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Prostate Cancer Microparticles in Men Undergoing Radical Prostatectomy

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Prostate Cancer Microparticles in Men Undergoing Radical Prostatectomy Western University Scholarship@Western Electronic Thesis and Dissertation Repository 12-12-2017 6:45 PM Prostate Cancer Microparticles in Men Undergoing Radical Prostatectomy Malcolm James Dewar The University of Western Ontario Supervisor Hon Leong The University of Western Ontario Graduate Program in Surgery A thesis submitted in partial fulfillment of the equirr ements for the degree in Master of Science © Malcolm James Dewar 2017 Follow this and additional works at: https://ir.lib.uwo.ca/etd Part of the Biological Phenomena, Cell Phenomena, and Immunity Commons, Male Urogenital Diseases Commons, Neoplasms Commons, Oncology Commons, Translational Medical Research Commons, and the Urology Commons Recommended Citation Dewar, Malcolm James, "Prostate Cancer Microparticles in Men Undergoing Radical Prostatectomy" (2017). Electronic Thesis and Dissertation Repository. 5140. https://ir.lib.uwo.ca/etd/5140 This Dissertation/Thesis is brought to you for free and open access by Scholarship@Western. It has been accepted for inclusion in Electronic Thesis and Dissertation Repository by an authorized administrator of Scholarship@Western. For more information, please contact [email protected]. Abstract Objectives: To determine changes in prostate microparticle (PMP) concentrations in men with prostate cancer (PCa) after digital rectal examination (DRE), after radical prostatectomy (RP), and at follow-up. Materials and Methods: 22 men were recruited before RP. Four blood specimens were collected – baseline (specimen 1), post-DRE (specimen 2), immediately post-RP (specimen 3), and follow-up (specimen 4). Pre- and post-DRE urine was collected (Specimen A and B respectively). Flow cytometric analysis of biofluids was performed with fluorescent-labeled antibodies against prostate-specific membrane antigen (PSMA) and polysialic acid. Total MP (TMP) and dual positive (PMP) events per µl of plasma or urine were recorded. Results: Median TMPs from specimen 1 to 4 were 3 005 500, 1 600 600, 976 353, and 3 951 400 events/µl, respectively (p<0.001). Median proportional PMPs increased from 9.6% to 17.3% after DRE (p<0.001), to 25.7% (p<0.001) post-RP, and decreased to 4.1% at follow-up (p=0.170). Urinary PMPs were unchanged after DRE. Conclusions: Compared to baseline levels, proportional PMP events were significantly increased following DRE, and further following RP. Keywords: Prostate Cancer, Biomarker, Liquid Biopsy, Diagnosis, Risk Stratification, Microparticle, Extracellular Vesicle, Exosome, Ectosome, Digital Rectal Examination ii Dedication To my wife Lucille, who has selflessly and lovingly supported me through this fellowship, this degree, and the writing of this thesis. Acknowlegements This study was made possible by a research grant received from the University of Western Ontario Department of Surgery Internal Research Fund. Thanks go to my supervisor, Dr. Hon Leong, for introducing me to the Master’s program, and for having the drive and the vision to enable this research. To Dr. Stephen Pautler, who welcomed me into his OR right at the beginning of the year. His infectious enthusiasm for the research kept me motivated To Dr. Joseph Chin, who has been nothing but kind and supportive. To Dr. Karla Williams, Sabine Brett, and Dr. Khurram Siddiqui, and others, whose previous work in the laboratory formed the basis of my study. Thanks to Karla for answering every small question that I had. iii Table of Contents Abstract ...................................................................................................................................... ii Dedication ................................................................................................................................. iii Acknowlegements ..................................................................................................................... iii Table of Contents ...................................................................................................................... iv List of Tables ............................................................................................................................. vi List of Figures ............................................................................................................................ vi List of Appendices ..................................................................................................................... vi List of Abbreviations ................................................................................................................ vii Introduction ..................................................................................................................... 1 Chapter 1: Literature Review ............................................................................................ 1 Methods ..................................................................................................................... 1 Current Biomarkers for Prostate Cancer .................................................................... 1 Prostate-Specific Antigen ....................................................................................... 2 Non-invasive biomarkers ........................................................................................ 5 Tissue biomarkers ................................................................................................. 14 Imaging ................................................................................................................. 19 Summary ............................................................................................................... 21 Extracellular Vesicles and Prostate Cancer .............................................................. 22 History ................................................................................................................... 22 Classification and Origins of EVs ........................................................................... 23 Qualitative and Quantitative Measurement ........................................................ 24 Structure and Function ......................................................................................... 26 Microparticles in Cancer ....................................................................................... 27 Microparticles and the Prostate ........................................................................... 29 Role as a Prostate Cancer Biomarker ................................................................... 31 Cell surface antigens ............................................................................................. 33 Chapter 2: Study Methodology ....................................................................................... 38 Background ............................................................................................................... 38 Objectives of the DRE Study ..................................................................................... 39 Recruitment .............................................................................................................. 39 Specimen Collection and Processing ........................................................................ 39 Antibody Conjugation .............................................................................................. 41 Problems with Anti-STEAP1 [J2D2] ....................................................................... 42 Nano-scale Flow Cytometry – Calibration, Thresholds, and Laser Settings ............. 43 Optimisation of Antibody Concentrations ............................................................... 47 Chapter 3: Influence of Digital Rectal Examination on Prostate Cancer Microparticles ................................................................................................................ 49 iv Background and Rationale ....................................................................................... 49 Aims and Objectives ................................................................................................. 49 Materials and Methods ............................................................................................ 49 Results ...................................................................................................................... 54 Chapter 4: Urinary Prostate Cancer Microparticles in Men with Prostate Cancer ............. 59 Background and Rationale ....................................................................................... 59 Aims and Objectives ................................................................................................. 59 Materials and Methods ............................................................................................ 60 Results ...................................................................................................................... 63 Chapter 5: Discussion ..................................................................................................... 65 Plasma Microparticles .............................................................................................. 65 Urine Microparticles ................................................................................................. 68 Challenges and Shortcomings .................................................................................. 69 Conclusions ...................................................................................................................
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