocarcinoma of the prostate, is the leading cause of cancer death in men in 46 countries, and it was estimated that by the end of 2018 there would approximately be 1.3 million new cases of prostate cancer worldwide. Currently, the Food and Drug Administration (FDA) approved biomarker for prostate cancer disease diagnostics Prostate Specific Antigen (PSA) is not specific to the disease itself but extends to other cases such as Benign Prostate Hyperplasia (BPH) a condition in which the prostate grows uncontrollably. This biomarker is then detected in blood samples via conventional methods which require a qualified individual to operate and are often time consuming. Examples of these methods are spectrophotometry and High Performance Liquid Chromatography (HPLC). Hence, a more efficient biomarker and method of detection is needed for prostate cancer disease diagnostics, as early detection of the disease means early treatment, which could ultimately save lives. Currently, an emerging biomarker for prostate cancer known as Alpha-Methyl CoA Racemase (AMACR) has shown to be more specific to the disease with advantages such as being a non-invasive biomarker. AMACR has been reported to be present in urine, and thus may be detected via a non-invasive method. This study proposed an economical, non-invasive electrochemical biosensor for the rapid detection of AMACR based on mercaptosuccinic acid capped tungsten telluride (MSA-WTe3) quantum dots (QDs). Nanomaterial has shown promise in terms of increasing the sensitivity and specificity of sensors. MSA-WTe3 QDs was successfully synthesized using easy, inexpensive method and was studied by various techniques such as High Resolution Transmission Electron Microscopy (HR-TEM) where the size was confirmed to be within the nanometer scale and was reported to be 2.65 nm with a good crystallinity. X-ray diffraction (XRD) confirmed the structural properties and chemical composition of the QDs and it is reported that the QDs are rich in both tellurium and tungsten and comprise of a hexagonal structure. Scanning Electron Microscopy (SEM) confirmed the successful immobilization of aptamer sequence specific to AMACR onto the electrode surface by showing a distinct conformational change when aptamers were introduced to the QDs under study. This study reports the successful detection of AMACR using an MSA-WTe3 QDs based aptasensor immobilized onto a screen printed glassy carbon electrode, with a detection limit of 0.35651 ng/mL and a limit of quantification calculated to be 1.08033 ng/mL.

Prostate cancer (PC), is initially androgen dependent due to the androgenic nature of the organ. Hence, initial therapy comprises androgen depletion via chemical castration in conjunction with an anti-androgen therapeutic. However, patients relapse and the tumours aggressively re-grow in a castrate resistant (CRPC) manner. In CRPC, androgen receptor (AR) signaling remains functional via numerous mechanisms hence the AR remains a viable therapeutic target. However, treatment with current AR targeting therapeutics also results in relapse indicating the potential of targeting AR signaling indirectly by targeting AR co-factors. Recently, KMT5A, a lysine methyltransferase, has been identified as an AR co-activator exclusively in models of CRPC. A number of KMT5A inhibitors have been identified recent years, which would enhance the possibility of targeting KMT5A in PC. This thesis aims to determine the signature of genes that are regulated directly by KMT5A or by combined activities of AR and KMT5A in PC cell lines and to further identify biomarkers for KMT5A activity. These aims were approached using Illumina Human HT-12 arrays to detect KMT5A gene expression profiles in an in vitro cell line model of androgen independent PC (LNCaP-AI cells). Microarray data analysis revealed a number of androgenregulated genes to be modulated by KMT5A concurrently, and other genes that were found to be regulated by KMT5A activity, and a further cohort of genes that were found to be regulated solely by KMT5A. CDC20 was selected for further study from the identified KMT5A regulated genes as a possible biomarker for KMT5A activity in aggressive PC. KMT5A was found to regulate CDC20 mRNA and protein expression. The enzymatic activity of KMT5A was demonstrated to affect CDC20 expression through the enrichment of the H4K20me1 mark at the CDC20 promoter in androgen-sensitive (LNCaP) and androgen-independent (LNCaP-AI) cells. The regulation of CDC20 by KMT5A expression, therefore identifies CDC20 as putative biomarker for KMT5A activity. KMT5A was also shown to influence CDC20 expression via p53. Knockdown of KMT5A inhibited the mono-methylation of p53 at K382 to enhance p53 activity, demonstrated by increased p21 expression which negatively regulated CDC20 ii expression. These findings were confirmed using commercially available KMT5A inhibitors Ryuvidine and UNC0379. In summary, KMT5A inhibition in PC cells using small molecule inhibitors may provide benefit to patients that have relapsed on AR- targeting therapeutics and as such requires further investigation as a potential therapeutic target. CDC20 was identified as a putative biomarker for KMT5A activity which may prove useful to detect effective KMT5A inhibition in these studies.

Background Prostate cancer (PCa) is the most frequently diagnosed malignant disease among adult males in the USA and the second leading cause of cancer deaths in men. Due to the lack of diagnostic tools that are able to differentiate highly malignant and aggressive cases from indolent tumors, overtreatment has become very common in the era of prostate specific antigen (PSA) screening. New diagnostic methods to determine biological status, malignancy, aggressiveness and extent of PCa are urgently needed. 1H High Resolution Magic Angle Spinning Nuclear Magnetic Resonance Spectroscopy (1H HRMAS MRS) can be used to establish PCa metabolomic profiles while preserving tissue architecture for subsequent histopathological analysis. Immunohistochemistry (IHC), as opposed to conventional histopathology methods, has the potential to provide objective, more accurate and quantitative knowledge of tissue pathology. This diagnostic- accuracy study sought to evaluate a novel approach to quantitatively identify metabolomic markers of PCa by exploring the potential of PCa immunomarkers to quantify metabolomic profiles established by 1H HRMAS MRS. Material and Methods 1H HRMAS MRS was performed on tissue samples of 51 prostate cancer patients using a 14.1 Tesla NMR spectrometer (BRUKER Biospin, Billerica, MA) with a rotor synchronized CPMG pulse sequence. Spectral intensities of 36 regions of interest were measured as integrals of curve fittings with Lorentzian-Gaussian line shapes. Immunohistochemistry (IHC) was carried out following the spectroscopy scan, using three prostate immunomarkers to identify cancerous and benign glands: P504S (Alpha-methylacyl-CoA-racemace), CK903 (high-molecular weight cytokeratin) and p63. The immunostaining quality following 1H HRMAS MRS was evaluated and compared to unscanned sections of the same sample, to verify the stability and accessibility of the proposed immunomarkers. IHC images were automatically and quantitatively evaluated, using a quantitative image analysis program (QIAP), to determine the percentage of cancerous and benign epithelia in the tissue cross- sections. The results of the program were validated by a correlation with the results of a quantitative IHC review and quantitative conventional histopathology analysis performed by an experienced pathologist. Ultimately, spectral intensities and the cancer epithelium percentage, obtained from quantitative immunohistochemistry, were correlated in order to validate PCa metabolomic markers identified by 1H HRMAS MRS. Patient outcomes and incidence of recurrence were determined by retrospective review of medical records five years after initial surgery. Categories of recurrence were correlated to spectral intensities to explore potential metabolomic markers of recurrence in the cohort. Results Immunostainings with P504S and CK903 showed excellent staining quality and accessibility following 1H HRMAS MRS, suggesting these markers to be suitable for the presented quantitative approach to determine metabolomics profiles of PCa. In contrast, the quality of p63 IHC was impaired after previously performed spectroscopy. IHC using the immunomarkers P504S and CK903 on adjacent slides was found to present a feasible quantitative diagnostic method to distinguish between benign and cancerous conditions in prostate tissue. The cancer epithelium percentage as determined by QIAP showed a significant correlation to the results of quantitative IHC analysis performed by a pathologist (p < 0.001), as well as to a quantitative conventional histopathology review (p = 0.001). The same was true for the benign epithelium percentage (p < 0.001 and p = 0.0183), validating the presented approach. Two metabolomic regions showed a significant correlation between relative spectral intensities and the cancer epithelium percentage as determined by QIAP: 3.22 ppm (p = 0.015) and 2.68 ppm (p = 0.0144). The metabolites corresponding to these regions, phosphocholine and citrate, could be identified as metabolomic markers of PCa in the present cohort. 45 patients were followed for more than 12 months. Of these, 97.8% were still alive five years after initial surgery. 11 patients (24.4%) experienced a recurrence during the follow- up time. The categories of recurrence showed a correlation to the spectral intensities of two regions, 2.33 – 2.3 ppm (p = 0.0403) and 1.28 ppm (p = 0.0144), corresponding to the metabolites phosphocreatine and lipids. Conclusion This study introduces a method that allows an observer-independent, quantitative analysis of IHC to help establish metabolomic profiles and identify metabolomic markers of PCa from spectral intensities obtained with 1H HRMAS NMR Spectroscopy. The immunomarkers P504S and CK903 have been found suitable IHC analysis following 1H HRMAS MRS. A prospective in vivo application of PCa metabolite profiles and metabolomic markers determined by the presented method could serve as highly sensitive, non- invasive diagnostic tool. This observer- independent, computer- automated, quantitative analysis could help to distinguish highly aggressive tumors from low-malignant conditions, avoid overtreatment and reduce risks and complications for cancer patients in the future. Further studies are needed to verify the identified PCa metabolomic markers and to establish clinical applicability
Einführung Prostatakrebs ist eine häufigsten Krebserkrankungen in den USA und die zweithäufigste malignom- assoziierte Todesursache männlicher Patienten weltweit. Seit der Einführung des Prostata- spezifischen Antigen (PSA)- Screeningtests wird diese Krebsart in früheren Stadien diagnostiziert und therapiert, wodurch die Mortalitätsrate in den letzten Jahren deutlich reduziert werden konnte. Da moderne diagnostische Methoden bislang jedoch nicht ausreichend in der Lage sind, suffizient zwischen hochmalignen und weniger aggressiven Varianten dieses bösartigen Krebsleidens zu unterscheiden, werden häufig auch Patienten aggressiv therapiert, deren niedriggradiges Prostatakarzinom keine klinische Relevanz gehabt hätte. Es besteht daher ein großes wissenschaftliches Interesse an der Entwicklung neuer diagnostischer Methoden zur akkuraten Bestimmung von biologischem Status, Malignität, Aggressivität und Ausmaß einer Prostatakrebserkrankung. \\\\\\\"1H High Resolution Magic Angle Spinning Nuclear Magnetic Resonance Spectroscopy\\\\\\\" (1H HRMAS MRS) ist eine vielversprechende diagnostische Methode, welche es ermöglicht, metabolomische Profile von Prostatakrebs zu erstellen, ohne die Gewebsstruktur der analysierten Proben zu zerstören. Durch anschließende histopathologische Begutachtung lassen sich die erstellten Metabolitprofile validieren und evaluieren. Im Gegensatz zu konventionellen histopathologischen Methoden können durch immunhistochemische Verfahren dabei objektivere, akkuratere und quantifizierbare histopathologische Erkenntnisse gewonnen werden. Die vorliegende Studie präsentiert einen neuentwickelten diagnostischen Ansatz zur quantitativen Bestimmung von metabolomischen Markern von Prostatakrebs, basierend auf der Durchführung von 1H HRMAS NMR Spektroskopie und quantitativer Immunhistochemie. Material und Methoden Einundfünfzig Gewebsproben von Prostatakrebspatienten wurden mittels 1H HRMAS MRS an einem 14.1 T BRUKER NMR Spektrometer unter Einsatz einer CPMG-Pulssequenz untersucht. Spektrale Intensitäten in 36 Metabolitregionen wurden gemessen. Anschließend wurden die analysierten Gewebeproben mit drei Immunfärbemarkern für sowohl malignes (P504S, Alpha-methylacyl-CoA-racemase) als auch benignes (CK903, High-molecular weight cytokeratin, und p63) Prostatagewebe angefärbt und quantitativ mit Hilfe eines Bildanalyseprogramms (QIAP) ausgewertet. Die Anwendbarkeit und Auswertbarkeit der genannten Immunomarker nach Spektroskopie wurde evaluiert und mit der Färbungsqualität von nicht- gescannten Schnitten verglichen. Die Resultate der automatischen Auswertung durch QIAP konnten durch einen erfahrenen Pathologen in einer quantitativen Analyse der Immunfärbungen sowie konventioneller histologischer Färbungen derselben Gewebsproben validiert werden. Die spektralen Intensitäten aus den Messungen mit 1H HRMAS MRS wurden mit den korrespondierenden Ergebnissen der quantitativen Auswertung der Immunfärbungen korreliert, um metabolomische Marker von Prostatakrebs zu identifizieren. Der klinische Verlauf und die Rezidivrate der Patienten wurden 5 Jahre nach der initialen Prostatektomie retrospektiv bestimmt. Rezidivkategorien wurden erstellt und mit den bestimmten spektralen Intensitäten korreliert, um metabolomische Marker für das Auftreten von Prostatakrebsrezidiven zu identifizieren. Ergebnisse Die Immunfärbungen mit P504S und CK903 zeigten exzellente Qualität und Auswertbarkeit nach vorheriger 1H HRMAS MRS. Beide Marker eigneten sich zur Durchführung von quantitativer Immunhistochemie an spektroskopierten Gewebeproben. Im Gegensatz dazu war die Qualität der Immunfärbungen mit p63 nach Spektroskopie vermindert. Quantitative Immunfärbungen unter Einsatz der Immunmarker P504S und CK903 stellten eine praktikable diagnostische Methode dar, um zwischen malignen und benignem Prostatagewebe zu unterscheiden. Der Anteil von bösartig verändertem Prostatagewebe, bestimmt durch QIAP, korrelierte signifikant mit den Ergebnissen der quantitativen Analyse der Immunfärbungen durch den Pathologen (p < 0.001), sowie mit der quantitativen Auswertung der konventionellen histopathologischen Färbung (p = 0.001). Ebenso ließ sich die Bestimmung des Anteils von benignem Gewebe mit QIAP zu den Ergebnissen der pathologischen Analyse korrelieren (p < 0.001 und p = 0.0183). Für zwei metabolomische Regionen konnte ein signifikante Korrelation zwischen relativen spektralen Intensitäten, bestimmt mit 1H HRMAS NMR Spektroskopie, und dem Anteil von malignem Epithelium in derselben Gewebeprobe, ermittelt durch QIAP, festgestellt werden: 3.22 ppm (p = 0.015) und 2.68 ppm (p = 0.0144). Die zu diesen Regionen korrespondierenden Metaboliten, Phosphocholin und Zitrat, konnten als potentielle metabolomische Marker für Prostatakrebs identifiziert werden. Die retrospektiven Analyse der klinischen Daten der Patienten fünf Jahre nach Prostatektomie ergab eine Überlebensrate von 97.8%. Elf dieser Patienten (24.4%) erlitten ein Rezidiv ihrer Erkrankung. Die bestimmten Rezidivkategorien korrelierten signifikant mit zwei metabolomischen Regionen (2.33 – 2.3 ppm, p = 0.0403 und 1.28 ppm, p = 0.0144), welche zu den Metaboliten Phosphokreatin und Lipiden korrespondierten. Schlussfolgerung Die vorliegende Studie präsentiert einen diagnostischen Ansatz zur objektiven und quantitativen Bestimmung metabolomischer Marker von Prostatakrebs unter Verwendung von 1H HRMAS MRS und Immunhistochemie. P504S und CK903 eignen sich als Immunmarker für quantitative Immunfärbungen nach vorheriger Durchführung von 1H HRMAS MRS. Die Metaboliten Phosphocholin und Zitrat konnten in der vorliegenden Patientenkohorte als potentielle metabolomische Marker für Prostatakrebs identifiziert werden. Eine mögliche in vivo Anwendung der gefundenen metabolomischen Marker könnte als hochsensitives, objektives und nicht- invasives diagnostisches Werkzeug der Prostatakrebsdiagnostik dienen. Der vorliegende untersucherunabhängige, automatisierte und quantitative diagnostischer Ansatz hat das Potential, zwischen hochmalignen und weniger aggressiven Krebsfällen zu unterscheiden und somit unnötige Risiken und Komplikationen für Prostatakrebspatienten zu reduzieren. Weitere Untersuchungen sind notwendig, um die identifizierten metabolomischen Marker zu verifizieren und eine klinische Anwendung zu etablieren

The diagnostic pathway for prostate cancer involves the blood test prostate specific antigen (PSA) which has high sensitivity but low specificity at age related reference ranges. The resultant clinical consequence is a large number of negative diagnostic studies (transrectal ultrasound guided biopsies - TRUS). There is a need for a secondary screening test to help improve on the current diagnostic pathway. Mechanical markers have been used previously to assess the prostate for disease with numerous ex-vivo reports of differences between benign and malignant prostates. There have been no in-vivo studies with direct elasticity assessment devices for prostate cancer detection. This thesis forms part of work in a collaborative study in conjunction with engineers who have created a microscale device, capable of dynamic elasticity assessment. The specific objectives of this thesis were to a) assess dynamic micromechanical markers for the detection and differentiation of clinically significant from insignificant prostate cancer b) to identify relationships between mechanical and histopathological variables in the ex-vivo and in-vivo environments and c) assess the potential for these markers to differentiate peri-prostatic tissues. A prospective study was set-up with full ethics and management approvals with patients undergoing a systematic mechanical assessment of their prostate using the E-finger device and after prostate excision a systematic ex-vivo mechanical assessment on a calibrated stage. The ex-vivo assessment allowed accurate histopathological and mechanical variable assessment in a controlled environment. 7-Tesla ex-vivo MRI scanning aided in assessing the limitations of mechanical assessment of the prostate. There were clear consistent differences between individual dynamic micromechanical markers for benign and tumour containing measurement areas in both environments. Modelling of these dynamic micromechanical markers yielded encouraging accuracy levels for the detection of prostate cancer and differentiation of significant from insignificant disease. There were associations between individual mechanical markers and important histopathological features associated with cancer (acinar size, tumour volume and reactive stroma). These markers showed promise and utility in the differentiation of prostate from bladder and rhabdosphincter. This work demonstrates the clear potential translational uses for dynamic micromechanical markers in the assessment of the prostate for cancer.

De sa découverte et avant d'arriver au chevet des patients, un biomarqueur parcours différentes étapes. Une qualification, une vérification, et une validation du potentiel clinique sont nécessaires pour faire le tri parmi les candidats. Ces étapes nécessitent la parfaite maîtrise de différentes méthodes expérimentales et d'analyse de résultats. Le nombre d'échantillons utilisés doit également être dimensionné pour répondre sans équivoque à la question clinique. Pour ces raisons, peu de marqueurs atteignent le stade de la validation clinique. Historiquement et encore aujourd'hui, l'oncologie reste l'un des principaux domaines d'utilisation des biomarqueurs. Les travaux de recherches présentés concernent deux principaux cancers. Le cancer colorectal où nous avons réalisé les étapes de la découverte du marqueur sérique HSP60 à sa vérification. Pour cela quatre techniques ont été utilisées, la 2D-DIGE, le WB, l'IHC et l'ELISA sur quatre cohortes indépendantes. Nous avons montré pour la première fois, que les doses d'HSP60 sérique sont significativement plus hautes chez les patients CCR et que au final, l'HSP60 avait plutôt un potentiel comme marqueur de pronostic. Pour le cancer de la prostate, nous avons démarré l'étude à la phase de vérification du potentiel de l'ANXA3 pour l'aide à la décision de réaliser une biopsie dans la zone grise de PSA (2,5-10 ng/ml), jusqu'à la validation clinique. Nous avons réalisé la deuxième étude clinique au monde sur ce marqueur en tant qu'outil d'aide au diagnostic sur une cohorte de 528 patients. Nous avons confirmé que l'ANXA3 est un marqueur du CaP, mais la performance obtenue a été inférieure à celle de la première étude
From discovery to the availability for the patient, a biomarker has to go through several phases with success. There are discovery, qualification, verification and clinical validation. Each phase needs adequate experimental methodologies and appropriate mathematical analysis. Cohort selection and size must allow to respond to one clinical question. The success rate is low. Very few markers reach clinical validation and even less become available for patients. Oncology has benefited from use of biomarkers for more efficient patient management and is among the fields that contribute to active translational research on biomarkers. The work presented in this thesis, concerns two main cancers. In the colorectal cancer, we followed the workflow to the discovery of HSP60 as a candidate marker, until its verification in sera. The study has been performed on four independent cohorts using four techniques, 2D-DIGE, WB, IHC and ELISA. For the first time, we demonstrated that serum HSP60 levels were significantly higher in CRC patients and we report that HSP60 has a better potential as a prognosis marker. In the case of prostate cancer, our work has started following the identification of ANXA3 as a potential marker and included verification and validation phases. We wanted to confirm the utility of ANXA3 as an aid for prostate biopsy decision in PSA grey zone (2.5-10 ng/ml). We carried out the second clinical trial on this marker for diagnosis purpose enrolling a cohort of 528 patients. We confirmed that ANXA3 is indeed an urinary marker for prostate cancer, but the performances were lower than in the first study

The widespread use of Prostate Specific Antigen (PSA) testing has resulted in the over detection and over treatment of potentially indolent disease due to the lack of specificity of PSA for prostate cancer (PCa). PROTEOMEX, a method of tumour associated antigen (TAA) identification, combines the separation of tumour proteins by conventional proteomic methods (2-DE and mass spectrometry) with serological screening using serum antibodies, to identify immunogenic proteins in cancer. This project aims to identify TAAs and/or biomarkers for PCa which on subsequent validation, can be utilised as an improved diagnostic screening test. In pilot studies, SDS PAGE, 2-DE and OFFGEL electrophoresis were performed to identify immunogenic urine TAAs using PCa and healthy control sera. Proteins within the serum-reactive spots were either identified by Liquid Chromatography coupled to Matrix-Assisted Laser Desorption/Ionisation Time-of-Flight mass spectrometry (MALDI-TOF MS) or Electrospray Ionisation mass spectrometry (ESI MS). Among the urinary proteins separated by SDS PAGE, 2-DE and OFFGEL respectively, exclusive autoreactivity was identified in PCa sera to serum albumin and existing PCa biomarkers - human prostatic acid phosphatase & zinc-alpha-2 glycoprotein. Differential autoantibody responses were also identified to various TAAs in the PC-3 and DU-145 PCa cell lines using PCa and healthy sera. The presence of a differential TAA and autoantibody PCa serum response to one of the proteins identified by MALDI-TOF, alpha enolase, was further verified in a subset of PCa samples using immunohistochemistry, Western blotting and ELISA. In a larger sample cohort, the cytoplasmic and nuclear alpha enolase expression in a PCa TMA was assessed, where statistical significance was observed between benign controls and PCa (p=0.000003 and p=0.003 respectively), although protein expression did not correlate with any important clinico-pathological variables. Alpha enolase autoantibody expression was statistically significant between PCa and healthy controls (p=0.0038), where its expression correlated with D’Amico risk classification, indicating that alpha enolase may serve as a potential indicator of biochemical recurrence in PCa. PROTEOMEX represents a valuable approach for the identification of tumour biomarkers which may have diagnostic and/or prognostic value in PCa. Further work should identifiy more TAAs and autoantibodies associated with PCa, alongside a validation of the diagnostic utility of the identified biomarkers from this study.

A critical problem in the clinical management of prostate cancer is that it shows high intra- and inter-tumoural heterogeneity. As a result, accurate prediction of individual cancer behaviour is not achievable at the time of diagnosis, leading to substantial overtreatment. It remains an enigma that, in contrast to other cancers, no molecular biomarkers which define robust subtypes of prostate cancer with distinct clinical outcomes have been discovered. In the first part of this study, using data from exon microarrays, we developed a novel method that can identify transcriptional alterations within genes. The alterations might be the result of chromosomal rearrangements, such as translocations, and deletions, or of other abnormalities, such as read-through transcription and alternative transcriptional initiation sites. Using data from two independent datasets we identify several candidate alterations that are constantly correlated with the biochemical failure or that are linked to the development of metastasis. In the second part of the study we illustrate the application of an unsupervised Bayesian procedure, which identifies a subtype of the disease in five prostate cancer transcriptome datasets. Cancers assigned to this subtype (designated DESNT cancers) are characterized by low expression of a core set of 45 genes. For the four datasets with linked PSA failure data following prostatectomy, patients with DESNT cancer exhibited poor outcome relative to other patients (p = 2.65 ・ 10−5, p = 4.28 ・ 10−5, p = 2.98 ・ 10−8 and p = 1.22 ・ 10−3). The DESNT cancers are not linked with the presence of any particular class of genetic mutation, including ETS gene status. However, the methylation analysis reveals a possible role of epigenetic changes in the generation of the DESNT subtype. Our results demonstrate the existence of a novel poor prognosis category of human prostate cancer and will assist in the targeting of therapy, helping avoid treatment-associated morbidity in men with indolent disease.

Background: Low-grade chronic inflammation has been implicated as a risk factor in prostate-related pathologies including benign hyperplasia and cancer. Sweet cherry containing the bioactive anthocyanin (ACN), has demonstrated tumor inhibitory action in model systems, specifically inhibition of inflammatory molecules and prostaglandin biosynthesis. Objective: To assess the urinary and plasma concentrations of ACN from the daily consumption of 3 cups of sweet cherries for 4 weeks and test the relationship of ACN levels and cherry consumption to inflammatory biomarkers in an at risk population. Results: Prostaglandin E2 Metabolite (PGEM) levels were reduced with cherry consumption in men with elevated baseline values. Conclusion: We conclude that 1c (142g) of sweet cherries 3 times daily for 4 weeks significantly reduced the COX-2 metabolite, PGEM, in men with elevated baseline levels. This was the first study to examine the chronic effects of daily sweet cherries on COX-2 inhibition in an at risk population.

Prostate cancer (PCa) is one of the most common cancer in men and affecting hundreds of thousands of men worldwide. The early detection of PCa have been proven to be advantageous for more efficient treatment against PCa. However, the conventional screening methods towards PCa are lack of selectivity and sensitivity, which leading to high false positive rate and overdiagnosis of PCa. The objective of this study is to (1) identify more sensitive and accurate diagnostic biomarkers towards PCa; (2) followed by developing new chemosensor towards the newly found biomarkers. In the first part of this study, based on the previously findings on urinary spermine as useful biomarker for PCa, a more comprehensive study on urinary polyamine levels was carried out and the important role of urinary spermine as biomarker for PCa detection was consolidated. In the second part of this work, molecularly imprinted polymer (MIP) towards the target analyte of urinary spermine was prepared for the capturing and extraction of spermine. The specific adsorption and selectivity towards spermine of the MIP were discussed and reported. In the third part of this study, a rapid detection method of spermine was developed via the use of aggregation-induced emission (AIE) active molecules. The presence of spermine would cause aggregation of this chemosensor for a "turn-on" detection of spermine under ultra-violet (UV) excitation, which would be useful in PCa diagnosis in the future.

Mestrado em Bioquímica, ramo de Bioquímica Clínica
Nowadays, prostate cancer is the third most common cause of death among men. Currently, there is no effective treatment when the tumor is diagnosed at an advanced stage. Given the high mortality rates associated with cancer, the identification and quantification of tumor biomarkers in human fluids and tissues have been the subject of intense research aiming more reliable diagnoses and to avoid excessive invasive treatments. In recent years, a special interest was also raised on the simultaneous analysis of several biomarkers for the same purpose, i.e., in order to make a more accurate diagnosis. Therefore, certain tumor markers, such as prostate specific antigen (PSA) widely used for the diagnosis of prostate cancer, should be considered together with other relevant biomarkers, like lactate dehydrogenase (LDH), which has recently been indicated as an excellent prognostic and monitoring tool of treatment of the same type of cancer. Considering the current methods for the quantification and purification of these biomarkers, and since both are proteins and are present in complex biological media, they usually exhibit low selectivity and may lead to false positives or negatives. In this context, it is crucial to develop efficient and selective analytical methods for the identification and quantification of several tumor biomarkers present in the same biological sample. For this purpose, aqueous biphasic systems (ABS) composed of ionic liquids (ILs) were investigated as an alternative extraction and purification technique for these biomarkers from synthetic and real biological fluids. In this work, novel ABS capable of extracting and concentrating PSA from human urine, and SAB capable of extracting and purifying LDH from human serum, in a single-step, were identified, allowing thus the identification and quantification of both biomarkers in biological fluids using more expedite analytical equipment, such as size-exclusion high-performance liquid chromatography (SE-HPLC).
O cancro da próstata representa nos dias de hoje a terceira causa de morte mais comum entre os homens, sendo que, atualmente, não existe nenhum tratamento eficaz quando o tumor é diagnosticado já num estado avançado. Tendo em conta as elevadas taxas de mortalidade associadas ao cancro, a identificação e quantificação de biomarcadores tumorais em fluidos e tecidos humanos têm sido alvo de uma investigação intensa no sentido de efetuar diagnósticos mais fiáveis e evitar tratamentos invasivos excessivos. Alguns biomarcadores tumorais, como o antigénio prostático específico (PSA) amplamente utilizado para o diagnóstico do cancro da próstata, podem ser avaliados em conjunto com outros biomarcadores relevantes, surgindo neste sentido a identificação e quantificação da enzima lactato desidrogenase (LDH), que recentemente tem sido sugerida como uma excelente ferramenta de prognóstico e monitorização do tratamento do mesmo tipo de cancro. Tendo em conta que ambos os biomarcadores são proteínas e que os fluidos humanos são matrizes muito complexas, os métodos atuais de identificação e quantificação apresentam pouca seletividade e podem conduzir a falsos positivos ou negativos. Neste sentido, torna-se fundamental desenvolver alternativas eficientes para a identificação e quantificação de vários biomarcadores tumorais presentes na mesma amostra. Para este efeito, avaliaram-se sistemas aquosos bifásicos (SAB) constituídos por líquidos iónicos (LIs) como uma técnica alternativa de extração e purificação de PSA e LDH a partir de fluídos biológicos sintéticos e reais. Neste trabalho identificaram-se SAB promissores capazes de extrair e concentrar PSA a partir de amostras de urina, e SAB capazes de extrair e purificar LDH a partir de amostras de soro humano, num único passo, permitindo portanto a sua identificação e quantificação de ambos os biomarcadores em fluidos humanos por métodos analíticos mais expeditos, tal como cromatografia líquida de alta eficiência por exclusão molecular (SE-HPLC).

Background: Prostate cancer is the most common cancer in Sweden. Due its highly variable behavior, multifocal nature, and insufficient diagnostic methods, prostate cancer is difficult to diagnose and prognosticate. Some patients have an aggressive lethal disease, but the majority of prostate cancer patients have slow-growing, non-lethal disease with long expected survival without treatment. Current diagnostic methods―serum levels of prostate-specific antigen (PSA) and histological grading of biopsied prostate tissue―often do not give the information required to be able to safely differentiate indolent tumors from potentially lethal ones. Many prostate cancers are difficult to detect by imaging, so tissue biopsy cannot be safely guided towards the tumor, and particularly not towards the most aggressive forms. To overcome this problem, multiple needle biopsies are taken from the organ, but biopsies are small and they sample less than 1% of the whole prostate. In this thesis, we explore the non-malignant prostate tissue adjacent to tumors, which is always sampled in biopsies, and we study adaptive changes in this tissue, which may provide new diagnostic and prognostic markers for prostate cancer. We have therefore proposed that this type of tissue should be termed TINT (Tumor Instructed/indicating Normal Tissue).  Methods: In our studies, we used orthotopic rat prostate cancer models with tumors of different aggressiveness. We also used clinical materials from patients diagnosed with prostate cancer at transurethral resection (1975‒1990); the majority of these men were followed with watchful waiting. Analyses were performed with whole-genome expression array, quantitative real-time PCR, immunohistochemistry, and western blotting.  Results: Using the animal model, we found that the presence of a tumor induces changes in gene expression in the surrounding tumor-bearing organ (TINT). The gene signature of TINT was linked to processes such as extracellular matrix organization, immune responses, and inflammation. We also showed that some of these adaptive TINT changes appear to be related to the aggressiveness and metastatic potential of the growing tumor, such as increases in macrophages, in mast cells, in vascular densities, and in vascular cell-proliferation. Some of these findings were confirmed by our observations in patient samples. We found that high staining of the extracellular matrix component hyaluronan in the stroma of the non-malignant prostate tissue was prognostic for short cancer-specific survival. We also found that an elevated proportion of C/EBP-beta positive epithelial cells in non-malignant (TINT) prostate tissue was associated with a good prognosis.  Conclusions: Using animal experiments and patient samples, we showed that the presence of prostate cancer induces changes in the tumor-bearing organ, alterations associated with tumor aggressiveness, and that grading of these changes in TINT can be used to predict outcome in prostate cancer patients.

El càncer de pròstata (CP), és la segona causa de mort per malaltia oncològica en els homes del món occidental. S’estima que un de cada sis homes desenvoluparà un càncer d’aquest tipus al llarg de la seva vida. El CP afecta com el seu nom indica, a la pròstata. La pròstata, és un òrgan glandular de l’aparell genital-urinari masculí. Té la mida d’una nou i es localitza sota de la bufeta, envoltant la uretra i davant del recte. La seva funció és secretar productes que s’afegiran al líquid seminal amb la finalitat de nodrir i protegir els espermatozous. El actual diagnòstic del CP es basa en una triada diagnòstica que consta de; l’anàlisi dels nivells de PSA (Prostate Specific Antigen) en sèrum, el tacte rectal (TR) i finalment la biòpsia prostàtica. Quan els nivells de PSA en sèrum es situen per sobre de 4 ng/mL i/o el TR és sospitós, l’uròleg pot estimar quina és la probabilitat que el pacient estigui afectat per un CP i per tant decidir la necessitat de practicar o no una biòpsia prostàtica, que permetrà establir el diagnòstic definitiu. La introducció del test del PSA, a finals dels anys 80, s'ha traduït en una millora del diagnòstic precoç del CP, moment en el qual les opcions de tractament per el són eficaces. No obstant això, malgrat aquesta detecció primerenca, la mortalitat per CP no ha disminuït significativament en els últims anys. El principal problema del PSA com a marcador d’screening és que aquest presenta un nivell d’especificitat baix (30% aprox) i alhora, un valor predictiu negatiu baix. Això es tradueix en que al voltant d'un terç de tots els homes sotmesos a una biòpsia seran positius per CP. Com a resultat dels seus persistents nivells sèrics de PSA, però els resultats negatius de la biòpsia, aquests homes es sotmeten a repetides biòpsies. Tot i el importants avenços en la investigació de biomarcadors de CP, alguns homes encara estan sobre-diagnosticats de CP “indolent”, mentre que d’altres moren de malaltia agressiva que s'ha diagnosticat massa tard. Aquesta situació es coneix com "dilema diagnòstic". És per tot això, que el càncer de pròstata, es beneficiaria de l’existència de nous marcadors d’screening més específics i alhora d’un diagnòstic menys invasiu. Per altra banda, una millora en el diagnòstic evitaria un gran nombre de biòpsies innecessàries i conseqüentment un important estalvi econòmic en el cost sanitari actual. La recerca de nous marcadors en el càncer de pròstata suposa un camp de treball important en la detecció precoç d’aquest tipus de càncer. Donada la situació de la pròstata a l’organisme, sota la bufeta i envoltant la uretra, les secrecions i inclús les mateixes cèl·lules prostàtiques, ja siguin normals o malignes, poden trobar-se presents en l’orina. És per això que considerem l’orina com una font important d’informació, a través de la qual es podria arribar a determinar quina situació s’està donant a l’òrgan en qüestió. Altres estudis evidencien l’existència de potencials biomarcadors en l’orina que podrien ajudar en la millora del diagnòstic del CP. A nosaltres ens ocupa l’estudi d’aquelles molècules de RNA així com de les molècules protèiques que es troben a l’orina. Suposem doncs, que un massatge prostàtic enriqueix la mostra d’orina de tot tipus de molècules proteiques. Així doncs, la nostra hipòtesi de treball recolza que, l’orina després d’un massatge prostàtic pot ser el fluid ideal per a la recerca de nous biomarcadors capaços de discriminar entre pacients amb o sense càncer de pròstata. L’objectiu principal de l’estudi és diagnosticar el CP assimptomàtic per us sistema minimament invasiu, que consisteix en l’anàlisi de l'ARN o de la proteïna en l'orina obtinguda després d’un massatge prostàtic i, superar la baixa especificitat de l’actual sistema d’screening (PSA en sèrum) mitjançant l'ús de biomarcadors addicionals (RNA o proteina) per tal de reduir el nombre de biòpsies innecessàries (reduir els costos socio-econòmics, així com la reducció dels efectes secundaris no desitjats). 1. ANÀLISI TRANSCRIPTÒMICA: 1a) “PSGR and PCA3 as Biomarkers for the Detection of Prostate Cancer in Urine” Rigau M et al., Prostate. 2010 Dec 1;70(16):1760-7. En el primer estudi, mitjançant l’anàlisi de transcripts per RTqPCR en 215 (34% amb CP) mostres de sediements d’orines de pacients (obtingudes després d’un massatge prostàtic), es va determinar la utilitat de PSGR (Prostate Specific G-coupled receptor), previament descrit com a sobre-expressat en mostres de teixit de CP, com un nou biomarcador, comparable a PCA3 (ja conegut), per a la detecció del CP en orines (taula 1). Per l'anàlisi de la corba ROC vàrem determinar els valors de l’Area sota la corba (AUC) de PSGR (AUC 0,68) i PCA3 (AUC 0,66). Fixant la sensibilitat a 95%, aconseguim un valor d’especificitat de 15% per PSGR i el 17% de PCA3. Alhora vàrem veure que al combinar els dos biomarcadors (PSGRvPCA3) mitjançant un anàlisi “multiROC”, els resultats van millorar significativament en termes de rendiment del biomarcador (AUC 0,73) com de la seva especificitat (34%). En conclusió, podem dir que PSGR presenta un comportament similar al biomarcador estàndard per CP en orina (PCA3), però, és necessàri la realització d’estudis futurs per millorar encara més el rendiment d'aquesta prova. 1b) “A Three-Gene panel on urine increases PSA specificity in the detection of prostate cancer” Rigau et al., Prostate. 2011 Apr 25. doi: 10.1002/pros.21390. En un segon estudi, vàrem estudiar la possibilitat de multiplexar diferents biomarcadors, per tal de ser capaços de guanyat especifcitat sense perdre sensibilitat en la detecció del CP. Donada l’heterogeneïtat del CP la idea d’utilitzar diferents marcadors per a la seva detecció es presenta atractiva. Mitjançant l’anàlisi de transcripts, per RTqPCR, en 215 (37% amb CP) mostres de sediements d’orines de pacients (obtingudes després d’un massatge prostàtic), vàrem determinar la utilitat de PSMA (Prostate Specific Membrane Antigen), un marcador descrit anteriorment com a sobre-expressat en el CP, com a biomarcadors per a la detecció de CP en orina. L’anàlisi de corbes ROC revela un rendiment similar PSMA (AUC 0,62), PSGR (AUC 0,65) i PCA3 (AUC 0,60) (Taula 1). Per tal de millorar l'eficàcia diagnòstica d'aquests biomarcadors es van combinar PSMAvPSGRvPCA3 (3M), utilitzant un anàlisi de multiROC. L’AUC del 3M (AUCm) (0,74) millora notablement, en comparació amb els valors individuals per a cada un dels marcadors. D'altra banda, si combinem 3M amb el valor de PSA en sang l’AUCm (0,77) va ser encara millor (taula 11). Si fixem la sensibilitat a 96%, l'especificitat del model 3M manté una especificitat del 34%, mentre que no es va observar augment en d’aquest valor quan ho combinem amb PSA en sang (34%) (Taula 11). El comportament d'aquests tres marcadors en una subpoblació de l’estudi que presentava nivells de PSA en sang entre 4-10 ng/mL i sense informació d’una biòpsia prèvia "zona gris del PSA" és de PSMA (AUC 0,74), PSGR (AUC 0,66) i PCA3 (AUC 0,61). Per al model combinat (3M), l’AUCm és de 0,82 (Taula 11). Fixant la sensibilitat al 96%, l'especificitat del model combinat (3M) manté una especificitat del 50%. En conclusió, aquests resultats demostren que la combiació de diferents marcadors proporciona una major especificitat a la prova sense comprometre el valor de sensibilitat. Expresat en el nombre de biopsies que s’haurien pogut estalviar, els valors se situen al voltant del 34% de biòpsies estalviades. 1c) “Behavior of PCA3 gene in the urine of men with high grade prostatic intraepithelial neoplasia” Morote and Rigau et al., World J Urol. 2010 Dec;28(6):677-80. El tercer estudi, està centrat en la utilitat, del ja conegut biomarcador per CP en orina (PCA3), per a l’identificació una lessió pre-maligna a la pròstata, així com la seva utilitat en la selecció dels homes amb biòpsies negatives, que requereixen la repetició del procediment. La troballa de les lesions de HGPIN (High grade preneoplastic intraepithelial lesion) és una indicació freqüent de la repetició de la biòpsia. Tot i la controvèrsia al voltant de la definició de HGPIN com a lessió pre-maligna i la necessitat de repetició de la biòpsia, encara avui en el nostre hospital s’indica el control exhaustiu del pacient, així com la repetició de la biòpsia en un periode de relativament curt de temps. Per a aquest estudi es van seleccionar un subgrup de pacients amb HGPIN (n=64) i com a grups control, es van seleccionar els homes amb CP (n=83) i homes amb patologia benigne de la pròstata (BP) (n=97). Després de l’anàlisi dels transcripts de PCA3 per RTqPCR en mostres de sediements d’orines de pacients (obtingudes després d’un massatge prostàtic), i de l'anàlisi de la corba ROC, es van obtenir els següents resultats; HGPIN vs CP (AUC 0,63) i BP vs PC (AUC 0,71). Aquests resultats demostren la capacitat dels transcripts de PCA3, en orina obtinguda deprés d’un massatge prostàtic, per a l’identificació d’homes amb CP així com la identificació de pacients amb HGPIN. 2. ANÀLISI PROTEÒMICA COMPARATIVA L’objectiu principal d’aquesta segona part és determinar un perfil proteòmic a l’orina capaç de diferenciar entre pacients amb CP i controls sans. Per dur a terme aquest objectiu ens vàrem plantejar fer una part inicial de discovery, que vàrem complementar amb “data mining” i finalment vàrem verificar mitjançant una tècnica proteòmica independent. 2a) Per la part de discovery es va dur a terme l’anàlisi comparativa de “Differential in Gel Electrophoresis (DIGE). Es van fer dos experiments independents, amb mostres de sobrenedants d’orines (on trobem la fracció soluble de les proteïnes). En el primer experiment es van comparar 6 mostres de proteïna total de pacients amb CP i 6 mostres controls. En el segon experiment es van comparar 9 mostres, de proteïna pre-tractada amb una tècnica de depleció per les proteïnes majoritàries, de pacients amb CP i 9 mostres de pacients control. Un total de 24 proteïnes (9 sobre- i 15 infra-expresades) vàren ser identificades per MALDI-TOF. Una anàlisi informàtic de les proteïnes va mostrar que la majoria d'aquestes proteïnes identificades es secreten els components de diverses xarxes ben conegudes, el càncer funcionals i la inflamació, com ara NFКB, PDGFBβ i β-catenina. D'altra banda, a causa del fet que hem utilitzat sobrenadants d'orina, on podem trobar proteïnes solubles secretades, la majoria de les proteïnes identificades (62%) es van localitzar en l'espai extracelular. Mitjançant una tècnica de proteòmica dirigida (Selected Reaction Monitoring: SRM) es van quantificar de forma relativa, sense l’adició d’estandards interns marcats, un total de 15 (dels 24 biomarcadors) en una població independent de 50 mostres (38% amb CP). Després d'una anàlisi de regressió logística de les dades obtingudes a través de l'assaig de SRM, es va obtenir un panell de set pèptids que corresponen a 5 proteïnes, capaços de distingir entre les mostres de CP i mostres controls amb una sensibilitat del 95% i una especificitat del 78 %. 2b) La segona part de l’estudi de proteómicas es basa en la verificació de les proteïnes prèviament qualificades i alhora l’estandarització dels mètodes de preparació de mostres per al seu posterior anàlisis mitjançant la tècnica de SRM. Les 5 proteïnes que formen part del panell identificat, juntament amb altres proteïnes biologicament rellevants, definides a la nostre fase de discovery, i juntament amb altres proteïnes descrites a la literatura, vàren ser escollides per una segona fase de qualificació i verificació en una població independent de 107 pacients, mitjançant un assaig de SRM en el qual es pretén quantificar de forma absoluta (adició de pèptids estàndards marcats), un total de 42 proteïnes. Prèviament a l’evaluació d’aquestes proteïnes, es va dur a terme una estandarització del protocol d’extracció de proteïna en mostres d’orina, amb l’objectiu de maximitzar la senyal obtinguda en els experiments de SRM. La precipitació amb Acetonitril (a temperatura ambient) seguida de la digestió de les proteïnes en solució amb condicions de ratio 1:10 (trypsine:protein), “over-night” i l’adició de “NAcetyl cysteine”, proporcionen els millors valors de quantificació absoluta. L’anàlisi estadístic de les dades generades d’aquesta segona fase de qualificació i verificació de les proteïnes està encara inacabat, de manera que ara per ara no podem conclou-re res. En conclusió, les dos linies experimentals que aquí es presneten (ARN i proteïnes) represeten resultats prometedors. No obstant això, estudis de validació en poblacions independents (del tipus estudi multicèntric) són necessaris per acabar amb un biomarcador o un panell de biomarcadors vàlid per al diagnòstic del CP. Per altra banda, el seguiment dels pacients ja analitzats poden relevalar dades interessant, com la utilitat pronòstica d’aquests biomarcadors. Els resultats obtinguts haurien de tenir una aplicació clínica ràpida que, juntament amb els paràmetres d’screening actual (nivells sèrics de PSA i DRE), podrien ajudar en la pressa de decisions per CP.
The prostate gland is a walnut-sized organ of the male urinary-genital tract that is located below the bladder surrounding the urethra and in front of the rectum. The most important disease affecting prostate is prostate cancer (PCa). PCa is the most common cause of cancer death, and it is the second most common cause of cancer death among men in the Western world. The risk of developing this type of cancer during a lifetime is estimated at 1 in 6 men in the US, and the risk of death due to this disease is 1 in 36. The current diagnosis of PCa is based on a triad consisting of diagnosis, analysis of the levels of PSA (Prostate Specific Antigen) in serum, the digital rectal examination (DRE) and finally the prostate biopsy (PB). When serum PSA levels are above 4 ng/mL and / or when DRE is suspected, the urologist can estimate how likely the patient is affected by PCa and therefore decided the need to practice or not a PB, which is the gold standard for PCa diagnosis. The introduction of PSA testing in the late 80s, has resulted in an improvement of early diagnosis of PCa, at which time options for treatment are effective. However, despite this early detection, mortality PCa has not decreased significantly in the last 50 years. The main limitation of serum PSA as a tumor marker is its lack of specificity (around 30%) at the cut-off value of 4 ng/mL, and also a low negative predictive value (NPV), which results in a high rate of negative biopsies. Elevated PSA levels can also be attributed to other factors such as benign prostatic hyperplasia (BPH), prostatitis, etc. As a consequence of the current screening parameters, around 2/3 of the approximately 1,300,000 biopsies made yearly in the United States and 390,000 in Europe are unnecessary. In contrast, the false positive rate of a biopsy is about zero, although the false negative rate in the first biopsy may oscillates around 20%. As a result of their persistently elevated PSA levels, but negative biopsy results, these men undergo repeated biopsies to rule out PCa. This situation is called the “PSA dilemma”. For these reasons, PCa would benefit from the existence of new markers for screening and also a more specific diagnosis less invasive. Furthermore, an improvement in diagnosis would avoid many unnecessary biopsies and consequently a significant savings in the cost of health today. The search for new markers in PCa is an important field of work in the early detection of this cancer. Urine has been defined as a liquid biopsy of the urogenital tract, and it can provide much more information about these organs (including the prostate) than a tissue biopsy. Urine obtained after DRE can easily serve as a mirror of what is happening within the prostate. Furthermore, urine collection can be accomplished without disruption of clinical standard practice. It can also be repeated several times throughout the course of the prostatic disease. For all of these reasons, urine can serve as a potential source of prostate disease biomarkers. Nevertheless, using urine for biomarker discovery represents an important technical challenge, both in transcriptomic and proteomic approaches. Although there are some studies that focus on those approaches and biomarker discovery and identification, there still exists some controversy regarding the standardization of collection procedures, sample processing, storage and normalization. We hypothesized that the utilization of targeted genomic and proteomic techniques on urine samples from patients suspected of having PCa can provide a pattern of biomarkers able to efficiently distinguish between the presence or absence of a prostate carcinoma and, further, can help to identify clinically significant prostate cancer patients. The main objective of this study is to diagnose asymptomatic PCa by non/minimally-invasive means using RNA or Protein in urine after prostate massage, and to overcome the low specificity of PSA by the use of additional biomarkers to reduce the number of unnecessary biopsies (reduce financial costs for society, reduction in unwanted secondary effects). 1. TRANSCRIPTOMIC APPROACH: In recent years, the explosion of genomic and transcriptomic approaches have resulted in increased biomarker discovery. The recent discovery of Prostate Cancer Gene 3 (PCA3) in urine as a biomarker for the detection of PCa and studies to determine its applicability in routine diagnosis represent a significant success for the scientific community in this field. First, we wanted to characterize a new urine candidate biomarker (PSGR) to be compared with PCA3, and second, we planned to use a panel of biomarkers, in order to improve diagnostic accuracy. Finally, we proposed to better characterize the well-known biomarker PCA3 as a tool for the early detection of pre-neoplastic PCa lesions, such as High grade prostatic intraepithelial neoplasia (HGPIN). 1a) “PSGR and PCA3 as Biomarkers for the Detection of Prostate Cancer in Urine” Rigau M et al., Prostate. 2010 Dec 1;70(16):1760-7. PSGR is a member of the G-protein coupled OR family. PSGR has previously been described to be highly prostate tissue-specific and over-expressed in PCa tissue. Our aim was to test whether PSGR could also be detected by RTqPCR in urine sediment obtained after prostate massage (PM). A total of 215 urine samples were collected from consecutive patients (34% with PCa), who presented for PB due to elevated serum PSA levels (> 4 ng/mL) and/or an abnormal DRE. These samples were analyzed by RTqPCR. By univariate analysis we found that PSGR and PCA3 were significant predictors of PCa. A Reciever Operator Characteristics (ROC) curve was used to assess the outcome predictive values of the individual biomarkers. We obtained the following Area Under the Curve (AUC) values: PSGR (0.68) and PCA3 (0.66). Both markers individually overcame the AUC value for serum PSA (0.60). Finally, we combined those markers to test if a combination of both biomarkers could improve the sensitivity of PCA3 alone. By using a multivariate extension analysis, multivariate ROC (MultiROC), the outcome predictive values of the paired biomarkers were assessed. We obtained an AUC value of 0.73 for the combination of PSGR and PCA3 (PSGRvPCA3). Then, we tested whether a combination of PSGR and PCA3 could improve specificity by fixing the sensitivity at 95%. We obtained specificities of 15% (PSGR) and 17% (PCA3) for each individual marker and 34% for PSGRvPCA3. In summary, a multiplexed model that included PSGR and PCA3 improved the specificity for the detection of PCa, especially in the area of high sensitivity. This could be clinically useful for determining which patients should undergo biopsy. 1b) “A Three-Gene panel on urine increases PSA specificity in the detection of prostate cancer” Rigau et al., Prostate. 2011 Apr 25. doi: 10.1002/pros.21390. Much evidence points to the fact that a single marker may not necessarily reflect the multifactorial and heterogeneous nature of PCa. The principle that underlies the combined biomarker approach is consistent with tests offered for the detection of PCa in tissue specimens and takes into consideration the heterogeneity of cancer development based on a diagnostic profile. The combined model that results from these combinations provides overall increased sensitivity without decreasing the specificity. Following the same approach than in our previous work, we combined three biomarkers to maximize individual specificities. Prostate Specific Membrane Antigen (PSMA), another well-known PCa biomarker, was used to test whether a combination of PSGR, PCA3 and PSMA was able to improve the specificity of the current diagnostic technique. We analyzed post-PM urine samples from 154 consecutive patients (37% with PCa), who presented for PB due to elevated serum PSA levels (>4 ng/mL) and/or an abnormal DRE. We tested whether the putative PCa biomarkers PSMA, PSGR, and PCA3 could be detected by RTqPCR in the post-PM urine sediment. By univariate analysis, we found that the PSMA, PSGR, and PCA3 scores were significant predictors of PCa. We then combined these findings to test if a combination of these biomarkers could improve the specificity of an actual diagnosis. Using a multiplex model (PSGRvPCA3vPSMA), the area under the MultiROC curve (AUCm) was 0.74, 0.77 with PSA and 0.80 with PSA density (PSAD). Fixing the sensitivity at 96%, we obtained a specificity of 34%, 34% with PSA and 40% with PSAD. Afterwards, we specifically tested our model for clinical usefulness in the PSA diagnostic ‘‘gray zone’’ (4–10 ng/mL) on a target subset of 82 men with no prior biopsy (34% with PCa) and a target subset of 77 men with the PSAD information (35% with PCa). Using a multiplex model, the AUCm was 0.82, 0.89 with PSAD. Fixing the sensitivity at 96%, we obtained a specificity of 50% and 62% with PSAD in the gray zone. This model would allow 34% of the patients to avoid unnecessary biopsies in the gray zone (42% when using PSAD). 1c) “Behavior of PCA3 gene in the urine of men with high grade prostatic intraepithelial neoplasia” Morote and Rigau et al., World J Urol. 2010 Dec;28(6):677-80. An ideal biomarker for the early detection of PCa should also differentiate between men with isolated HGPIN and those with PCa. PCA3 is a highly specific PCa gene, and its score in post-PM urine seems to be useful in ruling out PCa, especially after a negative PB. The biopsy finding of an HGPIN is a frequent indication that the PB should be repeated. The aim of this study was to determine the efficacy of post-PM urine PCA3 scores for ruling out PCa in men with previous HGPIN. The PCA3 score was assessed by RTqPCR in 244 post- PM urine samples collected from men subjected to PB (64-isolated HGPIN, 83-PCa, and 97-benign pathology findings (BP)). The median PCA3 score was 1.56 in men with BP, 2.01 in men with isolated HGPIN and 9.06 in men with PCa. A significant difference was observed among the three scores (p < 0.001) and also between HGPIN and PCa (p = 0.008); however, no differences were observed between HGPIN and BP (p = 0.128). The AUC in the ROC analysis was 0.71 in the subset of men with BP and PCa, while it decreased to 0.63 when only men with isolated HGPIN and PCa were included in the analysis. Finally, the median of the PCA3 scores was assessed in men with previously diagnosed unifocal HGPIN (2.63) and in men with previously diagnosed multifocal HGPIN (1.59). No differences were observed between unifocal and multifocal HGPIN (p = 0.56). In conclusion, the efficacy of post-PM urine PCA3 scores in ruling out PCa in men with HGPIN is less than in men with BP. For this reason, when HGPIN is found at PB, these results should be taken into consideration, in order to establish the clinical usefulness of the PCA3 score as a tool for avoiding unnecessary repeated biopsies. 2. PROTEOMIC APPROACH: The high-throughput proteomic analysis of urine samples has recently become a popular approach for the identification of novel biomarkers. Proteins secreted by cancer cells, also referred to as "cancer cell secretomes," are a promising source for biomarker discovery. A great advantage to these cancer-secreted proteins and/or their fragments is that in most cases, they enter body fluids, such as blood or urine, and therefore, can be measured via non-invasive assays. Since the protein products of PCa cells can be detected in urine, their use as a proximal body fluid in the detection of PCa is very attractive. 2a.“The Discovery and Qualification of a Panel of Urine Biomarkers for Prostate Cancer Diagnosis”. In this study we used DIGE proteomic analysis on 30 age-matched, post-PM urine supernatant specimens, in order to identify the differentially expressed proteins in patients with PCa. 24 potential biomarkers were identified, the majority of which were secreted proteins associated with several wellknown, functional cancer pathways. Qualification of 15 of the 24 identified biomarker candidates was then undertaken by relative quantification using an SRM-based assay (target) on 50 post-PM urine supernatant samples (38% with PCa). After statistical analysis, 7 peptides, corresponding to 5 different proteins, were selected. A multiplex ROC curve using those 7 peptides showed an AUC value of 0.93. Fixing the sensitivity at 95%, we achieved a specificity of 78%. 2b. “Qualification and Verification of Urine PCa Candidate Biomarkers with Selected Reaction Monitoring”. The qualification and verification of candidate biomarkers is a critical stage in the great biomarker discovery pipeline. Credentialed biomarkers that have successfully passed through this stage are considered verified biomarkers, which are of high value for translation into large-scale, clinical validation studies. The evaluation of biomarkers in body fluids necessitates the development of robust methods to quantify proteins in body fluids, using large sets of samples. In the present study, we performed the qualification of a set of 42 candidate biomarkers for PCa diagnosis on a set of 107 post- PM urine supernatant samples (36% with PCa) using SRM-based absolute quantification. Before that, urine sample preparation and analytical procedures were optimized for SRM methodology. We standardized preparation of the urine protein samples for SRM analysis by using 9 different protocols. Our final goal was to obtain a panel of biomarkers that alone, or in combination with the existing PCa biomarker, would help us to better define patients with PCa. In addition, due to the large number of samples and their pathological conditions, we would also be able to define candidate prognostic markers. However, this study has yet to be completed. In conclusion, the data presented in this dissertation represent a significant advance in the standard care for PCa diagnosis. Our two approaches (RNA- and Protein-based) have begun to yield promising results, as both have levels of specificity that exceed those of PSA. However, validation studies on larger, multi-centric cohorts of urine samples are needed to end up with a valid PCa biomarker. The obtained results should have a rapid application in the clinics and potentially influence, together with actual screening parameters (serum PSA and DRE), decisions that could improve the health system, as well as clinical, managerial and/or public practices for health outcomes in PCa.

Introduction: In this study we exploited the recent development of methods that have enabled the analysis of RNA present in urine exosomes of prostate cancer patients. We report RNA expression patterns that contain diagnostic and prognostic information for prostate cancer, and association with response to hormone treatment. Methods: First catch urine following digital rectal examination were collected from 662 men. 3 groups of patients were used: Low, Intermediate, and High-risk according to NICE stratification criteria, and two control groups: benign and advanced disease. 50-gene transcript expression analysis using NanoString technology was performed on 192 samples. Exosomal RNA Next-Generation Sequencing was performed on 18 samples for novel biomarker discovery. Results: Expression analysis showed that PCa-specific transcripts such as TMPRSS2/ERG fusion transcripts were identifiable in exosomes from PCa urine samples. LPD analysis highlighted expression levels of 15 transcripts with diagnostic potential (significantly up-regulated in cancer samples in comparison to benign control) and 17 transcripts with prognostic potential (differentialy expressed in high risk and advanced disease in comparison to lower grade disease). I also report two gene transcripts (SERPINB5/Maspin, HPRT) that were significantly differentially expressed in patients who failed to respond to hormone deprivation therapy for high risk/metastatic disease. Three genes (STEAP4, ARexons4_8 and NAALADL2) were significantly differentially expressed in patients who relapsed within 12 months of hormone treatment initiation. Next-Generation Sequencing of twenty samples identified 45 genes to be significantly differentially expressed between non-cancer and cancer samples (28 were up regulated and 17 down regulated). 33 out of the 45 genes showed a significant linear trend in association with cancer risk. Conclusions: Urine Exosomal RNA contains PCa specific transcripts. Gene expression analysis and Next Generation Sequencing identified genes that are significantly differentially expressed between cancer and non-cancer cases as well as prognostic genes and genes that can predict response to hormone treatment.

Whilst biomarker research is gaining momentum within the cancer sciences, disappointingly few biomarkers are successfully translated into clinical practice, which is partly due to lack of rigorous methodology. In this thesis, I aim to systematically study several quantitative magnetic resonance imaging (MRI) biomarkers (QIBs), at various stages of biomarker development for use as tools in the assessment of local and metastatic prostate cancer according to clinical need. I initially focus on QIBs derived from conventional multiparametric (mp) prostate MRI sequences, namely T2 weighted (T2W), apparent diffusion coefficient (ADC) and dynamic contrast enhanced (DCE). Firstly, by optimising analytical methods used throughout the thesis, deciding which approach is more reliable between single-slice region-of-interest vs. contouring the whole tumour volume using two different software packages. I then consider whether metric reproducibility can be improved by normalisation to different anatomical structures, and assess whether it is preferable to use statistics derived from imaging histograms rather than the current convention of using mean values. I combine multiple QIBs in a logistic regression model to predict a Gleason 4 component in known prostate cancer, which represents an unmet clinical need, as noninvasive tools to distinguish these more aggressive tumours do not currently exist. I subsequently ‘technically validate’ a novel microstructural diffusion-weighted MRI technique called VERDICT (Vascular, Extracellular and Restricted Diffusion for Cytometry in Tumours) to detect aggressive prostate cancer as part of a prospective cohort study. I assess the image quality, contrast-to-noise ratio, repeatability and performance of quantitative parametric VERDICT maps to discriminate between Gleason grades vs. the current best performing, but still imperfect tool of ADC. In the final two results chapters, motivated by the limited diagnostic accuracy of the prostate cancer staging modalities in current clinical use, I investigate the ability of mp whole-body (WB) MRI to stage aggressive cancer outside the prostate in patients with a high risk of metastases at primary diagnosis, and in biochemical failure following prostatectomy.

Prostate cancer is the most common noncutaneous cancer among men, yet current diagnostic methods are insufficient and more reliable diagnostic markers need to be developed. The answer that can bridge this gap and enable more efficient diagnoses may lie in microRNAs. These small, single stranded RNA molecules impact protein expression at the translational level and regulate important cellular pathways. Dysregulation of these small RNA molecules can have tumorigenic effects on cells and lead to many types of cancers. Currently the Prostate-Stimulating Antigen (PSA) is used as a diagnostic marker for prostate cancer. However, many factors can elevate PSA levels such as infections and certain medications, consequently leading to false positive diagnoses and unnecessary concern and over treatment with dire outcomes for the patient. Even worse, are the chances of false negative diagnoses, which result in prostate cancer not being diagnosed until its later stages. Therefore, although the use of the PSA level has had its uses in the clinic, it has failed to sufficiently bridge the gap or to distinguish indolent from aggressive disease. It has long been suggested in the literature that microRNAs are drastically altered throughout the course of cancer progression. Here, RNA sequencing was used to identify changes in miR expression profiles diagnostic for prostate cancer patients compared to non-patient controls. The RNA sequencing results were also used to identify normalization miRs to be used as endogenous controls. Confirmatory qRT-PCR was then used to corroborate these results for the top seven dysregulated miRs found from the RNA sequencing data. Data analysis of the Area Under the Curve (AUC) of the Receiver Operating Curves (ROC) of the selected miRs exhibited a better correlation with prostate cancer (AUC Range= 0.819- 0.950) than PSA (AUC of PSA=0.667). In summary, a panel of seven miRs are proposed, many of which have prostate specific targets, which would represent a significant improvement over current testing methods.

Background: Recent evidence suggests that the prostate cancer (PCa)-specific up-regulation of certain genes such as AMACR, EZH2, PSGR, PSMA and TRPM8 could be associated with an aberrant expression of non-coding microRNAs (miRNA). Methods: In silico analyses were used to search for miRNAs being putative regulators of PCa-associated genes. The expression of nine selected miRNAs (hsa-miR-101, -138, -186, -224, -26a, -26b, -374a, -410, -660) as well as of the aforementioned PCa-associated genes was analyzed by quantitative PCR using 50 malignant (Tu) and matched non-malignant (Tf) tissue samples from prostatectomy specimens as well as 30 samples from patients with benign prostatic hyperplasia (BPH). Then, correlations between paired miRNA and target gene expression levels were analyzed. Furthermore, the effect of exogenously administered miR-26a on selected target genes was determined by quantitative PCR and Western Blot in various PCa cell lines. A luciferase reporter assay was used for target validation. Results: The expression of all selected miRNAs was decreased in PCa tissue samples compared to either control group (Tu vs Tf: -1.35 to -5.61-fold; Tu vs BPH: -1.17 to -5.49-fold). The down-regulation of most miRNAs inversely correlated with an up-regulation of their putative target genes with Spearman correlation coefficients ranging from -0.107 to -0.551. MiR-186 showed a significantly diminished expression in patients with non-organ confined PCa and initial metastases. Furthermore, over-expression of miR-26a reduced the mRNA and protein expression of its potential target gene AMACR in vitro. Using the luciferase reporter assay AMACR was validated as new target for miR-26a. Conclusions: The findings of this study indicate that the expression of specific miRNAs is decreased in PCa and inversely correlates with the up-regulation of their putative target genes. Consequently, miRNAs could contribute to oncogenesis and progression of PCa via an altered miRNA-target gene-interaction.

Identification of novel biomarkers of breast and prostate cancers to supplement current diagnostic, prognostic and predictive tools is an important clinical need. Interaction between the host’s immune system and tumours is reflected in detectable changes in profiles of proteins in blood cells and therefore these cells can be a source for cancer biomarkers. In a previous investigation conducted in our laboratory, a panel of proteins differentially expressed in white blood cells (WBCs) of patients with primary and advanced breast cancer (BRCA and ABC cohorts, respectively) was identified using integrated approaches. The aim of the present study was to assess the biomarker potential of five proteins: Serpin B1, Lipocalin 2, Copine 3, Integrin a4 and 5LOX, using Western blot, immunofluorescence, flow cytometry and ELISA techniques. Serpin B1, Lipocalin 2, 5LOX and Copine 3 were present in neutrophils, whereas Integrin a4 was detected in T-lymphocytes. Significantly higher levels of Serpin B1 and Lipocalin 2, and lower levels of Integrin a4 were observed in WBCs in the BRCA cohort compared to healthy individuals, suggesting their utility as early breast cancer biomarkers. Levels of Serpin B1 and Lipocalin 2 increased and Integrin a4 decreased as the disease advanced, indicating their utility as biomarkers of poor prognosis. Lower basal levels of Serpin B1 in patients undergoing chemotherapy and endocrine treatment correlated with complete response to treatment. Additionally, the utility of BORIS as a diagnostic marker was investigated in plasma of breast cancer patients and urinary sediments and exosomes of prostate cancer patients. The presence of BORIS autoantibodies was demonstrated in plasma of breast cancer patients. The highest levels of BORIS mRNA were detected in urine of prostate cancer patient with the advanced disease. This indicates potential of the BORIS for breast cancer diagnosis and its utility for the prediction of the aggressiveness in prostate cancer.

Background and Aim: Prostate cancer (PCa) is the leading cancer diagnosis amongst South African men. The incidence of PCa is 68.0 per 100 000 Age Standardized Rate (ASR) and the mortality rates are 27.9 per 100 000 ASR; Globocan 2018. Diagnosis of PCa is based on a combination of digital rectal examination, prostate-specific antigen (PSA) and histology. Several biomarkers have been used to increase the sensitivity and specificity of PSA in distinguishing patients with PCa from those with benign prostatic hyperplasia (BPH). These include fractionated PSA, free/total PSA ratio, −2proPSA, prostate cancer antigen 3 and prostate health index amongst others. Biomarkers are needed to differentiate BPH from PCa due to a lack of specificity of these markers with PSA levels above 4.0 ng/ml. The aim of this study is to investigate gene expression patterns of South African men in 9 PCa and 10 BPH patients in order to distinguish between the two groups. Methods: Ethical approval was obtained (HREC 454/2012). Patients scheduled for transurethral resection of the prostate were recruited from the Western Cape. RNA was extracted from prostate tissue using the AllPrep DNA/RNA/miRNA Universal Kit (Qiagen). Complementary DNA was synthesized from RNA using the SuperScript IV VILO Master Mix (Thermo Fischer Scientific). Gene expression was analyzed with the Human Prostate Cancer RT2 Profiler PCR Array and SYBR Green Master Mix. Data were analyzed with the GeneGlobe RT2 and miScript PCR Array Data Analysis Centre from Qiagen. Results: The cohort included patients from different ethnic groups namely, Caucasians, Mixedand African ancestry. The PCa group has an age range from 56 to 75 years (mean 65) while the BPH group was slight older ranging from 60 to 76 years (mean 68). PSA levels range from 24 to 5000 ng/ml (mean 1252 ng/ml, median 185) for the PCa group and 11 to 58 ng/mL (mean 25 ng/ml, median 22) for the BPH group. The following genes were downregulated 2-fold in the PCa group with p values s <0.05; IGF1, PTEN, GSTP1, SOCS3, EGR3, GPX3, TIMP3, ZNF185, DKK3, PTGS2, FOXO1, ARNTL, TNFRSF10D, CCND1, and DLC1, upregulated genes included; CDH1, MKI67, TMPRSS2, ERG, CDKN2A, FASN, and AR but were not statistically significant. At a fold change threshold of 1.5, the following additional genes were downregulated in the PCa group with p values <0.05; DAXX, EGFR, RASSF1, SOX4, and TIMP2, upregulated genes were ACACA, AR, CDKN2A, ERG and FASN but were also not statistically significant. The study shows similarly differentially expressed genes as seen in international studies. Of note PTEN, MKI67 and FASN which are associated with poor prognosis. EGR3 was downregulated in our study and this has been associated aggressive disease and predict relapse after PCa treatment. This could explain the high mortality demonstrated in South African epidemiological studies. Conclusion: We identified a group of differentially expressed genes that have potential in distinguishing PCa and BPH patients with PSA values above 10 ng/ml. A larger population study is needed to further evaluate the clinical significance of our findings.