The discovery of immunophenotypes associated with the diagnosis of human Prostate Cancer

Abstract

Current tests for prostate cancer are unreliable because they are not specific enough to distinguish between benign prostatic hyperplasia (BPH) and prostate cancer. The aim of this thesis was to develop a diagnostic test for prostate cancer with improved sensitivity and specificity that required only a small sample (5-10mL) of venous blood. Antibody microarrays were used to capture populations of peripheral blood mononuclear cells in order to distinguish healthy individuals from patients with BPH or clinically significant or insignificant prostate cancer. A total of 31 statistically significant CD antigens that are differentially expressed in patients with intermediate and low-grade prostate cancer compared to BPH. The patterns provided a diagnostic “fingerprint” of the disease state of each individual. A cross-validation analysis revealed a high sensitivity and specificity, with an average area under the Receiver Operator Curve of 0.79. To validate these profile changes, a panel of eight CD markers were selected and analysed on T cells, B cells, NK cells, monocytes or platelets using flow cytometry. In parallel, work to identify a proteomic “signature” of prostate cancer was undertaken using secreted exosomes derived from prostate cell lines (PC3, Du145 and LNCaP) or primary prostate epithelial cells (PrECs) and exosomes isolated from plasma of patients with varying stages of prostate cancer or BPH. Exosomes were confirmed to be within the exosome size range of 30-150nm using nanoparticle tracking analysis and transmission electron microscopy. Scanning electron microscopy revealed exosomes captured on a polycarbonate surface using cancer-related and exosome markers. Mass spectrometry identified Annexins A1, A2, A4 and A6, tumour associated calcium signal transducer, CD5, CD9, CD44, CD49f, CD59, CD147, ICAM-1, EpCAM, enolase-1, PSMA-3 and PSMA-7, which may promote tumour progression and act as angiogenic factors that promote tumour vascularisation. The assessment of CD antigens and exosome proteins suggest that a diagnostic test for prostate cancer should not be restricted to one biomarker. Antibody microarrays may provide a vehicle to assess the antigen changes occurring as a result of prostate disease. It may also help understand the multifaceted complexity underlying the cross talk between the immune system and the prostate cancer microenvironment